Mass Spectrometry-Based Global Proteomic Analysis of Endoplasmic Reticulum and Mitochondria Contact Sites

Home , ACO1, Annexin A11, Annexin A2, Annexin A4, Annexin A5, Annexin A6

Chloe N. Poston Bachelor of Science, Clark Atlanta University, Atlanta, GA 2007 Master of Arts, Brown University, Providence, RI 02912

A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Department of Chemistry at Brown University

Providence, Rhode Island

May 2013

This dissertation by Chloe N. Poston is accepted in its present form by the Department of Chemistry as satisfying the dissertation requirements for the Degree of Doctor of Philosophy

Date

Dr. Carthene R. Bazemore-Walker, Director

Recommend to the Graduate Council

Date

Dr. Sarah Delaney, Reader

Date

Dr. J. William Suggs, Reader

Approved by the Graduate Council

Date

Peter Weber, Dean of the Graduate School

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Curriculum Vitae

EDUCATION

2010 Brown University Graduate School, Providence, RI Master of the Arts, Chemistry

2007 Clark Atlanta University, Atlanta, GA Bachelor of Science, Chemistry Provost Scholar

PUBLICATIONS C.N. Poston, E. Duong, Y. Cao, C.R. Bazemore-Walker, Proteomic Analysis of Lipid Raft- enriched Membranes Isolated from Internal Organelles, Biochemical and Biophysical Research Communications 415(2) 2011, pg. 355-360.

RESEARCH EXPERIENCE Thesis Research: Mass spectrometry based proteomic investigations of internal organelle membranes Accomplishments:  Characterized the protein content of lipid enriched detergent resistant membranes found in mitochondria and mitochondria associated membranes  Demonstrated the potential influence of detergent resistant membranes in protein and small molecule exchange across internal membranes  Identified bona fide mitochondria associated membrane proteins by protein correlation profiling

Summer 2007 Brown University Accomplishments:  Determined an optimal detergent-free organic based solvent for proteins to be analyzed by ESI LC/MS/MS

Summer 2006 University of Pennsylvania Accomplishments:  Studied quantum dots to evaluate the frequency and life-time of fluorescence

Summer 2005 Georgia Institute of Technology Accomplishments:  Optimized a synthetic approach for silica based nanotubes with hydroxy groups tethered to the outer surface  Characterized nanotube formation using transmission electron microscopy, X-ray diffraction, and infrared spectroscopy

Summer 2004 Skidaway Institute of Oceanography iv

Accomplishments:  Designed and implemented a waste removal system using aluminosilicate zeolytes  Evaluated ammonia concentration, water temperature, and mortality fish daily  Created a hydroponic garden using ammonia waste water from fish tanks

AWARDS AND RECOGNITION  NSF GK-12 Fellowship 2011-2012  ASBMB Travel Award 2011  FASEB MARC Travel Award 2010  Ford Foundation Fellowship Honorable Mention 2008

TEACHING EXPERIENCE AND TRAINING  Brown Summer High School, Life Sciences 2011  Methods in Teaching Science Training 2011  Teaching Assistant, Organic Chemistry 2007-2010

ORGANIZATION AFFILIATIONS  American Society of Biochemistry and Molecular Biology  American Chemical Society  Delta Sigma Theta Sorority, Inc

Abstract

Mass spectrometry has been a long-standing analytical tool for chemists. Tandem mass spectrometry-based proteomics capitalizes on the sensitivity, accuracy, and efficiency of this classic technique in order to investigate the protein content of specific sub-cellular regions. In this work we apply tandem mass spectrometry (MS/MS) to the analysis of mitochondria-associated endoplasmic reticulum membranes (MAMs). MAMs are a point of communication between the ER and the mitochondria that facilitate Ca2+ trafficking, protein folding, and energy metabolism. They have been implicated in the regulation of apoptosis and more recently neurodegenerative disorders including Alzheimer’s disease and Parkinson’s. The involvement of the MAM in these diseases has yet to be determined, largely because proteins localized to the MAM have yet to be elucidated. We address this gap in knowledge with our tandem mass spectrometry-based global characterization of MAMs from mouse brain tissue. We employ a gel-based LC/LC- MS/MS method that allows for the analysis of hydrophobic membrane proteins. Our analysis reveals 1,212 unique proteins in the MAM that are involved in cell signaling, small molecule trafficking, and protein processing. Bio-informatic analysis of our identified MAM proteins supports postulates that the MAM may play a role in the pathology of Alzheimer’s disease, schizophrenia, and dyskinesia. Our data also suggests that lipid rafts within the MAM are involved in signaling and transport within the region, and may play a role in the MAM’s relationship to neurodegenerative disease. The completion of this work provides a much-anticipated catalog of proteins at the MAM, which can be used for targeted studies to elucidate pathways and novel therapeutic targets in the region.

Preface and Acknowledgements

In 1676, Sir Isaac Newton wrote “if I have seen further, it is by standing on the shoulders of giants”. I, too, contend that I would not have completed this work without the giants in my life who have continuously supported my academic endeavors. I am most grateful to Carthene R. Bazemore-Walker for being a teacher, a mentor, a motivator, and a role model throughout this process. I also wish to acknowledge the members of the Bazemore-Walker group for fostering a collegial and collaborative research environment every single day. I am also appreciative of Professors Sarah Delaney and J. William Suggs for taking time away from their busy schedules to serve as my committee members. Throughout this program, I have been fortunate enough to have the steadfast and unwavering support of my friends and family. They have been the voices of inspiration and positivity that keep me focused. It is my hope that I can one day be a giant that holds up the next generation of scientists, and I dedicate this work to that next generation.

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Table of Contents

CHAPTER 1: INTRODUCTION ...... 1 1.1 Mass Spectrometry-Based Proteomics ...... 2 1.1.1 Electrospray Ionization ...... 3 1.1.2 Tandem Mass Spectrometry ...... 4 1.1.3 Computing and Data Processing ...... 5 1.2 Relevant Tandem Mass Spectrometers ...... 6 1.2.1 QSTAR Elite ...... 6 1.2.2 Triple TOF 5600 ...... 7 1.2.3 Orbitrap Velos ...... 8 1.3 Mitochondria – Associated Endoplasmic Reticulum Membranes ...... 10 1.3.1 Calcium Trafficking at the MAM ...... 11 1.3.2 Structural Linkage Between ER and Mitochondria ...... 12 1.4 Research Contribution ...... 13 1.5 Figures ...... 14 Figure 1.1: Bottom-up Proteomic Workflow...... 14 Figure 1.2: PEPTIDE IDENTIFICATION FROM MASS SPECTRA...... 15 Figure 1.3.A: General Schematic of a Quadrupole Time-of-Flight Tandem Mass Spectrometer ...... 16 Figure 1.3.B: General Schematic of an Orbitrap Velos Mass Spectrometer ...... 16 Figure 1.4: WIDELY ACCEPTED PROTEINS AND FUNCTIONS IN THE MAM ...... 17 1.6 References ...... 18

CHAPTER 2: COMPREHENSIVE CHARACTERIZATION OF MITOCHONDRIA-ASSOCIATED ENDOPLASMIC RETICULUM MEMBRANES FROM BRAIN TISSUE ...... 22 2.1 Introduction ...... 23 2.2 Experimental Procedures ...... 25 2.2.1 Materials ...... 25 2.2.2 MAM Isolation for Mouse Brains ...... 25 2.2.3 Microsome Fractionation ...... 26 2.2.4 Western Blot Analysis ...... 26 2.2.4 Gel-Assisted Digestion ...... 27 2.2.5 Offline HPLC Fractionation ...... 27 2.2.6 LC-MS/MS and Data Analysis ...... 28 2.2.7 Protein Correlation Profiling Sample Preparation ...... 29 2.2.8 Protein Correlation Profiling LC-MS/MS Analysis and Quantitation ...... 30 2.3 Results ...... 31 2.3.1 Isolation and Identification of MAM Proteins ...... 31 2.3.2 Quantitative Validation of Biologically Relevant MAM Proteins ...... 33 2.3.3 MAM Proteins Conserved Across Tissues ...... 35 2.4 Discussion ...... 36 2.4.1 Isolation Validation ...... 36 2.4.2 Comparison to Previous Reports ...... 37 2.4.3 MAM Molecular Functions ...... 38 2.4.4 Relationship to Neurological Diseases ...... 39 2.5 Conclusion ...... 40 2.6 Figures ...... 41 Figure 2.1: MAM Isolation Schematic ...... 41 Figure 2.2: Localization and Biological Relevance of Identified Proteins ...... 42 Figure 2.3: Significant Disease Pathways ...... 43 Figure 2.4: Protein Correlation Profiling ...... 44

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Figure 2.5: Biological Significance of MAM Proteins Conserved Across Tissues ...... 45 2.7 References ...... 46

CHAPTER 3: PROTEOMIC INVESTIGATION OF LIPID RAFT-ENRICHED MEMBRANES ISOLATED FROM INTERNAL ORGANELLES ...... 52 3.1 Introduction ...... 53 3.2 Experimental Procedures ...... 54 3.3.1 Materials ...... 54 3.3.2 Cell Culture and Whole Cell Lysate Preparation ...... 55 3.3.3 DRM Enrichment ...... 55 3.3.4 MAM Isolation ...... 56 3.3.5 Lipid Raft Isolation from MAM ...... 56 3.3.6 SDS-PAGE and Western Blot Analysis ...... 57 3.3.7 Gel-Assisted Digestion ...... 57 3.3.8 High pH Reversed-Phase (RP) HPLC ...... 58 3.3.9 LC-MS/MS and Data Analysis ...... 58 3.3 Results ...... 60 3.3.1 Isolation and Evaluation of Detergent Resistant Membranes ...... 60 3.3.2 Proteomic Characterization of DRMs ...... 61 3.3.3 Isolation and Analysis of Lipid Rafts from MAM ...... 63 3.3.4 Evaluation of Proteins Identified Only in the Lipid Raft ...... 64 3.3.5 Statically Validated Lipid Raft-Enriched Proteins ...... 65 3.4 Discussion ...... 68 3.5 Conclusion ...... 69 3.6 Figures ...... 70 Figure 3.1: DRM Isolation and Western Blot Validation ...... 70 Figure 3.2: DRM Protein Localization ...... 71 Figure 3.3: Biological Significance of DRM Proteins ...... 72 Figure 3.4: Lipid Raft Isolation and Western Blot Validation...... 73 Figure 3.5: Biological Significance of Proteins Identified Only in the Lipid Raft ...... 74 Figure 3.6: Predicted Palmitoylated Sites in MAM Proteins ...... 75 Figure 3.7: Biological Significance of Lipid Raft-Enriched MAM Proteins ...... 76 3.5 References ...... 78

CHAPTER 4: CONCLUSION ...... 82 4.1 Summary of Results ...... 83 4.2 Future Work ...... 85

APPENDIX ...... 88 Figure A1. Immunoassay of MAM fractions ...... 89 Table A1. Complete Listing of Proteins Identified in the MAM From Mouse Brain ...... 90 Table A2.Complete Listing of DRM Proteins from NG108-15 Cells ...... 120 Table A3. Proteins Only Identified in the Lipid Raft of MAMs ...... 123 Table A4. Lipid Raft-Enriched Proteins in the MAM ...... 128 Table A5. Proteins Identified in Mouse Liver ...... 132

Table of Abbreviations

AD Alzheimer's Disease CM Crude mitochondria DC Direct current 2DIGE 2 dimensional difference gel electrophoresis DRM Detergent resistant membranes ER Endoplasmic reticulum ESI Electrospray ionization FDR False discovery rate GO Gene Ontology GRP75 Growth receptor protein 75 IDA Information dependent acquisition IP3R 1,4,5 inositol triphosphate receptor LR Lipid raft m/z Mass to charge MAM Mitochondria-associated endoplasmic reticulum membranes RF Radio frequency RYR Ryodine receptors TOF Time of flight VDAC Voltage dependent anion channel XIC Extracted ion chromatogram

CHAPTER 1

INTRODUCTION

Mass Spectrometry-Based Proteomics

Mass spectrometry (MS) based proteomics is the study of protein structure

derived from characteristic mass shifts that are indicative of specific peptides, proteins, or

chemical modifications. Proteomic approaches to biological questions are particularly

informative because they do not rely on previous knowledge of proteins in a specific

cellular region or tissue. Traditional biological techniques to study protein localization

largely depend on antibody-based studies. This is especially limiting because it requires

some idea of the proteins in the region and necessitates a specific antibody for each

protein, which can often be difficult and expensive to obtain. Because proteomics is

based on protein masses, all detected masses can be compared against databases of

characteristic masses in order to identify thousands of proteins in a single experiment (1,

2). The protein databases used in proteomics have been generated as a result of the

human genome project, and allow for functional information about identified proteins to

be cross referenced, ultimately providing the opportunity for a truly global analysis of

any subcellular region.

Originally, the study of proteins by mass was conducted using two-dimensional

difference gel electrophoresis (2DIGE). In this technique, proteins were separated by pI

in the first dimension, and by molecular weight in the second dimension (3, 4). The resulting gel was stained either with silver or fluorescent dye and a series of spots corresponding to proteins could be visualized. This method allowed for comparison of different treatment populations based on the appearance, disappearance, or migration of the protein on the gel. Although this technique was quite useful, the mass accuracy was poor, variation in protein migration was high and spots visualized by staining techniques

could contain multiple proteins, making it difficult to distinguish specific proteins of

interest (5, 6). The development of mass spectrometry techniques that allowed for the

analysis of large biomolecules overcame many of the issues inherent to gel-based

analysis and allowed for sequencing of hundreds of proteins in one experiment. These techniques include: electrospray ionization (ESI), the coupling of powerful mass

analyzers to create tandem mass spectrometers, and powerful computing algorithms to

integrate data acquisition, database searching, and statistical validation.

Electrospray Ionization

The basis for electrospray ionization was described in the early 1900s, but it was

Fenn and coworkers who first reported its use as an ionization source for large

biomolecules (7). Electrospray ionization takes molecules in the liquid phase and directs

them towards a charged tip, usually 1-10μm in diameter. A voltage is applied at the tip,

which imparts the molecules with charges. Then, a gas is used to evaporate solvent from

the droplet, leading to Coulombic repulsion, and resulting in a fine mist of ions are that sprayed into the inlet of a mass analyzer (8). There are several advantages to employing an ESI source for the analysis of proteins and peptides. First, ESI produces multiply charged ions for MS analysis. Larger charge states effectively reduce the overall mass to charge ratio or m/z, thus expanding the mass range of the instrumentation. For analysis of tryptic peptides, the charge state is usually +2 or +3 with some variation due to peptide size. ESI is also favorable because it can be coupled to an online separation workflow.

This greatly reduces losses due to sample handling, and nicely lends itself to automation

with high performance liquid chromatography. The work described in this text capitalizes

on both of these properties in order to analyze highly complex peptide mixtures.

Tandem Mass Spectrometry

The technology for tandem mass spectrometry was developed throughout the 70s

and it’s potential for large scale analysis of complex samples was appreciated by 1980

(9). As the name suggests, the technique involves coupling two mass analyzers, usually with each mass analyzer utilizing different properties to provide mass selection. The first mass analyzer selects the intact parent or precursor ion based on a user-defined analysis window by applying either an RF voltage or direct current potential. Ions that are within the selected analysis window are guided to a chamber where molecules of an inert gas, usually nitrogen or helium, collide with the molecules to generate a series of characteristic fragment or daughter ions that are accelerated to and detected by a second mass analyzer (10). These characteristic fragment ions are identified by a combination of their elution time and mass shift and searched against a protein database in order to identify the components of the original uncharacterized mixture (Figure 1.2).

In proteomics experiments, the complex mixture is that of proteins or peptides.

Fragmentation patterns along with m/z ratios can be compared to known protein sequences (2). Because peptides are assigned to spectra based on how well the predicted fragmented pattern matches the experimental result, protein identifications must be accompanied by a false discovery rate (FDR) and a measure of confidence (11-13). Most studies require that protein identifications be filtered to contain at least 2 unique peptides with an FDR below 5%. Due to the large number of spectra generated in each

experiment, the searching and statistical validation of protein identification is often

automated by integrative proteomics software such Protein Pilot or MASCOT (14, 15).

Computing and Data Processing

Mass spectrometry-based proteomics has been a major catalyst in the

development of key analytical techniques, which have allowed for the global

identification of proteins and peptides with reasonable throughput. Computational

biologists have joined in this endeavor by generating software targeted to facilitate the

comparison of thousands of spectra to constantly curated protein databases (14, 15). This

has been instrumental in protein and peptide identification. Robust statistical validation,

and quantitative comparison can be performed in an automated fashion using software

packages that use the database searching output to further validate protein identifications.

While there are several types of these programs, this text primarily uses ProteoIQ

(NuSep, Bogart, GA) for statistical validation. Not only are these software tools

powerful, they are user friendly and no longer require extensive knowledge of

computational code.

Bioinformatics has also provided the means by which to efficiently ascertain the

function, interactions, and localization of proteins identified in an analysis, allowing proteomic researchers to delve beyond the question of “what proteins are present in this

region?” and move further toward “what are the functions of these proteins at this region and do they interact?” from a mass spectrometry-based experiment. The work described here utilizes Ingenuity Pathway Analysis or IPA (www.ingenuity.com) for functional

analysis. IPA is an integrative software package which draws on curated pathway

5 databases and literature references to asses potential interactions, biomarkers, and pathways within a submitted protein list. The development of programs similar to IPA has allowed researchers to have a better idea of the biological significance of their proteins lists, while providing some evidence to pursue targeted studies on specific proteins. The combination of tandem mass spectrometry and enhanced bioinformatics provides the platform through which known protein information can be verified, new proteins can be identified, and functional information about these proteins can be ascertained for thousands of proteins from a single experiment.

Relevant Tandem Mass Spectrometers

QSTAR Elite

The coupling of quadrupole and reflectron time-of-flight (TOF) instruments allows for the mass selectivity of the quadrupole mass analyzer to be combined with high mass accuracy of the TOF (Figure 1.3 A). Quadrupole mass analyzers resolve ions using tightly regulated electronics to select for specific ions. The quadrupole has two sets of parallel rods: one set has a positive direct current (DC) potential and the other a negative

DC potential. Both sets of quadrupoles carry a radio frequency or RF potential. Both the

DC and RF potentials control the flight path of ions that are injected into the quadrupole mass analyzer. Ions above or below the selected m/z will collide with the poles and not be transmitted to the detector for mass analysis(16). Conversely, ions that are stable in the selected DC and RF window will maintain a stable flight path through the quadrupole.

The quadrupole mass analyzer is comprised of three sets of quadrupoles. Upon injection, ions travel through the first set of quadrupoles called q0, which focus ions for

further transmission, while removing unstable ions from the beam(16). The next set of

quadrupoles, known as q1, serves as a mass filter and can be used to select for specific

precursor ions, or to scan for all precursor ions in a given mass range. Ions that are within

the selected range are transmitted to a third quadrupole, q2, which serves as a collision

cell where ions collide with an inert gas to produce characteristic fragment ions, which

are detected and analyzed by the TOF mass analyzer.

The TOF instrument separates ions according to mass by applying the same

acceleration voltage to a group of ions and allowing them to travel through an

electromagnetic field (16). Analyte ions of lower mass will travel to the detector faster

than ions of higher mass. The mechanism of the TOF instrument theoretically precludes

any upper mass limit, although practically, the upper mass limits are governed by the

abilities of the detector and the previous quadrupoles. Early in their development, TOF

instruments were restricted by their inability to discern and detect ions with varying

kinetic energy, leading to less than ideal mass resolution. This was precluded by the addition of the reflectron (17), which removes variations due to differences in kinetic energy, while increasing the length of the flight tube. Fragment ions travel through the

TOF to a multi-channel plate detector.

Triple TOF 5600

The Triple TOF 5600 is an updated version of the QSTAR Elite from AB-Sciex

(Framingham, MA) (18). Improvements include a focusing lens at the inlet, which

increases ion transmission from the electrospray source. Electronics have also been

improved across the quadrupoles to focus ion transmission during the first mass filter.

Analyte ions are accelerated from the quadrupole mass analyzer to the TOF mass

analyzer, like in the QSTAR Elite, however the detector at the end of the flight tube can accommodate and detect ions at a rate comparable to the duty cycle of the instrument.

The combination of enhanced ion optics and detection allows for more than 50 ions per

minute to be detected, ultimately increasing the number of peptides and proteins detected

in an experiment.

Orbitrap Velos

The LTQ Orbitrap Velos is the state of the art hybrid mass analyzer (Figure 1.3

B). This instrument couples a two-dimensional linear ion trap with a 3-dimensional orbitrap(19). Unlike the quadrupole-TOF mass analyzers, trapping instruments isolate ions in time, not space. This means that electronics allow ions to accumulate in the trap, then RF potential is applied to eject ions that do not resonate at the frequency of the selected m/z values. The accumulation of ions allows lower abundance peptides to be detected because these ions can accumulate in the trap in order to generate more robust spectra, and ultimately increasing peptide and protein identifications.

The first mass analyzer of the Orbitrap Velos is a linear ion trap, known as the

LTQ from Thermo-Fisher (Waltham, MA). The two-dimensional linear ion trap is composed of two rods similar to that of the quadrupole instrument. An RF potential is applied to the rods and the ions are trapped between the rods by a DC voltage at the end caps of the rods. Mass information is obtained by selective ejection either in a radial or axial direction. In a fashion similar to the quadrupole mass analyzer, the DC and RF can be increased over time to allow for a precursor ion scan of the ions in the trap. In the case

of the Velos tandem mass spectrometer, ions are usually ejected in the axial direction and

accelerated toward the second mass analyzer, an orbitrap (19).

The orbitrap mass analyzer is a derivation of the three-dimensional Paul trap (20)

reported by Markarov in 2000 (21). This trapping instrument capitalized on electrostatics

in order to cause injected ions to oscillate. The trap consist of a “spindle-shaped” inner

electrode about 8 mm in diameter that is enclosed by a “barrel-shaped” outer electrode.

An electrostatic potential is applied to the inner electrode, which causes charged ions to travel around the spindle and back and forth along the z-axis. The frequency of the ions oscillations generates a broadband current that is readily comparable to a harmonic oscillator, allowing for Fourier transformation into mass spectra of the product ions.

The instrument has significantly improved the original design (the LTQ Orbitrap), yielding an increase in peptide and protein identifications when compared to other commercially available tandem mass spectrometers. The addition of a series of stacking rings with varied RF, known as an “s-lens”, at the orifice of the instrument guides ions from an ESI source into the instrument to increase ion transmission. The distance between the orifice and the LTQ has also been reduced to improve overall ion transmission. More powerful pumps were added to this instrument, making the pressure in the trap ~10-11 Torr. This allows ions to remain stable for longer periods of time and prevents premature fragmentation. These improvements together have reduced the overall duty cycle of the instrument, allowing for the analysis of 1,000,000 ions in 1 sec.

Furthermore, analysis with BSA protein standards show reliable detection of up to 50 peptides at attomole loading amounts (19). The application of this instrument for the identification of low abundance proteins in the presence of complex mixtures has made

the LTQ Orbitrap Velos the new “workhorse” in many well-regarded proteomics laboratories.

Mitochondria –associated Endoplasmic Reticulum Membranes

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) are

cholesterol and lipid-enriched portions of the ER that are physically connected to the

mitochondria (22). Portions of the ER were observed to be in contact with mitochondria

by electron microscopy of teleosts in 1958 by Copeland and Dalton (23). The

serendipitous finding led the authors to postulate, “this contact may have some functional

significance” (23). Following this initial description, other electron microscopy studies on

the mitochondria reported images of the endoplasmic reticulum membrane in contact

with mitochondrial membranes (24, 25). With the advent of electron tomography, a

method that allows 3D visualization of the organelles, a better image of ER/mitochondria

contact sites became available (24, 26). The combination of electron microscopy and

electron tomography images depicting ER and mitochondria contact sites led to the

apposition of the two organelles being widely accepted.

The field remained stagnant for several years until investigation in mitochondria

and endoplasmic reticulum was revived by interest in the lipid synthesis of phosphatidyl

choline (PtdCln). Research on the mechanism of PtdCln synthesis revealed that

phosphatidyl serine (PtdSer) is transported from the ER to the mitochondria where it is

decarboxylated to generate phosphatidyl ethanolamine (PtdEtn) (27-29). What remained

unclear about this mechanism was how the phosphatidyl serine was transported from the

ER to the mitochondria. Three mechanisms were under consideration: vesicle transport of

the lipid between the two membranes; lipid transport facilitated by transport proteins

localized in the outer mitochondrial membrane; and transport of proteins through tight

associations between ER and mitochondria. While investigating these mechanisms, Jean

Vance and her co-workers isolated what they called “fraction X” on a self-forming

Percoll gradient (22). “Fraction X” was described as a portion of the ER that pellets with

the mitochondria during centrifugation. In an effort to better understand “fraction X”,

enzymatic activity assays were performed. Vance et. al reported a higher phosphatidyl

serine synthase activity in “fraction X” when compared to mitochondria and microsomes

(22). This was the first definitive description of what is now known as mitochondria

associated ER membranes or MAMs as an entity separate from the bulk ER. This report

also provided a reliable method to isolate the MAM from bulk ER and mitochondria (22).

Calcium Trafficking at the MAM

The MAM has emerged as a Ca2+ signaling hub for transport of the ion to and

from the mitochondrion. Calcium ion channels such as ryodine receptors (RYR),

uniporters, voltage dependent anion channel 1 (VDAC1) and 1,4,5 inositol triphosphate

receoptors (IP3R) are enriched in the MAM (30-32). The MAM is also rich in calcium

dependent chaperones like the sigma-1 receptor, BiP (GRP78), calnexin, and calreticulin

that facilitate proper protein folding in the ER (33-35). In fact, the protein bridge

mentioned above is composed of two calcium ion channels (IP3R and VDAC1) and a

calcium dependent chaperone (GRP75), and acts as a tunnel for Ca2+ transport (36). Ca2+ flows from ER stores, through the MAM, and into the mitochondria where it facilitates

ATP production (36). Ca2+ then flows out of the mitochondrion to prevent accumulation

of the ion, which can induce Bcl-2 mediated apoptotic pathways (37). The distance

between the MAM and mitochondria is dynamic, with the association becoming tighter when the mitochondrion requires more Ca2+ (38). Any alteration in these highly regulated

calcium trafficking processes can cause mitochondrial dysfunction or initiate the

unfolded protein response (UPR) (39).

Structural Linkage Between ER and Mitochondria

Lipid synthesis studies made early advances toward understanding how the MAM

and mitochondria are physically connected. Mitofusin 2, a protein responsible for

regulating mitochondrial fusion, was among the first proteins shown to physically link

the MAM to the mitochondria. Human embryonic fibroblasts cells negative for mitofusin

2 showed no association between the ER and mitochondria; however upon injection of

mitofusin 2, the association was restored (38, 40). The same group showed that ER

mitofusin 2 interacts with mitochondrial mitofusin 1 or mitofusin 2 in order to link the

MAM to the mitochondria. Supporting the hypothesis that the MAM is the trafficking site

of phosphatidyl serine, the synthesis of phosphatidyl choline was disrupted upon the

dissociation of the two organelles (38). A second complex called ERMES consisting of 4

interacting proteins was also reported to physically link the MAM and mitochondria (41).

In this report, the authors show that the 4 genes (2 on the mitochondrial membrane and 2

on the MAM) interact like a zipper to connect the two membranes (41). Other proteins

since reported to physically link the MAM to the mitochondria include IP3R and the

voltage dependent anion channel 1 (VDAC1) (42, 43). The physical linkage between the

MAM and mitochondria is a dynamic one. Electron tomography studies revealed the

distance between the membranes as small as 10 μm and as large as 90 μm (44).

Research Contribution

The MAM is regarded as a trafficking site for small molecules, a regulator of protein

synthesis, and a mediator of apoptosis. Previous research has focused on specific molecules and their functions within the MAM using antibody-based techniques. In this work, we increase the global understanding of the mitochondria-associated endoplasmic

reticulum membrane by providing a comprehensive catalog of proteins located in the

region. We also delve into the functions and biological processes regulated by MAM

residents. Knowledge of these proteins and their functions and processes will propel the

recently revived study of the MAM forward towards the elucidation of new signaling

pathways, the discovery of novel biomarkers, and the acceptance of alternative disease

pathologies centered at this membrane contact site.

Figures

Figure 1.1: Shotgun proteomic workflow. An unknown protein mixture is digested with an enzyme such as trypsin to generate peptides. Peptides are then separated into fractions using an offline HPLC system. Each fraction is loaded onto an online chromatographic system, which ionizes peptides and sprays them into a tandem mass spectrometer. Spectra are compared to known peptide sequences in order to match peptides to proteins and ultimately provided a list of protein candidates likely to be in the unknown sample.

Figure 1.2: Peptide identification from mass spectra. As peptides elute from the online column, they are sprayed into the mass spectrometer. The first mass analyzer performs a precursor ion scan to detect peptide ions. Ions that meet user define characteristics (a specific mass range and number of counts) are fragmented within a collision cell to generate b and y ions, which are detected by the second mass spectrometer. The combination of the precursor ion m/z and the tandem mass spectra are used to sequence previously uncharacterized peptides.

Figure 1.3. (A) General schematic of a quadrupole time-of-flight tandem mass spectrometer (16) are accelerated though the first set of quadroples (q0)) to the second set of quadrupoles (q1) where a precursor ion scan is performed. A third set of quadrupoles (q2) guides ions to the collision cell where fragment ions are generated by collisions with an inert gas. The fragment ions are accelerated through the TOF mass analyzer. (B) General schematic of an Orbitrap Velos tandem mass spectrometer (19). Ions emitted from an electrospray source are focused through the s-lens and guided through a series of multipoles to the linear ion trap where ions are accumulated and precursor ion selection is performed. Precursor ions are fragmented and transported to the orbitrap mass analyzer for detection.

Figure 1.4: Widely accepted proteins and functions in the MAM. Mitochondria are physically linked to the MAM portion of the ER through the ERMES complex. Calcium ions are trafficked through IP3Rs, RYRs, and VDAC ion channels to facilitate the conversion of ADP to ATP. Newly produced ATP is transported back to the MAM to facilitate canx and calr mediated protein folding. Lipid precursors like ptdser require transport from the ER to the mitochondria where they are converted to intermediates, which are transported back to the ER where the final lipid is produced and secreted.

References

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CHAPTER 2

COMPREHENSIVE CHARACTERIZATION OF MITOCHONDRIA-ASSOCIATED ENDOPLASMIC RETICULUM MEMBRANES FROM BRAIN TISSUE

Introduction

Electron microscopy studies first observed the mitochondrion and endoplasmic reticulum to be in close contact in a variety of organisms (1-4). In depth investigation of the region now known as the mitochondria-associated ER membrane or MAM began after a reliable method to isolate the MAM was reported by Vance in 1990 (5). Though

Vance and her colleagues were interested in the lipid synthesis capabilities of the MAM, the elucidation of the PtdCln synthetic pathway provided a key breakthrough in understanding MAM function: small molecules are transported from the ER to the mitochondrion through the MAM (2, 6-8). Knowledge of the MAM has significantly increased over the last 20 years. Structural studies have identified the association of calcium–dependent proteins inositol 1, 4, 5-triphosphate receptors (IP3R), growth receptor protein 75 (GRP75), and the voltage dependent anion channel 1 (VDAC1) as a protein bridge that physically links the MAM to the mitochondrial membrane. (9, 10).

Further, the association between the MAM and the mitochondria is regulated by the protein chaperone, mitofusin 2, and silencing of this protein leads to dissociation of the two organelles(10). Most recently, two proteins, calnexin and thioredoxin containing protein TMX, have been shown to target the MAM based on post-translational palmitoylation, hinting at the dynamic nature of the MAM region (11).

The MAM has emerged as a Ca2+ signaling hub for transport of the ion to and from the mitochondrion. Calcium ion channels such as ryoodine receptors (RYR), uniporters, VDAC1 and IP3R are enriched in the MAM (12-14). In addition, calcium dependent chaperones like the sigma-1 receptor, BiP (GRP78), calnexin, and calreticulin that facilitate proper protein folding in the ER are prominent in the MAM (15-17). Ca2+

flows from ER stores, through the MAM, and into the mitochondria where it facilitates

ATP production (18). Ca2+ then flows out the mitochondrion to prevent accumulation of

the ion, which can induce Bcl-2 mediated apoptotic pathways (19). The distance between the MAM and mitochondria is dynamic, with the association becoming tighter when the

mitochondrion requires more Ca2+ (10). Any alteration in these highly regulated calcium

trafficking processes can cause mitochondrial dysfunction or initiate the unfolded protein

response (UPR) (20).

Current hypotheses link the MAM to neurodegenerative conditions such as,

Alzheimer’s disease (AD) (21). When mutated by the Alzheimer’s disease state, AD precursors presenilin 1 (PS1) and presenilin 2 (PS2) result in a disruption of Ca2+ homeostasis that leads to mitochondrial dysfunction, and ultimately the activation of apoptotic pathways (22-24). Both PS1 and PS2 are enriched in the MAM in mouse neurons (25). In addition, an increase in reactive oxygen species (ROS) production due to

AD can cause improper protein folding, triggering the UPR (20, 26-28). Because protein

folding is regulated by chaperones enriched in the MAM, this region may play a role in

neuronal cell death related to AD pathologies (26, 29-32). Further, the MAM-enriched

acyltransferease ACAT1 regulates amyloid precursor proteins and the ultimate formation

of amyloid-β plaques, a hallmark of AD (33-37). A clear mechanism to connect the

activities of the MAM to AD and other neurodegenerative diseases remains to be

elucidated primarily because the field lacks a complete understanding of the proteins

localized within this region.

To that end, we report a mass spectrometry-based proteomic characterization of

MAM from mouse brain tissue. In contrast to several studies on the MAM, our study in

tissue allows us to provide a snapshot of the MAM without any metabolic alteration that can occur with transformed cells. We also employ a quantitative validation method in

order to distinguish true MAM proteins from contaminating proteins in order to furnish a

robust list of proteins and MAM-related biological processes for future studies of the

MAM.

Experimental Procedures

Materials

Standard laboratory chemicals were obtained from Thermo Fisher Scientific

(Rockford, IL). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO)

with the exception of the following: protease inhibitor cocktail was from Roche Applied

Science (Indianapolis, IN); sequencing-grade modified-trypsin was from Promega

(Madison, WI). Primary antibodies (anti-calnexin, anti-histone H3, anti-Na+/K+ ATPase)

and all secondary antibodies were purchased from Santa Cruz Biotechnologies (Santa

Cruz, CA)

MAM Isolation from Mouse Brains

Mouse brains were extracted from 20 day-old C57 male wild-type mice kindly provided by Mark Cervas. The MAM was isolated by a previously described method

(38). Briefly, brains were homogenized by 6-8 strokes of a motorized Potter-Elvehjm glass homogenizer on ice. All centrifugations were performed at 4 °C. Nuclei and unbroken cells were pelleted at 500 xg for 5 min twice. Crude mitochondria (CM) were separated from microsomes by centrifugation at 10,000 xg for 10 min and washed twice.

CM was resuspended in 0.5 mL of mitochondria re-suspension buffer (30 mM HEPES,

20 mM Mannitol, 5 mM EDTA, pH 7.4) and layered on top of 30% Percoll (8 mL),

followed by mitochondrial re-suspension buffer (4 mL). After centrifugation at 95,000 xg

for 30 min, the MAM band was aspirated from the gradient. The MAM was diluted 5X

and centrifuged to remove mitochondrial contamination at 10,000 xg for 10 min. To

pellet the MAM, the supernatant was centrifuged at 100,000 xg for 1 hr. The final MAM

pellet was resuspended in 250 μL of 2% SDS. Protein concentration was determined by

BCA assay. Biological replicates were prepared from 2 different brains.

Microsome Fractionation

Microsomes isolated from mouse brains during the MAM isolation were further

fractionated for Western blot analysis. The supernatant resulting from crude mitochondria enrichment was centrifuged at 20,000 xg for 30 min. The ER (pellet) was isolated from the cytosol (supernatant) by centrifugation at 100,000 xg for 1hr. All fractions were

solubilized prior to analysis by 2% SDS. Protein concentrations were determined by BCA assay.

Western Blot Analysis

Protein fractions separated by SDS-PAGE (10% Bis/Tris hand-poured gels) were electrophoretically transferred to a PVDF membrane (0.2 μm, Millipore, Billerica, MA)

at 4 ˚C for 90 min, 35 V) using the BioRad Mini Protean Trans-Blot Module. HRP-

conjugated secondary antibodies allowed for detection and visualization of specific

26 antigens by ECL (SuperSignal Chemiluminescent Substrate; Thermo Fisher Scientific).

Blots were imaged using a Syngene GeneGnome (Frederick, MD) bio-imaging system.

Gel-Assisted Digestion

MAM proteins were digested with trypsin using a gel-assisted method (39, 40).

Briefly, a polyacrylamide gel was created by adding 18.5 μL of a 19:1 mixture of 40%

(v/v) acrylamide solution/bis-acrylamide, 2.5 μL of 10% (w/v) ammonium persulfate, and 1 μL of 100% TEMED to 50 μg of protein in a Eppendorf tube. After polymerization, the gel/protein matrix was cut into small pieces and washed twice with

50% (v/v) acetonitrile in 25 mM ammonium bicarbonate (pH 8.0), followed by a single wash with neat acetonitrile. Dried gel pieces were rehydrated with trypsin solution and incubated at 37 °C for 18 h. Peptides were extracted using 0.1% acetic acid, 50% acetonitrile/0.1% acetic acid, and 100% acetonitrile sequentially. Extracts were pooled and concentrated via centrovap to ~2μL.

Offline HPLC Fractionation

Peptide digests were reconstituted in 0.1% acetic acid and loaded onto an Agilent

ZORBAX 300Extend-C18 column (150 × 2.1 mm i.d., 3.5 μm) using an Agilent 1200 binary HPLC system. Peptides were separated by gradient elution from 0-50% mobile phase B in 50 min with a constant flow rate of 0.2 mL/min. Mobile phase A was composed of 1% methanol in 20 mM ammonia (pH 7.5) while mobile phase B consisted of a 90/10 mix of acetonitrile/1% methanol in 20 mM ammonia. UV absorbance was

monitored at 214 nm. Fractions were collected every minute and concentrated by vacuum

centrifugation to ~2 μL.

LC-MS/MS and Data Analysis

LC-MS/MS analysis was performed on two technical replicates of each of two

biological replicates for a total of four complete analyses. Each offline HPLC fraction

was reconstituted in 30 μL of 0.1% acetic acid. To reduce analysis time, 3 μL of three

fractions (i+10+20) were combined and analyzed by nanoLC-MS/MS using a Tempo

MDLC system coupled to a QSTAR Elite hybrid quadrupole time-of-flight mass

spectrometer (ABSciex, Foster City, CA) with an ionization voltage setting of 1600 V.

The peptides were eluted at a flow rate of 70-80 nl/min onto analytical columns (75 μm

ID, 7 cm length of Monitor 100Å-Spherical Silica C18; Column Engineering Inc.,

Ontario, CA) using the following gradient: 0-15% solvent B in 5 minutes and 15-35% B

in 40 minutes. Solvent A consisted of 0.1% formic acid and 2% acetonitrile in water and

solvent B contained 0.1% formic acid and 2% water in acetonitrile. MS data were

acquired in information-dependent acquisition mode with Analyst QS 2.0 (ABSciex) with

Smart IDA enabled. MS cycles were comprised of one full scan (m/z range = 300-2000, 1

sec accumulation) followed by sequential MS/MS scans of the four most abundant ions

(+2 to +4 charge state, minimum ion count = 100, automatic collision energy, exclusion

time = 15 sec, maximum accumulation time = 2 sec).

Each of the concatenated fractions was also analyzed by nanoLC-MS/MS using

an Agilient 1200 system coupled to an LTQ Orbitrap Velos instrument (Thermo

Scientific) with an ionization voltage setting of 1600 V. The peptides were eluted at a

flow rate of 70-80 nl/min onto New Objective™ analytical columns (75 μm ID, 7 cm

length) using the following gradient: 0-15% solvent B in 5 minutes and 15-35% B in 40

minutes. Solvent A consisted of 0.1% formic acid and 2% acetonitrile in water and

solvent B contained 0.1% formic acid and 2% water in acetonitrile. MS data were

acquired in information-dependent acquisition mode with Xcalibur 2.0

(ThermoScientific) with lock mass enabled. MS cycles were comprised of one full scan

(m/z range = 300-2000) followed by sequential MS/MS scans of the four most abundant

ions (+2 to +4 charge state, minimum ion count = 500, automatic collision energy,

exclusion time = 30 sec, maximum)

Tandem mass spectra were searched using both the Paragon Algorithm of Protein

Pilot 4.0 and MASCOT v. 2.0 (Matrix Sciences) against a mouse UniprotKB database

(downloaded 9/2011) concatenated with a reverse decoy database. Protein identifications

were required to have a minimum of 2 peptides with an FDR less than 1%. Protein localization and pathway analysis data were obtained using Ingenuity Pathway Analysis

(Ingenuity® Systems, www.ingenuity.com).

Protein Correlation Profiling Sample Preparation

Mouse brain homogenate was thawed at 4 °C and centrifuged for 10 min at 500 xg to pellet unbroken cells and nuclei. The post-nuclear supernatant was centrifuged for 2

hr at 200,000 xg. The resulting pellet containing all insoluble proteins was resuspended in

mitochondrial re-suspension buffer (0.5 mL of 30 mM HEPES, 20 mM Mannitol, 5 mM

EDTA, pH 7.4) and layered on top of 30% Percoll solution (8mL), followed by

mitochondrial re-suspension buffer (4mL). The self-forming gradient was centrifuged at

95,000 xg for 30 min. Fractions of 500 μL were collected from the top of the gradient.

Protein content was determined by BCA assay and digested using trypsin. To remove

Percoll, mannitol, and other contaminating solutes, each fraction was loaded onto an

Agilent ZORBAX 300 Extend-C18 column (150 × 2.1 mm i.d., 3.5 μm) using an Agilent

1200 binary HPLC system. Peptide was eluted using the following gradient: 0-8.5 %B in

20 min; 8.5-90 %B in 2 min; and 90-0%B in 2 min. One sharp peak corresponding to peptide elution was collected, concentrated using a CentraVap to ~2 μL and reconstituted in 50 μL of 0.1% AcOH.

Protein Correlation Profiling LC-MS/MS Analysis and Quantitation

Quantitation was adapted from the previously described method (41). There was no offline separation prior to analysis. Peptides were loaded onto a 75m I.D. New

Objective C-18 column and directly sprayed into a Triple TOF 5600 (AB-SCIEX,

Framingham, MA) quadrupole time of flight tandem mass spectrometer. The peptide elution gradient was as follows: 0-15%B in 5 min and 15-35% B in 40 min. Solvent A consisted of 0.1% formic acid and 2% acetonitrile in water and solvent B contained 0.1% formic acid and 2% water in acetonitrile. Fractions surrounding the MAM band were analyzed twice: once in information dependent acquisition (IDA) mode where the top 40 ions per MS scan were selected for MS/MS and once with an inclusion list for peptides known to reside in the MAM based on our previous shotgun experiments. Proteins were required to be identified in all fractions, and have at least 3 peptides at an FDR below 1% in order to be included in the quantitation. Spectra from the IDA experiment were searched with the Paragon algorithm in ProteinPilot 4.0. Peak areas were calculated from

30 the XIC of each peptide in each fraction using PeakView™ Software (AB Sciex).

Intensities were normalized across the fractions for each peptide and then averaged for the corresponding protein according to the previously described method (42). The MAM trend line used as a reference for the Mahalanobis distance calculations was obtained using data from the targeted analysis. All other peak areas were extracted from the IDA experiment.

Results

Isolation and Identification of MAM Proteins

The MAM was isolated from crude mitochondria on a 30% Percoll gradient as previously described (Figure 2.1 A) (38). ER, plasma membrane, and cytosolic fractions were separated by differential centrifugation as described previously (38). To evaluate our isolation method, fractions were probed using markers for MAM (calnexin) and nuclei (histone H3). Western blot results showed an enrichment of calnexin in the ER and the MAM, validating our isolation method (Figure 2.1 B). Minimal nuclear contamination was observed in the MAM fraction as evidenced by Western blot results of

Histone H3 (Figure 2.1 B).

Tandem mass spectrometry analysis of the MAM fraction using both QStar Elite and Orbitrap Velos mass analyzers yielded a list of 1,212 proteins. The final list required that protein be identified from analysis on either instrument with an FDR <1% with at least two unique peptides. We increased the number of identification by using a validated and robust method to solubilize and digest the lipid-rich MAM fraction (39, 40). Protein identifications were required to have a minimum of 2 unique peptides below a 1% FDR 31

(Table A1). The Gene Ontology (GO) database was searched to obtain organelle

association information for each protein. Of those 1,212 proteins, 19% (227 proteins) are

associated with the ER, 19% (232 proteins) are associated with mitochondria and 24%

(286 proteins) are associated with the plasma membrane (Figure 2.2 A). Our protein

localization is consistent with a previous study (43) and further validates our MAM

isolation method, as 38% (459 proteins) are associated with the ER or mitochondria. The

large number of proteins associated with the plasma membrane is mostly like due to

membrane contact sites between the ER and plasma membrane known as the plasma

membrane-associated membranes or PAM (44). This could also be the result of a larger

membrane network between the ER, mitochondria, and plasma membrane (3). Other notable organelle associations include the cytoskeleton (5%, 66 proteins), golgi

apparatus, (6%, 73 proteins), and ribosomes (3%, 31 proteins), which assist in

mitochondrial movement, protein processing, and protein synthesis respectively (45, 46).

To understand the types and functions of the proteins identified, we employed

Ingenuity Pathway Analysis (IPA) (Ingenuity® Systems, www.ingenuity.com). For all

analyses, each protein was required to have published evidence to substantiate its

categorization. Of the proteins for which this information was available, 355 are

enzymes, 164 are transporters, 81 are kinases, 34 are ion channels, and 31 are

phosphatases (Figure 2.2 B). These protein types are indicative of known MAM functions

such as energy metabolism, small molecule trafficking, ion transport, and cellular

signaling (32, 47, 48). Functional information for identified proteins was obtained by

comparing our MAM protein list to known signaling pathways. Significance was

measured using Fisher’s T-test, which relies on p‐values to determine whether the

null hypothesis should be accepted or rejected. Molecular functions with a p‐value <

0.01 were considered significant The most significant pathways in our list of identified

proteins were mitochondrial dysfunction, oxidative phosphorylation, and the citrate cycle

(Figure 2.2 C), highlighting the consistency of our approach with previous studies on the

MAM (43). We also identified significant pathways that are specific to brain tissue such

as synaptic long-term potentiation, axonal guidance in signaling, and CREB signaling in neurons.

Comparison of identified proteins to known neurological diseases and disorders using IPA reveals movement disorders, Huntington’s disease, and chorea as the most significant pathways (Figure 2.3). These disorders have yet to be attributed to the activity of the MAM and targeted analysis of the proteins in these pathways could lead to a new appreciation for an MAM-based mechanism for these diseases. We also found

Alzheimer’s disease, schizophrenia, dementia, and neuropathy to be significant within our MAM list. Interestingly, these diseases have been linked to altered Ca2+ homeostasis

and mitochondrial dysfunction, all of which are regulated by the MAM (21, 49, 50). Each

of the neurological disease states reported here require further substantiation, but their

presence as significant pathways enriched in our MAM data highlights the potential role

the MAM may play in these types of disorders.

Quantitative Validation of Biologically Relevant MAM Proteins

Protein identification via mass spectrometry is a highly sensitive method. Our

shotgun MS/MS approach lends itself to the identification of contaminating proteins that

are found even at low abundance. To address this problem, we used protein correlation

profiling to distinguish true MAM proteins from contaminating proteins (41, 42, 51). We enriched for MAM from tissue homogenate and collected fractions from the Percoll gradient (Figure 2.4 A). Each fraction was analyzed by LC-MS/MS and searched against a concatenated target-decoy mouse database.

Normalized peak areas were plotted across fractions for each peptide (Figure 2.4

B), and normalized areas were averaged for protein correlation. Both Western blot (data not shown) and extracted ion chromatograms confirm that fraction 13 was enriched in

MAM protein. Markers for the ER (peptidyl prolyl isomerase a or ppia) and the mitochondria (ATP5a1) are most abundant in fraction 12, and not fraction 13, where as the MAM marker calnexin is most abundant (Figure 2.4 C). A trend line for MAM proteins was established by averaging the normalized intensity of 3 MAM proteins

(calnexin, calreticulin, and VDAC2). All other proteins were compared to this reference set using the Mahalanobis distance (DM) to distinguish true MAM proteins from

contaminants (42, 52).

In order to identify new MAM residents, we calculated DM for proteins in our

data set that met the following criteria: a minimum of 3 unique peptides at a 95% confidence interval; an FDR of < 1%; identification across all five of the assayed fractions; and previous identification in the discovery-based analysis. The application of these cut-offs yielded 60 quantifiable proteins (Table 2.1). Calculated Mahalanobis distances for these proteins range from 0.00174 – 0.36374, with the smaller DM values corresponding to a definitive localization to the MAM. This is confirmed by ACAT1, a well-characterized MAM resident, which has a DM of 0.0108. Median DM values are

consistent with MAM-associated proteins like the mitochondrial voltage dependent anion

channel 1(Dm = 0.16893). Proteins with high Dm values like ATP synthase subunit beta

and synapsin (Dm = 0.35497 and 0.36374 respectively) are likely contaminant artifacts;

however they may associate only temporarily with the MAM in response to specific

signaling events, resulting in an increased Mahalanobis distances.

MAM Proteins Conserved Across Tissues

To understand MAM functions that are conserved across tissues, we investigated

the protein composition of MAM isolated from liver (Table A5) and compared it to brain.

A total of 293 proteins were found in common, including MAM residents ACAT1,

calnexin, GRP78 (BiP), GRP75, SERCA, VDAC1, VDAC2, and VDAC3 (Figure 2.5 A).

Organelle associations from GO annotations reveal that most conserved proteins are

localized in the mitochondria (37%), endoplasmic reticulum (34%), and the plasma

membrane (11%) (Figure 2.5 B). IPA was used to elucidate statistically significant biological processes from this list of conserved proteins (Figure 2.5 C). The 20 most significant pathways include mitochondrial dysfunction, oxidative phosphorylation,

citrate cycle, small molecule biochemistry, molecular transport, fatty acid metabolism,

and protein folding (6, 32, 53-57). These findings show that these biological processes are not tissue specific, and may relate MAM function to diseases and disorders outside of the brain.

We continued to investigate conserved proteins by isolating and analyzing the

MAM from mouse heart tissue. We compared the proteins identified from mouse heart and mouse brain after mass spectrometry analysis on a QSTAR Elite Q-TOF tandem mass spectrometer only. The Orbitrap Velos was not used in this study. Proteins were required to have at least two unique peptides with FDR <1%. Applying these cut-offs

generated a list of 303 and 359 MAM protein from the heart and brain respectively; 80 of which were shared between the two tissues. Interestingly, there are no conserved proteins across the brain, liver, and heart based on this preliminary comparison. This observation is likely due to our analysis on only one type of mass analyzer, a Q-TOF, as opposed to combining data from multiple mass analyzers as discussed in the comprehensive brain

MAM characterization above. For this reason, the results presented here are preliminary.

A complete data set including analysis from at least two orthogonal mass spectrometry instruments will certainly increase the number of identifications and presumably conserved proteins in the MAM from the heart, brain, and liver.

Discussion

Isolation Validation

MAM isolation via Percoll gradient is the most widely accepted method for

analysis of this sub-cellular region (5, 38). We used a modified isolation technique that

yielded reliable and reproducible MAM fractions from tissue homogenates. Our Western

blot data confirms that our MAM sample is devoid of gross contamination and enriched

in MAM proteins. This is further substantiated by the identification of several proteins

that have been repeatedly shown to localize in the MAM such as GRP78 (Bip), VDAC1,

calnexin, calreticulin, GRP75, and SERCA. Organelle associations of the 1212 proteins

we identified showed that together ER and mitochondrial proteins comprise 38% of the

total. Proteins associated with other organelles should not be discounted as contaminants

in our list because the plasma membrane, golgi apparatus, cytoskeleton, and ribosome all have tight associations with the ER or mitochondria (2, 3, 58). The presence of these

36 proteins in an MAM fraction supports emerging theories of organelle interconnectivity

(59). It is also important to note that the organelle associations reported here are based on

GO annotation, which was unavailable for 13% of our proteins.

Comparison to Previous Reports

The first report of a proteomic study on the MAM was published in 2011 (43). In that quantitative comparison, the authors report the identification of 991 “confident unique proteins” using SILAC quantitation. Here, we report the identification of 1, 212 proteins localized to the MAM. The marked increase of protein identification is due to our previously reported robust proteomic workflow, which allows for the identification of recalcitrant membrane proteins that are normally not identified due to difficulties with solubility (39, 40). It is also of interest to note that the previously published study was focused on the identification of modulated proteins in relationship to viral infection. The focus of our work is to elucidate fundamental characteristics of proteins at the MAM in order to guide future studies. As a result of this contrast, only those MAM proteins that exhibited a response to viral infection were reported in the previous work. Our work provides a global snapshot of the MAM without any perturbation of the system, making it a potentially better reference point for MAM residents. As further validation of our work, we did identify PDIA3, Erp44, BiP, ACAT1, calnexin, calreticulin, VDAC1, VDAC2,

VDAC3, Hspa9, Mfn1, Erlin1 and Erlin-2, all of which are considered to be MAM marker proteins (32, 57, 60). Another MAM marker, Sig1R, did not meet our stringent filters, but was detected in our samples nevertheless. We did not detect Ero1-alpha, which is thought to be MAM-localized, as a result of this proteins cell state-dependent

association with the MAM (61). Our protein catalog, taken together with existing data, is

a tremendous step toward understanding the MAM both at steady state and under stressed

conditions.

One of the biggest challenges inherent to the global study of the MAM is the co-

isolation of bulk ER and mitochondrial proteins. We addressed this issue by employing

protein correlation profiling to further validate our list of MAM proteins. This method

quantitatively discriminates MAM residents (those with the lowest DM) from loosely

associated or contaminating proteins (those with highest DM). Proteins with the lowest

DM values are the most viable candidates for MAM markers. This is substantiated by the

presence of vesicle associated membrane protein 2 (vamp2), which has the lowest DM, and is reported to by palmitoylated in neurons (62), a post-translational modification that targets proteins to the MAM (11). Antibody-based studies of vamp2, and other candidates reported here will lead to new biomarkers of the region.

MAM Molecular Functions

Statistically –validated pathway analysis using IPA revealed the significant molecular function attributable to our MAM catalogue of proteins. Consistent with previous reports, proteins in the MAM are involved in mitochondrial dysfunction, oxidative phosphorylation, lipid synthesis, and Ca2+ trafficking (2, 5, 18, 32, 53). These

molecular functions were conserved in MAM fractions isolated from mouse liver in

addition to mouse brain, suggesting they are germane to the MAM and are not tissue

specific. Interestingly, many of these pathways are interconnected, and several MAM

proteins are involved in more than one function. For example, GRP78 (BiP) is a protein

chaperone that also binds IP3R to regulate Ca2+ transport from the MAM to mitochondria

(63). Because many MAM proteins are multifunctional, complex mechanisms to regulate

the activity of the MAM may be in place. It is also possible that post-translational

modification and phosphorylation-based signaling may be at the root of this regulation as

both functions were also found to be significant functions within the MAM. For example,

calnexin has been shown to target the MAM portion of the ER due to palmitoylation (11)

and other proteins (like the aforementioned vamp2) may be regulated in a similar fashion.

In addition, we identified 81 kinases and 31 phosphatases in the MAM, which supports the case for phosphorylation based activity at the MAM.

Relationship to Neurological Diseases

MAM dysfunction has been postulated to be an alternative explanation for

Alzheimer’s disease (AD) pathology (21). While we do not identify PS1 and PS2 in our

MAM fraction, we did observe Alzheimer’s disease as a statistically over-represented significant pathway (Figure 2.3). This is likely due to the presence of proteins involved in lipid metabolism, the unfolded protein response, and Ca2+ trafficking, as each of these

processes have been widely implicated AD (22, 27, 28, 64, 65). We also found tauopathy,

the accumulation of tau proteins in the brain, as a significantly-enriched pathway in our

data set. This is particularly interesting because the accumulation of tau tangles in the

brain has been directly related to AD pathology (66). Our findings support the postulate

that AD is a disease of the MAM and a comparative study of healthy and AD brain MAM

will further substantiate these claims.

Comparison of the identified MAM proteins to known neurological disease

pathways relates our list of MAM proteins to movement disorders (chorea and

Parkinson’s disease), genetic disorders (Huntington’s disease), and neurodegenerative diseases (schizophrenia, dementia, seizures). These disorders have not yet been attributed

to the MAM. Our data serves as a reference set for targeted studies that can relate the

functions of the MAM to the neurological diseases above. Further investigation of this

relationship may lead to a better understanding of disease pathologies and the elucidation

of novel therapeutic targets.

Conclusion

The data presented in this work provides a robust list of proteins that are localized

at the MAM from brain tissue. We report statistically significant biological processes, molecular functions, and neurological disorders over-represented in our list of proteins, providing a global look at the importance of the activity of the MAM. Understanding the proteome of this ER subdomain in tissue has great potential for biomedical application, as the biological state is not modified due to transfection or transformation, which is often the case with work conducted in vitro in cells. Targeted and comparative analysis of the

MAM will ultimately lead to a better understanding of MAM protein interactions and disease states.

Figures

Tissue Homogenate A

500 x g, 5min

P1 Supernatant

9,000 x g, 10min

Crude Supernatant Mitochondria (ER, PM, Cyto)

MAM 95,000 x g, 30min

30% Percoll

Crude MAM Mitochondria 9,000 x g, 10min

Contaminating Supernatant Mitochondria (MAM} 100,000 x g, 1 hr

Pure MAM Supernatant

Nuc Cyto PM ER MAM B Histone H3

Calnexin

Figure 2.1. MAM isolation schematic. (A) The MAM was isolated from brain tissue by differential centrifugation, followed by flotation on a self-forming Percoll gradient. (B) Fractions enriched in nuclei (nuc), cytosol (cyto), plasma membrane (pm), MAM, and ER were probed with the MAM marker calnexin, and the nuclear marker, histone H3. Intense bands for calnexin were observed in the ER and MAM fractions. No nuclear contamination was observed in the MAM.

Figure 2.2. Localization and biological relevance of identified proteins. A total of 1212 proteins were identified in the MAM fraction. (A) Organelle association for each protein was determined from GO annotation. Unreported proteins had no association. Ingenuity Pathway Core Analysis was used to identify protein types (B) and biological processes (C). Proteins without GO annotation (n = 484) are not shown here. The 15 most significant biological processes are shown. Processes with a p < 0.01 were considered significant.

20 value) ‐ 15 (p

log ‐ 10

Figure 2.3. Significant disease pathways. Identified proteins were compared against known neurological disease pathways using Ingenuity Pathway Core Analysis. The 15 most significant disease pathways are shown here. Pathways were considered significant if p < 0.01.

Figure 2.4. Protein correlation profiling. (A) Total membranes were pelleted from tissue homogenate and loaded onto a Percoll gradient. Fractions were collected and analyzed by LC-MS/MS. (B) Peak areas from XICs of 3 peptides from calnexin were normalized and plotted across fractions to create a profile. (C) Normalized areas for 3 peptides were averaged to provide protein correlation profiles. Profiles for mitochondrial (atp5b), ER (ppia), and MAM (canx) are shown with the MAM reference trend line.

Figure 2.5. Biological significance of MAM proteins conserved across tissues. (A) Brain MAM proteins were compared to liver MAM proteins. Of the 1212 proteins, found in the brain MAM, 293 were also identified in the liver MAM. (B) Organelle associations for these 293 proteins were obtained from GO annotation. (C) IPA provided insight into the significant biological processes of MAM proteins conserved across both tissues. Processes were considered significant if p < 0.01.

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CHAPTER 3

PROTEOMIC INVESTIGATION OF LIPID RAFT-ENRICHED MEMBRANES ISOLATED FROM INTERNAL ORGANELLES

Introduction

Lipid rafts (LRs) are classically defined as cholesterol-and sphingolipid-enriched

microdomains found in plasma membranes (PMs) (1). They are thought to provide a

more ordered environment within the cell membrane due to the presence of lipid species

that, together with cholesterol, stabilize the structure through close packing (2). Rafts are

characteristically insoluble in cold, non-ionic detergents and can be experimentally

isolated as detergent-resistant membranes (DRMs) (3). A key characteristic of LR-

enriched microdomains is that they sequester select proteins an.d act as organizing

scaffolds for numerous processes (e.g. molecule trafficking) (1, 4-6).

Although LRs were initially thought to reside solely in the plasma membrane,

increasing evidence suggests the presence of raft-like regions in internal organelles (7-

11). Membrane rafts derived from within the cell would most likely exhibit different

biochemical properties and contain a unique set of proteins since these regions have

distinctive lipid compositions and decreased levels of cholesterol (12). Although rafts

from these internal structures have not yet been characterized in a global way, significant

progress is evident. Membrane rafts isolated from mitochondria contain the voltage-

dependent anion channel 1 (VDAC1) and the fission protein hFis and can recruit other

proteins when the cell death program is initiated (10, 11). Raft-like domains in the endoplasmic reticulum (ER) are characterized by the presence of two proteins that only

localize to the ER, the prohibitins erlin-1 and erlin-2 (9). In addition, lipid raft-enriched

membranes isolated from the ER sub-structure known as the mitochondria-associated

membrane (MAM) were recently shown to contain the novel ligand-responsive sigma-1

receptor molecular chaperone and the type 3 inositol 1,4,5-trisphosphate receptor (IP3R3)

(13).

The MAM has garnered much attention recently as a signaling focal point because

it specifically interacts with the mitochondrial membrane in order to integrate and

coordinate Ca2+ signaling (13-15). Deregulation of Ca2+ trafficking can result in improper

protein folding, metabolic disruption, and apoptosis (16-19) and recent studies suggest

that membrane rafts are involved in coordinating the protein interactions required for

proper Ca2+ exchange between the MAM and mitochondria (14, 20). For this reason, we

comprehensively characterized lipid rafts isolated from MAM. Lipid rafts were isolated

via sucrose density centrifugation, solubilized and trypsinized using gel-assisted digestion

(21), and analyzed via two-dimensional reversed-phased (RP/RP) tandem mass

spectrometry (MS/MS). Our analysis allowed for the confident identification of proteins

known to reside in the MAM and that facilitate crosstalk between the mitochondrion and

ER. This new knowledge provides further insight into the biological processes that may be regulated by intracellular lipid rafts.

Experimental Procedures

Materials

Standard laboratory chemicals were obtained from Thermo Fisher Scientific

(Rockford, IL). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO)

with the exception of the following: FBS was from Atlanta Biologicals (Lawrenceville,

GA); protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN);

sequencing-grade modified-trypsin was from Promega (Madison, WI). Primary

antibodies (anti-flotillin-2 and anti-histone H3) and all secondary antibodies were

54 purchased from Santa Cruz Biotechnologies (Santa Cruz, CA) except for the anti-IP3R3 primary antibody, which was obtained from BD Sciences (Franklin Lakes, NJ).

Cell Culture and Whole Cell Lysate Preparation

NG108-15 cells (ATCC, Manassass, VA) were cultured at 37 °C, 5% CO2 in

MEM (pH 7.4) containing 10% FBS, 0.1 mM hypoxanthine, 400 nM aminopterin, and

0.016 mM thymidine. Cells were considered confluent at 80% flask coverage. Attached cells were dissociated with 1.5 mM EDTA solution, washed twice with PBS, and resuspended in 0.32 M sucrose and 10 mM Tris-HCl (pH 7.4) containing a cocktail of protease inhibitors (Roche). Cells were manually lysed by 40 strokes of a Dounce homogenizer. Nuclear debris was removed from the whole cell lysate by low speed centrifugation (500 × g, 5 min, 4 °C).

DRM Enrichment

Bulk DRMs from the crude mitochondrial fraction (P2, Fig. 3.1 A) were isolated using a modified version of a published protocol (3, 14). Care was taken to maintain a homogeneous solution of Triton X-114 throughout the isolation procedure by keeping the temperature at 4°C at all times (22). Briefly, mitochondria/MAMs were obtained from whole cell lysates by centrifugation at 16,000 × g for 45 min. The pellet was resuspended in 150 mM NaCl, 0.5% TritonX-114 in 10 mM Tris (pH 7.4). After a 1 h end over end rotation period, the sample was centrifuged at 12,000 × g for 30 min to reduce plasma membrane contamination. The supernatant was removed and then centrifuged at 100,000

× g for 1 h. The resulting pellet was taken as the DRM fraction. DRM proteins were

solubilized in a mixture of 2% SDS and 10 mM Tris (pH 7.4). Aliquots of each fraction

from each stage of the enrichment procedure were analyzed by Western blotting.

MAM Isolation

The MAM was isolated by a previously described method. (23) Briefly, brains

(generously donated from the Mark Cervas) extracted from wild-type CB57 mice were

homogenized by 6-8 strokes of a motorized Potter-Elvehjm glass homogenizer on ice. All

centrifugations were performed at 4 °C. Nuclei and unbroken cells were pelleted at 500 xg for 5 min twice. Crude mitochondria (CM) was separated from microsomes by centrifugation at 10,000 xg for 10 min and washed twice. CM was resuspended in 0.5 mL

of mitochondria re-suspension buffer (30 mM HEPES, 20 mM Mannitol, 5 mM EDTA,

pH 7.4) and layered on top of 30% Percoll (8 mL), followed by mitochondrial re-

suspension buffer (4 mL). After centrifugation at 95,000 xg for 30 min, the MAM band was aspirated from the gradient. The MAM was diluted 5X and centrifuged to remove mitochondrial contamination at 10,000 xg for 10 min. To pellet the MAM, the supernatant was centrifuged at 100,000 xg for 1 hr. The final MAM pellet was resuspended in 250 μL of 2% SDS. Protein concentration was determined by BCA assay.

Lipid Raft Isolation from MAM

Pure MAM was isolated from crude mitochondria as previously described (23).

Crude mitochondria were layered on top of a 30% Percoll gradient and centrifuged for 30 min at 95,000 x g. A dense band corresponding to pure MAM was aspirated from the gradient and diluted 5X. Mitochondrial contamination was pelleted by centrifugation at

10,000 xg for 10 min. The resulting supernatant was centrifuged at 100,000 xg for 1 hr to

pellet the MAM. The pellet was resuspended in 500 μL 0.5% TritonX-114 in 10 mM

Tris-HCl, 150mM NaCl, pH 7.4. The suspension was incubated at 4 °C for 1 hr. Sucrose was added to the MAM suspension for a total concentration of 80% sucrose. The suspension was placed in the bottom of a 4 mL ultraclear polyallomer tube (Beckman

Coulter, Brea CA). A 30% sucrose solution (3mL) was poured on top of the MAM suspension followed by 5% sucrose to fill the remainder of the tube. The gradient was centrifuged for 18 hr at 150,000 xg at 4 °C to isolate lipid rafts. Fractions of 500 μL were collected from the top of the gradient. Protein concentrations were determined by BCA assay.

SDS-PAGE and Western Blot Analysis

Protein samples separated by SDS-PAGE (NuPAGE Novex 10% Bis/Tris gels;

Invitrogen Corp., Calsbad, CA) were electrophoretically transferred to a PVDF membrane (0.2 μm, Millipore, Billerica, MA) at 4 ˚C for 90 min (45 V) using the

Invitrogen XCell II Blot Module. HRP -conjugated secondary antibodies allowed for detection and visualization of specific antigens by ECL (SuperSignal Chemiluminescent

Substrate; Thermo Fisher Scientific). Blots were imaged using a Syngene GeneGnome

(Frederick, MD) bio-imaging system.

Gel-Assisted Digestion

Fractions with positive immunoassay results for the lipid raft marker flotillin-2 were digested with trypsin using a gel-assisted method (24). Briefly, a polyacrylamide gel

57 was created by adding 18.5 μL of a 19:1 mixture of 40% (v/v) acrylamide solution/bis- acrylamide, 2.5 μL of 10% (w/v) ammonium persulfate, and 1 μL of 100% TEMED to 50

μg of protein in a Eppendorf tube. After polymerization, the gel/protein matrix was cut into small pieces and washed twice with 50% (v/v) acetonitrile in 25 mM ammonium bicarbonate (pH 8.0), followed by a single wash with neat acetonitrile. Dried gel pieces were rehydrated with trypsin solution and incubated at 37 °C for 18 h. Peptides were extracted using 0.1% acetic acid, 50% acetonitrile in 0.1% acetic acid, and neat acetonitrile sequentially. Extracts were pooled and concentrated by vacuum centrifugation to ~2 μL.

High pH Reversed-Phase (RP) HPLC

Peptide digests were reconstituted in 0.1% acetic acid and loaded onto an Agilent

ZORBAX 300Extend-C18 column (150 × 2.1 mm i.d., 3.5 μm) using an Agilent 1200 binary HPLC system. Peptides were separated by gradient elution from 0-80% mobile phase B in 20 min with a constant flow rate of 0.1 mL/min. Mobile phase A was composed of 1% methanol in 20 mM ammonia (pH 10.5) while mobile phase B consisted of a 90/10 mix of acetonitrile/1% methanol in 20 mM ammonia. UV absorbance was monitored at 214 nm. Fractions were collected every minute and concentrated by vacuum centrifugation to ~2 μL.

LC-MS/MS and Data Analysis

DRM fractions were reconstituted in 50 μL of 0.1% acetic acid and analyzed by nanoLC-MS/MS using a Tempo MDLC system coupled to a QSTAR Elite hybrid

quadrupole time-of-flight mass spectrometer (ABSciex, Foster City, CA) with an

ionization voltage setting of 1800 V. The peptides were eluted at a flow rate of 70-80

nl/min onto analytical columns (50 μm ID, 10 cm length of Monitor 100Å-Spherical

Silica C18; Column Engineering Inc., Ontario, CA) using the following gradient: 0-30% solvent B in 40 minutes; 30-60% B in 40 minutes; and 60-95% B in 20 minutes. Solvent

A consisted of 0.1% formic acid and 2% acetonitrile in water and solvent B contained

0.1% formic acid and 2% water in acetonitrile. MS data were acquired in information-

dependent acquisition mode with Analyst QS 2.0 (ABSciex) with Smart IDA enabled.

MS cycles were comprised of one full scan (m/z range = 300-2000, 1 sec accumulation) followed by sequential MS/MS scans of the four most abundant ions (+2 to +4 charge state, minimum ion count = 100, collision energy = 40.0 V, exclusion time = 15 sec, maximum accumulation time = 2 sec).

Concatenated lipid raft fractions were analyzed by nanoLC-MS/MS using an

Agilient 1200 system coupled to an LTQ Orbitrap Velos instrument (Thermo Scientific). with an ionization voltage setting of 1600 V. The peptides were eluted at a flow rate of

70-80 nl/min onto New Objective™ analytical columns (75 μm ID, 7 cm length) using the following gradient: 0-15% solvent B in 5 minutes and 15-35% B in 40 minutes.

Solvent A consisted of 0.1% formic acid and 2% acetonitrile in water and solvent B contained 0.1% formic acid and 2% water in acetonitrile. MS data were acquired in information-dependent acquisition mode with Xcalibur 2.0 (ThermoScientific) with lock mass enabled. MS cycles were comprised of one full scan (m/z range = 300-2000) followed by sequential MS/MS scans of the four most abundant ions (+2 to +4 charge state, minimum ion count = 500, automatic collision energy, exclusion time = 30 sec,

59 maximum. Both MASCOT and the Paragon algorithm within ProteinPilot 2.0.1

(ABSciex) and a Uniprotein (updated 6/13/2012) database was used to search the

MS/MS files. A 95% confidence threshold and 1% FDR for protein matches was used to filter the data, which corresponded to an unused protein score ≥ 1.3.

Results

Isolation and Evaluation of Detergent Resistant Membranes

As a preliminary study, we isolated lipid raft enriched detergent resistant membranes (DRMs) from a combined mitochondria and mitochondria-associated ER membrane sample. Our isolation was based on a previously reported method (3) that capitalizes on the insolubility of lipid raft-enriched membranes in non-ionic detergents at low temperatures (Figure 3.1 A). To confirm that lipid rafts are indeed enriched in

DRMs, we assayed for lipid raft marker, flotillin-2 via western blot (Figure 3.1 B). When compared to nuclear, microsomes, and detergent soluble (TS) fractions, we observe the strongest signal for flotillin-2 in the DRM fraction, suggesting that lipid rafts are enriched in this fraction. We also assayed for inositol-1-4-5-triphosphate receptor type 3 (IP3R3), a calcium channel, which is found in the MAM (25). While we do observe a signal across all of the fractions, the signal is most abundant in the DRM. As a negative control, we assayed for histone H3, a nuclear biomarker, for which we observe no signal in the DRM fraction. Based on our Western blot data, we conclude that our DRM fraction is enriched in lipid rafts and MAM proteins and therefore suitable for our LC-MS/MS analysis.

Proteomic Characterization of DRMs

Solubilized DRM proteins were subjected to our multiplexed analysis strategy that includes gel-assisted digestion (26) and RP/RP-MS/MS. During sample processing, detergent is removed from the sample, proteins are in-gel digested with trypsin, peptides are fractionated offline by RP-HPLC at pH 10 and peptide fractions are analyzed by RP-

LC-MS/MS at pH 2. A total of 4 analyses were conducted: two technical replicates for each of two biological replicates. The MS/MS spectra were searched using ProteinPilot software and a combined rat/mouse IPI database and 2,033 unique peptides representing

447 proteins were identified. We required that each protein: be substantiated by the detection of 2 or more unique peptides at the 95% confidence level; have a protein unused score of 2.6 or greater; and have a protein confidence level of >95%. Spectra were also searched against a decoy database and an FDR of 1% was determined.

Applying these filters resulted in the identification of 250 high-confidence proteins

(Table A2).

The majority of our 250 high-confidence proteins (146 proteins) are bona fide mitochondrial or ER proteins (Figure 3.2 A). Proteins without a reported location comprise the third largest group followed closely by PM proteins. The proteins annotated as residing in the PM may actually have multiple subcellular locations that are not yet described in the GO database, a problem noted in other publications (21, 27). The low percentage of protein identifications from the Golgi, nucleus and other subcellular locations suggests that these proteins are residual contamination. Almost 52% of the 250 high confidence identifications are membrane proteins, mostly of mitochondrial, ER, or

PM origin (Figure 3.2 B). Our manual review of the literature indicates that ~20% (49

proteins) of the 250 proteins have been described as components of LRs or DRMs

previously. Importantly, we detected the known internal LR marker proteins IP3R3

(specific for the MAM) (14), erlin-2 (specific for the ER) (28), and VDAC1 (specific for

the mitochondria) (10). Additionally, 74% (184 proteins) of the 250 proteins have

previously been noted as MAM-resident or -associated proteins. In sum, these results

provide strong evidence that our isolation method preferentially recovered DRM proteins from mitochondria and MAMs and that the sample was enriched in LR proteins.

Further classification of the 250 proteins according to biological process and molecular function revealed that the proteins segregate into categories congruous with the

central role of mitochondria in metabolism and the transport/protein processing activity of the MAM (Figure 3.3). Protein processing (24%), metabolic processes (22%), and transport (10%) are the top three biological activities represented (Figure 3.3 A). Proteins localized to the MAM that participate in different aspects of protein processing as well as components of the mitochondrial electron transport chain (ETC) and the tricarboxylic acid (TCA) cycle were detected. In addition, enzymes tasked with cholesterol, glucose, lipid, and glycerophospholipid metabolism were found.

Particularly noteworthy is the detection of members of the Ca2+ macromolecular

complex that resides at the ER-mitochondrion interface and exerts control over Ca2+ signaling and transport between the two organelles (29, 30). The proteins in this complex that were detected include IP3R3 (gene symbol Itpr3), VDAC1 (Vdac1), Grp75 (Hspa9),

Bip/Grp78 (Hspa5), ERp57 (Pdia3), calnexin (Canx), calreticulin (Calr), and the adenine nucleotide transporters ANT1 (Slc25a4) and ANT2 (Slc25a5). Furthermore, the functional classification of the 250 proteins separate into roughly two categories –

62 enzymes and binding proteins (Figure 3.3 B). This ‘big picture’ view of molecular function supports the roles of the identified proteins in the biological processes that occur in the mitochondrion and the MAM regions, respectively.

Isolation and Analysis of Lipid Rafts from MAM

The MAM was isolated as described in Chapter 2 (23). Once purified MAM was obtained, lipid rafts were isolated. To confirm the enrichment of lipid rafts, fractions were collected from the top of the gradient and assayed for the lipid raft marker, flotillin-2.

(Figure 3.4 B). A total of 8 fractions were collected, and only two fractions in the low density region of the gradient show a strong signal for flotillin-2, suggesting that these fractions correspond to the lipid raft. Western blot data was considered reproducible after

3 replicates.

A total of 4 analyses were conducted: two technical replicates for each of two biological replicates. The MS/MS spectra were searched against a concatenated forward/reverse mouse database using both MASCOT and the Paragon Algorithm. We required that each protein be substantiated by: the detection of 2 or more unique peptides with an FDR below 1%; have a protein unused score of 2.6 or greater; have a protein

FDR below 1%; and be identified by both search algorithms. The application of these stringent cut-offs revealed a final list of 545 proteins identified in lipid rafts isolated from pure MAM.

Evaluation of Proteins Identified Only in the Lipid raft

Of the 545 proteins identified in the lipid raft sample, 133 are solely identified in the lipid raft when compared to the MAM sample, with no spectral counts for these proteins detected in the MAM fraction (Table A3). These proteins are of particular interest because they are likely low-abundance lipid raft proteins that are not detected in the presence of the relatively high-abundance proteins associated with the MAM and could only be detected when enriched. The biological enrichment of lipid rafts from the

MAM removes this high-abundance background, ultimately uncovering new information about lipid rafts in this region. Protein localization based on Gene Ontology (GO) annotation reveals 47% of the 133 lipid raft proteins are localized either to the mitochondria (28%) or ER (19%), substantiating the MAM as the origin of these lipid rafts (Figure 3.5 A). We also observe proteins localized to the cytoplasm (19%), cytoskeleton (7%), golgi apparatus (4%), and plasma membrane (1%). There was no localization annotation in the database for 22% of these proteins.

Proteins thought to localize to the cytoplasm, cytoskeleton, golgi apparatus, and plasma membrane may undergo reversible s-acylation, which has been shown to temporarily localize proteins to the MAM (31). The addition of palmitoic acid to cysteine residues increases the hydrophobicity of the protein and causes preferential localization to membrane regions in the cell. Reversible s-acylation has been shown to control protein localization both to the MAM and to lipid rafts (31-33). It follows then, that proteins enriched in lipid rafts isolated from the MAM would contain sites of palmitoylation. To assess this, we used CSS-PALM (34) an algorithm which predicts sites of palmitoylation based on amino acid sequence motifs and literature findings.(Figure 3.6).

We compared the number of predicted palmitoylation sites on proteins in the

MAM to that of proteins found only in the lipid raft. Our findings support reports that s- acylation targets proteins in the MAM: 60% (738 proteins) of proteins identified in the

MAM contain at least one predicted palmitoylation site with 3 proteins having up to 26 predicted sites. We find 75% (100 proteins) of proteins identified only in the lipid raft isolated from the MAM contain at least one palmitoylation site with the number of predicted sites on one protein as high as 53. Although this result is based on site prediction, and requires validation for each individual protein sequence, our result demonstrates a trend for the localization of s-acylated proteins in the lipid raft portion of the MAM.

Statistically Validated Lipid Raft-Enriched Proteins

While the identification of proteins found solely in lipid rafts isolated from the

MAM provides some insight into the residents of this region, it does not account for the proteins that may join (or leave) the lipid raft environment, as these proteins would be found in both the bulk MAM and lipid raft enriched samples. To gain further information on these “temporary” lipid raft residents, we employ a quantitative comparison of proteins identified in the bulk MAM and in lipid rafts enriched from the MAM. Our quantitative analysis of lipid rafts from the MAM is based on the correlation of spectral count to the abundance of identified proteins. Spectral count analysis is a label-free mass spectrometry-based quantitation technique which directly correlates the number of times a particular ion is sampled during the precursor ion scan, to its abundance in the sample

(35). It is advantageous because it does not require chemical modification or excess

sample manipulation which can ultimately lead to sample loss, while providing reliable

absolute quantitation for identified peptides.

Because this work requires a comparative analysis of the bulk MAM and lipid raft

enriched MAM fractions, we used fold changes in spectral count as a measure of

enrichment for each protein, with a protein required to exhibit a 50% or greater fold

increase in spectral count to be considered lipid raft-enriched. In order to provide a more

robust report of lipid raft-enriched proteins from the MAM, we added a second

dimension of statistical validation to our data set by analyzing our spectral count data using QSpec (35). Briefly, QSpec pools spectral count data across all proteins in both data sets to generate two hypothetical distributions corresponding to the null hypothesis and there being significant change between the two data sets. Then, the spectral count data for each protein is fit to both of the distributions, and a measure of significance known as a Bayes factor is calculated. The QSpec algorithm is especially advantageous for proteomic experiments because it employs G-test and Bayes factor statistics, which do not require several replicates in order to determine the significance of a fold change.

Based on QSpec analysis we report 44 proteins with both 50% fold changes with a Bayes factor greater than 10, suggesting that these changes are significant (Table A4).

Notably, among these proteins are VDAC1 and VDAC2, two calcium ion channels reported to localize both at the MAM and lipid rafts (Table 3.1). Gene ontology annotation reveals that these 8 proteins are composed of transporters (ATP5A1, ATP5B,

ATP1A3, and SLC25A4), ion channels (VDAC1 and VDAC 2), an enzyme (NDUFB10), and a kinase (CAMK2B). The presence of transporters and ion channels as statistically validated lipid raft-enriched proteins illustrates the essential role that lipid rafts play in

the MAM. Presumably these proteins can associate with and dissociate from the lipid raft

in the MAM as necessary to facilitate functions in the MAM.

We further assessed the molecular functions and diseases associated with our 44

statistically validated proteins by analyzing our data set with IPA (Figure 3.7). This

comparison was statistically validated by Fisher’s T-test, with a significance threshold of

p < 0.01. We found that mitochondrial dysfunction (p = 1.24E-07), oxidative

phosphorylation (p = 1.85E-02), and ubiquinone biosynthesis (p = 1.98E-03) are the top

three most significant molecular functions within this data set (Figure 3.7). We also

found that molecular transport (p = 4.69E-07), nervous system development (p = 7.19E-

07) and cell to cell signaling (p = 7.19E-07) were the most statistically significant biological processes represented in our lipid raft enriched protein list.

Because the MAM has been postulated to be at the root at several neurodegenerative diseases, including Alzhiemer’s disease, we used IPA to probe for any associations between our lipid raft data and neurological diseases. This a analysis revealed proteins enriched in lipid rafts from the MAM to be involved in diseases and

disorders such as Huntington’s disease (p = 7.15E-4), schizophrenia (p = 2.99E-4) and

mood disorders (p = 1.83E-6). While the Alzhiemer’s disease pathway does not meet our

significance threshold of p < 0.01, nine of our fourty-four proteins are a part of this

disease pathway. Taken together, these data suggest that the formation of the lipid raft

within the MAM may be a requirement for cellular survival mechanisms, such as

transport and ATP production, as well as signaling activities at the MAM which relate the

region to neurodegenerative diseases.

Discussion

Our preliminary work with DRMs in neuronal cells showed that lipid rafts are

present in a combined MAM/mitochondria fraction. These novel lipid microdomains

differ from classically-defined LRs in that they can localize within internal organelles (9,

10, 36, 37), be cholesterol-independent (38, 39), remain stable over extended timeframes

(40, 41), and/or exhibit a higher density on sucrose gradients (42). To better understand protein composition inherent only to the MAM, we chose to isolate lipid rafts from the

MAM via sucrose density gradient, a well documented method for LR isolation (43-45).

(Figure 3.4 A). We specifically chose to use Triton X-114 detergent for this isolation

because DRMs derived from internal organelles are preserved in its presence (14, 46).

The proteomic analysis of membrane proteins is particularly challenging as this

subset of proteins are difficult to keep in solution and the addition of harsh detergent is

often detrimental to ionization for mass spectrometry analysis. We solved this problem

by subjecting lipid raft proteins to our multiplexed analysis strategy that includes gel-

assisted digestion (26) and RP/RP-MS/MS (47, 48). During sample processing, detergent

was removed from the sample, proteins were in-gel digested with trypsin, and peptides

were fractionated offline by RP-HPLC at pH 7.5. Then, peptide fractions were analyzed

by RP-LC-MS/MS at pH 2, providing an orthogonal separation and increasing peptide

identification.

Our bioinformatic analysis of statistically validated proteins enriched in lipid rafts

isolated from the MAM highlighted the involvement of these proteins in molecular

functions, such as oxidative phosphorylation, lipid metabolism, and Ca2+transport. This

suggests that the formation of lipid raft scaffolds in the MAM is involved in facilitating

theses functions at this region. We also found that several neurodegenerative diseases

including Alzhiemer’s and Huntington’s disease are significantly represented in our data

set, supporting postulates that lipid raft formation in the MAM is at least partially

involved in mechanisms which link processes regulated by the MAM to

neurodegenerative diseases.

Concluding Remarks

The work reported here combines an optimized membrane protein proteomics

workflow with bioinformatics in order to gain key insights into lipid rafts within the

MAM. In addition to providing a catalog of proteins found in lipid rafts in the MAM, the

data presented here is meant to substantiate current claims that link lipid rafts in the

MAM to apoptosis, cell signaling and ion trafficking on the molecular level, while

providing more data to support implications that this region plays a role in neurological diseases. Targeted analysis to confirm the lipid raft-dependence of these pathways could ultimately lead to the elucidation of new therapeutic targets for disorders and a more clear understanding of how lipid rafts and the MAM are linked to these disease states.

Figures

Figure 3.1. DRM isolation and Western blot validation. (A) Isolation schematic for DRMs from NG108-15 cells. (B). Western blot validation of DRM fraction. Abbreviations: Whole cell lysate (WCL), Nuclear fraction (Nuc), Microsomes (Mic), Triton soluble fraction (TS).

Figure 3.2. DRM protein localization. (A) Total protein subcellular localization based on Gene Ontology annotation (GO). (B) Subcellular localization of membrane proteins based on GO annotation. Proteins in unreported categories do not have annotation in the GO database.

Actin Filament Movement 5 Apoptosis 2 Biosynthesis 11 Calcium Ion Homeostasis 8 Cellular Response 2 Cholesterol Homeostasis 2 Cristae Formation 1 Lipid Biosynthesis 5 Lipid Metabolism 8 Metabolic Processes 54 Microtubule Activity 2 mRNA processing 2 Protein Processing 59 Signal Transduction 8 Transcription 2 Translation 16 Transport 24 Unreported 39 0 10203040506070

Antiporter 3 Elongation factor 1 Hydrolase 3 Ion channel 2 Isomerase 1 Kinase 1 Ligase 6 Lyase 1 Micotubule motor activity 1 Oxidoreductase 34 Peptidase 4 Protein Binding 64 Receptor 3 Small Molecule Binding 64 Structural 12 Symporter 2 Transferase 11 Unreported 35 Vesicle 2 0 10203040506070

Figure 3.3. Biological significance of DRM proteins. (A) Biological processes and (B) molecular functions of DRM proteins based on Gene Ontology (GO) annotation. Proteins in unreported categories do not have annotation in the GO database.

Pure MAM A 0.5% Tx‐114, 1hr, 4° C

Protein Suspension Collect 500μL fractions

0.5 mL 5% Sucrose 150,000 x g, 18Hr LR

3 mL 30% Sucrose LC‐MS/MS Analysis

0.5mL 80% Sucrose , MAM Suspension

Lipid Raft B

5% 30% 80%

Figure 3.4. Lipid raft isolation and Western blot validation. (A) Lipid rafts are isolated from pure MAM pellets isolated form brain tissue (see Figure 2.1). (B) Lipid raft fractions from sucrose density gradients are determined by enrichment of flotillin-2, a lipid raft biomarker.

Figure 3.5. Biological significance of proteins identified only in the lipid raft. (A) Localization and (B) protein type based on Gene Ontology (GO) annotation. Unreported proteins do not have localization annotation. Significant (C) Molecular function and (D) neurological diseases from Ingenuity Pathway Analysis. Significance threshold was Log (p-value) >2.

Lipid Raft 30 MAM

20 Number of Sites Predicted Palmitoylation 10

0 0 100 200 300 400 500 600 700 800 Number of Protein IDs

Figure 3.6. Predicted palmitoylated sites in MAM proteins. Comparison of predicted palmitoylation sites for proteins found only in the lipid raft and MAM proteins. Site predictions were obtained using CSS- PALM software.

Figure 3.7. Biological significance of lipid raft-enriched MAM proteins. (A) Localization and (B) protein type based on Gene Ontology (GO) annotation. Unreported proteins do not have localization annotation. Significant (C) Molecular function and (D) neurological diseases from Ingenuity Pathway Analysis. Significance threshold was Log (p-value) >2.

Table 3.1 Statistically validated lipid raft-enriched proteins Lipid Bayes Symbol* Entrez Gene Name* Control2 Type(s)4 Raft1 Factor3 ATP5A1 ATP synthase, H+ transporting, mitochondrial 446 58 2.09E+38 Transporter F1 complex, alpha subunit 1, cardiac muscle ATP5B ATP synthase, H+ transporting, mitochondrial 220 36 2.26E+15 Transporter F1 complex, beta polypeptide VDAC1 Voltage-dependent anion channel 1 112 6 1.40E+15 Ion channel ATP1A3 ATPase, Na+/K+ transporting, alpha 3 351 84 1.06E+15 Transporter polypeptide SLC25A4 Solute carrier family 25 (mitochondrial 63 10 22606.046 Transporter carrier; adenine nucleotide translocator), member 4 VDAC3 Voltage-dependent anion channel 3 20 2 43.834 Ion channel NDUFB10 NADH dehydrogenase (ubiquinone) 1 beta 18 2 24.926 Enzyme subcomplex, 10, 22kDa CAMK2B Calcium/calmodulin-dependent protein kinase 30 7 12.762 Kinase II beta *Gene name and symbol based on Entrez database; 1. Protein spectral counts in lipid raft sample; 2. Protien spectral counts in MAM samples; 3. Bayes Factor significance reported by QSpec, significance threshold 10; 4. Protein type as reported by the Gene Ontology database

References

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CHAPTER 4

CONCLUSION

Summary of Results

Mitochondria-associated endoplasmic reticulum membranes have re-emerged as a

region of interest due to their role in small molecule trafficking, calcium homeostasis,

and protein synthesis (1-3). Major breakthroughs in the study of the MAM have been

slow and steady because investigations have focused on one or two proteins or processes

at a time, and all of the proteins known to localize at the MAM have yet to be

determined. Antibody-based studies of this region are often difficult because they require

specific and often expensive antibodies for each protein of interest. With the work

reported herein, we advance the field of MAM study by contributing robust global

characterization of ER/mitochondria contact sites using a tandem mass spectrometry-

based proteomic workflow. This method is advantageous because it allows for the

identification of both known and previously unreported proteins in the MAM in a single experiment. Further, the large-scale characterization of MAM proteins lends itself to elucidation of pathways under the control of the MAM. With this in mind, we pursued the global proteomic characterization of the ER/mitochondria contact from mouse brain tissue.

Proteomic analysis of membrane proteins is particularly challenging due to the hydrophobic nature of these proteins. In this work we overcame this hurdle with our gel- based LC/LC-MS/MS workflow that allows the protein to be dissolved in strong detergents that would otherwise prevent ESI ionization for MS detection or destroy column/solute interactions during reverse-phase chromatography. In addition, we increased the number of proteins identified by analyzing our peptide digests using both a

quadrupole time-of-flight and an LIT-orbitrap mass analyzer, removing bias for the

detection of specific ions based on the ion activation mechanisms of each instrument.

Shotgun proteomic analysis combined with robust statistical analysis of raw data yielded 1,212 proteins from the brain MAM. Among these proteins were known MAM residents: calnexin, calreticulin, VDAC1, VDAC2, VDAC2, and IP3R-1. We also found a number of oxidoreductases, ion transporters, and protein chaperones. Proteins representing these classes of proteins have been found to localize to the MAM region.

We also employ protein correlation profiling to quantitative validate the presence of

MAM marker proteins in our isolated MAM fraction. Our bioinformatics analysis of

these proteins revealed significantly over-represented signaling pathways that may be

facilitated by the activities of the MAM, such as oxidative phosphorylation, signal

regulation and apoptosis regulation. In addition, we found several neurological disease

pathways to be statistically significant when compared to our list of MAM proteins.

These include schizophrenia, movement disorders, and Alzheimer’s disease. Our findings

suggests that the activity in the MAM may in influence the pathways and diseases

mentioned above, making the region a potential hot spot for therapeutic targets.

Motivated by reports (4, 5) that the MAM contained lipid-rich, detergent resistant

microdomains, we isolated lipid rafts from the MAM and differentiated these proteins

from the bulk MAM using the label–free mass spectrometry based method, spectral

counting. A total of 133 proteins were found to be unique to lipid raft, while 44 proteins

were statistically validated to be enriched in lipid rafts isolated from the MAM. Quite

interestingly, protein transporters and ion channels are enriched in the lipid raft,

suggesting that lipid rafts in the MAM are crucial regulators of these widely accepted

MAM functions. In addition, the statistically significant neurological diseases identified in our list of MAM proteins are also significant in lipid rafts, again highlighting the potential role of lipid rafts in the MAM. The data presented here emphasizes the importance of lipid rafts in the MAM.

Future Work

Because this work provided a comprehensive evaluation of the MAM proteins, functions, and implications in neurological disease, there are several new questions to pursue. Molecular pathways considered to be statistically significant based on our bioinformatics analysis could be confirmed by knockout studies to clearly determine the role that each protein plays in the MAM. The extent of phosphorylation-based signaling at the MAM could also be elucidated by a phospho-proteomic analysis of the region.

One of the most promising applications of this research is the potential to uncover new pathologies for neurodegenerative diseases. Alzheimer’s disease has the most evidence to date to have a connection to the activity of the MAM. A quantitative proteomic comparison between healthy brain tissue and brain tissue expressing an

Alzhiemer’s disease phenotype would illuminate the proteins in the MAM that are altered in the diseased state, ultimately leading to direct links between the MAM and

Alzheimer’s disease.

In sum, future studies should focus on validating the predicted mechanisms through which the MAM regulates cellular processes presented here, and elucidating the link between the MAM and disease states. While we focus on the MAM in the brain here, the region has been postulated to play a pathological role in Type II diabetes and

85 cardiovascular disease, suggesting that similar studies on the MAM from liver and heart tissue could be insightful. The complete elucidation of the processes and diseases regulated by activities of the MAM will require the continued effort of analytical chemists, molecular biologists, and computational biologists.

References

1. Simmen, T., Lynes, E. M., Gesson, K., and Thomas, G. (2010) Oxidative protein folding in the endoplasmic reticulum: Tight links to the mitochondria-associated membrane (MAM). Bba-Biomembranes 1798, 1465-1473. 2. Rizzuto, R., Pinton, P., Giorgi, C., Siviero, R., and Zecchini, E. (2008) Calcium and apoptosis: ER-mitochondria Ca(2+) transfer in the control of apoptosis. Oncogene 27, 6407-6418. 3. Hayashi, T., Rizzuto, R., Hajnoczky, G., and Su, T. P. (2009) MAM: more than just a housekeeper. Trends in Cell Biology 19, 81-88. 4. Hayashi, T., and Fujimoto, M. (2010) Detergent-resistant microdomains determine the localization of sigma-1 receptors to the endoplasmic reticulum- mitochondria junction. Mol Pharmacol 77, 517-528. 5. d'Azzo, A., Sano, R., Annunziata, I., Patterson, A., Moshiach, S., Gomero, E., Opferman, J., and Forte, M. (2009) GM1-Ganglioside Accumulation at the Mitochondria- Associated ER Membranes Links ER Stress to Ca(2+)-Dependent Mitochondrial Apoptosis. Mol Cell 36, 500-511.

APPENDIX

Nuc Cyto PM ER MAM

Ero1-alpha

Histone H3

Cox IV

Na+/K+ ATPase

Calnexin

Figure A1. Immunoassy of MAM fractions. Western blot analysis of fractions from MAM isolation of nuclear (Nuc), cytosol (Cyto), plasma membrane (PM), endoplasmic reticulum (ER), and MAM. Fractions were assayed for ER marker Ero1-aplha, nuclear marker Histone H3, mitochondira marker CoxIV, plasma membrane marker Na+/K+ ATPase, and MAM marker calnexin. Minimal contamination from these organelles was observed in the MAM fraction.

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) 1700009N14Rik RIKEN cDNA 1700009N14 gene transporter 4930544G11Rik RIKEN cDNA 4930544G11 gene enzyme AAK1 AP2 associated kinase 1 kinase AARS alanyl-tRNA synthetase enzyme ABAT 4-aminobutyrate aminotransferase enzyme ABCA2 ATP-binding cassette, sub-family A (ABC1), member 2 transporter ABCB7 ATP-binding cassette, sub-family B (MDR/TAP), member 7 transporter ABCB8 ATP-binding cassette, sub-family B (MDR/TAP), member 8 transporter ABCB9 ATP-binding cassette, sub-family B (MDR/TAP), member 9 transporter ABCD3 ATP-binding cassette, sub-family D (ALD), member 3 transporter ABCF1 ATP-binding cassette, sub-family F (GCN20), member 1 transporter ABHD12 abhydrolase domain containing 12 unreported ABHD16A abhydrolase domain containing 16A unreported ABHD6 abhydrolase domain containing 6 enzyme ABLIM1 actin binding LIM protein 1 unreported ABLIM2 actin binding LIM protein family, member 2 unreported ABR active BCR-related unreported ACAA1 acetyl-CoA acyltransferase 1 enzyme ACAA2 acetyl-CoA acyltransferase 2 enzyme ACAD9 acyl-CoA dehydrogenase family, member 9 enzyme ACADL acyl-CoA dehydrogenase, long chain enzyme ACADVL acyl-CoA dehydrogenase, very long chain enzyme ACAT1 acetyl-CoA acetyltransferase 1 enzyme ACLY ATP citrate lyase enzyme ACO2 (includes aconitase 2, mitochondrial enzyme EG:11429) ACOT2 acyl-CoA thioesterase 2 enzyme ACOT7 acyl-CoA thioesterase 7 enzyme ACOX1 acyl-CoA oxidase 1, palmitoyl enzyme ACP1 (includes acid phosphatase 1, soluble phosphatase EG:11431) ACSBG1 acyl-CoA synthetase bubblegum family member 1 enzyme ACSL1 acyl-CoA synthetase long-chain family member 1 enzyme ACSL3 acyl-CoA synthetase long-chain family member 3 enzyme ACSL6 acyl-CoA synthetase long-chain family member 6 enzyme ACTC1 actin, alpha, cardiac muscle 1 enzyme ACTG1 actin, gamma 1 unreported ACTN1 actinin, alpha 1 unreported ACTN2 actinin, alpha 2 transcription regulator ACTN4 actinin, alpha 4 unreported ACTR1A ARP1 actin-related protein 1 homolog A, centractin alpha (yeast) unreported

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) ACTR1B ARP1 actin-related protein 1 homolog B, centractin beta (yeast) unreported ACTR2 ARP2 actin-related protein 2 homolog (yeast) unreported ADAM22 ADAM metallopeptidase domain 22 peptidase ADAM23 ADAM metallopeptidase domain 23 peptidase ADAP1 ArfGAP with dual PH domains 1 unreported ADCY9 adenylate cyclase 9 enzyme ADD1 adducin 1 (alpha) unreported ADD1 adducin 1 (alpha) unreported ADD2 adducin 2 (beta) unreported ADD3 adducin 3 (gamma) unreported AFG3L2 AFG3 ATPase family gene 3-like 2 (S. cerevisiae) peptidase AGAP2 ArfGAP with GTPase domain, ankyrin repeat and PH domain 2 enzyme AGK acylglycerol kinase kinase AHCY adenosylhomocysteinase enzyme AHCYL1 adenosylhomocysteinase-like 1 enzyme AHCYL2 adenosylhomocysteinase-like 2 enzyme AHSA1 AHA1, activator of heat shock 90kDa protein ATPase homolog 1 unreported (yeast) AIFM1 apoptosis-inducing factor, mitochondrion-associated, 1 enzyme AIFM3 apoptosis-inducing factor, mitochondrion-associated, 3 enzyme AK1 adenylate kinase 1 kinase AK3 adenylate kinase 3 kinase AKAP12 A kinase (PRKA) anchor protein 12 transporter AKAP2/PALM2 A kinase (PRKA) anchor protein 2 unreported -AKAP2 AKAP5 A kinase (PRKA) anchor protein 5 unreported ALB albumin transporter ALCAM activated leukocyte cell adhesion molecule unreported ALDH1L1 aldehyde dehydrogenase 1 family, member L1 enzyme ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) enzyme ALDH3A2 aldehyde dehydrogenase 3 family, member A2 enzyme ALDH3B1 aldehyde dehydrogenase 3 family, member B1 enzyme ALDH5A1 aldehyde dehydrogenase 5 family, member A1 enzyme ALDH6A1 aldehyde dehydrogenase 6 family, member A1 enzyme ALDH9A1 aldehyde dehydrogenase 9 family, member A1 enzyme ALDOA aldolase A, fructose-bisphosphate enzyme ALDOC aldolase C, fructose-bisphosphate enzyme ALS2 amyotrophic lateral sclerosis 2 (juvenile) unreported AMPH amphiphysin unreported ANK1 ankyrin 1, erythrocytic unreported ANK2 ankyrin 2, neuronal unreported ANK2 ankyrin 2, neuronal unreported ANKRD63 ankyrin repeat domain 63 unreported ANLN anillin, actin binding protein unreported ANXA2 annexin A2 unreported ANXA5 annexin A5 unreported 91

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) ANXA6 annexin A6 unreported AP1B1 adaptor-related protein complex 1, beta 1 subunit transporter AP2A1 adaptor-related protein complex 2, alpha 1 subunit transporter AP2A2 adaptor-related protein complex 2, alpha 2 subunit transporter AP2B1 adaptor-related protein complex 2, beta 1 subunit transporter AP2M1 adaptor-related protein complex 2, mu 1 subunit transporter AP2S1 adaptor-related protein complex 2, sigma 1 subunit transporter AP3B2 adaptor-related protein complex 3, beta 2 subunit transporter AP3D1 adaptor-related protein complex 3, delta 1 subunit transporter APOE apolipoprotein E transporter APOO apolipoprotein O unreported APOOL apolipoprotein O-like unreported ARAF v-raf murine sarcoma 3611 viral oncogene homolog kinase ARCN1 archain 1 unreported ARF3 ADP-ribosylation factor 3 enzyme ARF6 ADP-ribosylation factor 6 transporter ARHGAP23 Rho GTPase activating protein 23 unreported ARHGAP35 Rho GTPase activating protein 35 transcription regulator ARHGAP39 Rho GTPase activating protein 39 unreported ARHGAP5 Rho GTPase activating protein 5 enzyme ARHGDIA Rho GDP dissociation inhibitor (GDI) alpha unreported ARHGEF11 Rho guanine nucleotide exchange factor (GEF) 11 unreported ARHGEF2 Rho/Rac guanine nucleotide exchange factor (GEF) 2 unreported ARL2 ADP-ribosylation factor-like 2 enzyme ARL8B ADP-ribosylation factor-like 8B enzyme ARPC2 actin related protein 2/3 complex, subunit 2, 34kDa unreported ARPC4 actin related protein 2/3 complex, subunit 4, 20kDa unreported ASAP1 ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 unreported ASNA1 arsA arsenite transporter, ATP-binding, homolog 1 (bacterial) transporter ASPA aspartoacylase enzyme ASS1 argininosuccinate synthase 1 enzyme ATAD3A/ATA ATPase family, AAA domain containing 3A unreported D3B ATCAY ataxia, cerebellar, Cayman type unreported ATG9A ATG9 autophagy related 9 homolog A (S. cerevisiae) unreported ATL1 atlastin GTPase 1 enzyme ATP13A1 ATPase type 13A1 transporter ATP1A1 ATPase, Na+/K+ transporting, alpha 1 polypeptide transporter ATP1A2 ATPase, Na+/K+ transporting, alpha 2 polypeptide transporter ATP1A3 ATPase, Na+/K+ transporting, alpha 3 polypeptide transporter (includes EG:232975) ATP1A3 ATPase, Na+/K+ transporting, alpha 3 polypeptide transporter (includes EG:232975) ATP1B1 ATPase, Na+/K+ transporting, beta 1 polypeptide transporter 92

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) ATP1B2 ATPase, Na+/K+ transporting, beta 2 polypeptide transporter ATP2A1 ATPase, Ca++ transporting, cardiac muscle, fast twitch 1 transporter ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 transporter ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 transporter ATP2A3 ATPase, Ca++ transporting, ubiquitous transporter ATP2A3 ATPase, Ca++ transporting, ubiquitous transporter ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 transporter ATP2B2 ATPase, Ca++ transporting, plasma membrane 2 transporter ATP2B3 ATPase, Ca++ transporting, plasma membrane 3 transporter ATP2B3 ATPase, Ca++ transporting, plasma membrane 3 transporter ATP2B4 ATPase, Ca++ transporting, plasma membrane 4 transporter ATP2B4 ATPase, Ca++ transporting, plasma membrane 4 transporter ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha transporter subunit 1, cardiac muscle ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta transporter polypeptide ATP5C1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma transporter polypeptide 1 ATP5C1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma transporter polypeptide 1 ATP5D ATP synthase, H+ transporting, mitochondrial F1 complex, delta transporter subunit ATP5F1 ATP synthase, H+ transporting, mitochondrial Fo complex, subunit B1 transporter Atp5h (includes ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d enzyme EG:100039281) Atp5h (includes ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d enzyme EG:100039281) ATP5J ATP synthase, H+ transporting, mitochondrial Fo complex, subunit F6 transporter ATP5O ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit transporter ATP6V0A1 ATPase, H+ transporting, lysosomal V0 subunit a1 transporter ATP6V0D1 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d1 transporter ATP6V1A ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A transporter ATP6V1B2 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B2 transporter ATP6V1C1 ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C1 transporter ATP6V1D ATPase, H+ transporting, lysosomal 34kDa, V1 subunit D transporter (includes EG:299159) ATP6V1E1 ATPase, H+ transporting, lysosomal 31kDa, V1 subunit E1 transporter ATP6V1F ATPase, H+ transporting, lysosomal 14kDa, V1 subunit F transporter ATP6V1G2 ATPase, H+ transporting, lysosomal 13kDa, V1 subunit G2 transporter ATP6V1H ATPase, H+ transporting, lysosomal 50/57kDa, V1 subunit H transporter ATP8A1 ATPase, aminophospholipid transporter (APLT), class I, type 8A, transporter member 1 ATP9A ATPase, class II, type 9A transporter ATXN2 ataxin 2 unreported AUH (includes AU RNA binding protein/enoyl-CoA hydratase enzyme EG:11992) BAG5 BCL2-associated athanogene 5 unreported

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) BAIAP2 BAI1-associated protein 2 kinase BAIAP2 BAI1-associated protein 2 kinase BASP1 brain abundant, membrane attached signal protein 1 transcription regulator BCAP31 B-cell receptor-associated protein 31 transporter BCAS1 breast carcinoma amplified sequence 1 unreported BCAS3 breast carcinoma amplified sequence 3 unreported BCR breakpoint cluster region kinase BDH1 (includes 3-hydroxybutyrate dehydrogenase, type 1 enzyme EG:100037356) BIN1 bridging integrator 1 unreported BLVRA biliverdin reductase A enzyme BRSK1 BR serine/threonine kinase 1 kinase BRSK2 BR serine/threonine kinase 2 kinase BSG (includes basigin (Ok blood group) transporter EG:12215) BSN bassoon (presynaptic cytomatrix protein) unreported BTBD17 BTB (POZ) domain containing 17 unreported C10orf35 chromosome 10 open reading frame 35 unreported C11orf2 chromosome 11 open reading frame 2 unreported C18orf8 chromosome 18 open reading frame 8 unreported C1orf95 chromosome 1 open reading frame 95 unreported C1QBP complement component 1, q subcomponent binding protein unreported C22orf28 chromosome 22 open reading frame 28 enzyme C2CD2L C2CD2-like unreported C2CD4C C2 calcium-dependent domain containing 4C unreported C2orf43 chromosome 2 open reading frame 43 enzyme C2orf55 chromosome 2 open reading frame 55 unreported CA14 carbonic anhydrase XIV enzyme CA2 carbonic anhydrase II enzyme CACNA2D1 calcium channel, voltage-dependent, alpha 2/delta subunit 1 ion channel CACNA2D2 calcium channel, voltage-dependent, alpha 2/delta subunit 2 ion channel CACNA2D3 calcium channel, voltage-dependent, alpha 2/delta subunit 3 ion channel CACNG8 calcium channel, voltage-dependent, gamma subunit 8 ion channel CADM1 cell adhesion molecule 1 unreported CADM4 cell adhesion molecule 4 unreported CALB2 calbindin 2 unreported Calm1 (includes calmodulin 1 unreported unreporteds) CALU calumenin unreported CAMK2A calcium/calmodulin-dependent protein kinase II alpha kinase CAMK2B calcium/calmodulin-dependent protein kinase II beta kinase CAMK2D calcium/calmodulin-dependent protein kinase II delta kinase CAMK2D calcium/calmodulin-dependent protein kinase II delta kinase CAMK2G calcium/calmodulin-dependent protein kinase II gamma kinase CAMK2G calcium/calmodulin-dependent protein kinase II gamma kinase

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) CAMKV CaM kinase-like vesicle-associated kinase CAND1 cullin-associated and neddylation-dissociated 1 transcription regulator CANX calnexin unreported CAP1 CAP, adenylate cyclase-associated protein 1 (yeast) unreported CAP2 CAP, adenylate cyclase-associated protein, 2 (yeast) unreported CAPZA2 capping protein (actin filament) muscle Z-line, alpha 2 unreported CAPZB capping protein (actin filament) muscle Z-line, beta unreported CARKD carbohydrate kinase domain containing unreported CASC4 cancer susceptibility candidate 4 unreported CASK calcium/calmodulin-dependent serine protein kinase (MAGUK family) kinase CASKIN1 CASK interacting protein 1 transcription regulator CCDC88A coiled-coil domain containing 88A unreported CCT2 chaperonin containing TCP1, subunit 2 (beta) kinase CCT3 chaperonin containing TCP1, subunit 3 (gamma) unreported CCT4 chaperonin containing TCP1, subunit 4 (delta) unreported CCT6A chaperonin containing TCP1, subunit 6A (zeta 1) unreported CCT7 chaperonin containing TCP1, subunit 7 (eta) unreported CCT8 chaperonin containing TCP1, subunit 8 (theta) enzyme CD200 CD200 molecule unreported CD47 CD47 molecule unreported CD81 CD81 molecule unreported CD82 CD82 molecule unreported CDC37 cell division cycle 37 homolog (S. cerevisiae) unreported Cdc42 cell division cycle 42 homolog (S. cerevisiae) enzyme CDC42BPA CDC42 binding protein kinase alpha (DMPK-like) kinase CDC42BPB CDC42 binding protein kinase beta (DMPK-like) kinase CDH13 cadherin 13, H-cadherin (heart) unreported CDH2 cadherin 2, type 1, N-cadherin (neuronal) unreported CDIPT CDP-diacylglycerol--inositol 3-phosphatidyltransferase enzyme CDK16 cyclin-dependent kinase 16 kinase CDK5 cyclin-dependent kinase 5 kinase CDS2 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2 enzyme CEP170 centrosomal protein 170kDa unreported CFL1 cofilin 1 (non-muscle) unreported CHCHD3 coiled-coil-helix-coiled-coil-helix domain containing 3 unreported CHCHD6 coiled-coil-helix-coiled-coil-helix domain containing 6 unreported CHID1 chitinase domain containing 1 unreported CISD1 CDGSH iron sulfur domain 1 unreported CIT citron (rho-interacting, serine/threonine kinase 21) kinase CKAP4 cytoskeleton-associated protein 4 unreported CKAP5 cytoskeleton associated protein 5 unreported CKB creatine kinase, brain kinase CKM creatine kinase, muscle kinase

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) CKMT1A/CKM creatine kinase, mitochondrial 1B kinase T1B CKMT2 creatine kinase, mitochondrial 2 (sarcomeric) kinase CLASP1 cytoplasmic linker associated protein 1 unreported CLASP2 cytoplasmic linker associated protein 2 unreported CLCN6 chloride channel, voltage-sensitive 6 ion channel CLIC4 chloride intracellular channel 4 ion channel CLTC clathrin, heavy chain (Hc) unreported CLTC clathrin, heavy chain (Hc) unreported CLU clusterin unreported CMAS cytidine monophosphate N-acetylneuraminic acid synthetase enzyme CMPK1 cytidine monophosphate (UMP-CMP) kinase 1, cytosolic kinase CNKSR2 connector enhancer of kinase suppressor of Ras 2 unreported Cnnm1 cyclin M1 unreported CNOT1 CCR4-NOT transcription complex, subunit 1 unreported CNP 2',3'-cyclic nucleotide 3' phosphodiesterase enzyme CNRIP1 cannabinoid receptor interacting protein 1 unreported CNTN1 contactin 1 enzyme CNTN2 contactin 2 (axonal) unreported CNTNAP1 contactin associated protein 1 unreported COPB2 coatomer protein complex, subunit beta 2 (beta prime) transporter CORO1A coronin, actin binding protein, 1A unreported COX2 (includes cytochrome c oxidase subunit II enzyme EG:140540) COX4I1 cytochrome c oxidase subunit IV isoform 1 enzyme COX5A cytochrome c oxidase subunit Va enzyme (includes EG:12858) Cox5b cytochrome c oxidase, subunit Vb enzyme COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 (liver) enzyme CPD carboxypeptidase D peptidase CPM carboxypeptidase M peptidase CPNE4 copine IV unreported CPNE5 copine V unreported CPNE6 copine VI (neuronal) transporter CPNE8 copine VIII unreported CPNE9 copine family member IX unreported CPT1C carnitine palmitoyltransferase 1C enzyme CRMP1 collapsin response mediator protein 1 enzyme CRMP1 collapsin response mediator protein 1 enzyme CRYM crystallin, mu enzyme CS citrate synthase enzyme CSNK2A1 casein kinase 2, alpha 1 polypeptide kinase CSRP1 cysteine and glycine-rich protein 1 unreported CTNNA2 catenin (cadherin-associated protein), alpha 2 unreported CTTN cortactin unreported

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) CUL5 cullin 5 ion channel CYB5R3 cytochrome b5 reductase 3 enzyme CYC1 cytochrome c-1 enzyme Cycs cytochrome c, somatic transporter CYFIP1 cytoplasmic FMR1 interacting protein 1 unreported CYFIP2 cytoplasmic FMR1 interacting protein 2 unreported CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6 enzyme CYP46A1 cytochrome P450, family 46, subfamily A, polypeptide 1 enzyme DAG1 (includes dystroglycan 1 (dystrophin-associated glycoprotein 1) transmembra EG:114489) ne receptor DBC1 deleted in bladder cancer 1 peptidase DBI diazepam binding inhibitor (GABA receptor modulator, acyl-CoA unreported binding protein) DBNL drebrin-like unreported DCAKD dephospho-CoA kinase domain containing unreported DCLK1 doublecortin-like kinase 1 kinase DCLK2 doublecortin-like kinase 2 kinase DCTN1 dynactin 1 unreported DCTN2 dynactin 2 (p50) unreported DDAH1 dimethylarginine dimethylaminohydrolase 1 enzyme DDOST dolichyl-diphosphooligosaccharide--protein glycosyltransferase enzyme DDX1 DEAD (Asp-Glu-Ala-Asp) box helicase 1 enzyme DDX17 DEAD (Asp-Glu-Ala-Asp) box helicase 17 enzyme DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked enzyme DDX5 DEAD (Asp-Glu-Ala-Asp) box helicase 5 enzyme DDX6 DEAD (Asp-Glu-Ala-Asp) box helicase 6 enzyme DECR1 2,4-dienoyl CoA reductase 1, mitochondrial enzyme DHX15 DEAH (Asp-Glu-Ala-His) box polypeptide 15 enzyme DHX9 DEAH (Asp-Glu-Ala-His) box polypeptide 9 enzyme DIP2B DIP2 disco-interacting protein 2 homolog B (Drosophila) unreported DIRAS2 DIRAS family, GTP-binding RAS-like 2 enzyme DIRC2 disrupted in renal carcinoma 2 unreported DLAT dihydrolipoamide S-acetyltransferase enzyme DLD dihydrolipoamide dehydrogenase enzyme DLG1 discs, large homolog 1 (Drosophila) kinase DLG2 discs, large homolog 2 (Drosophila) kinase DLG4 discs, large homolog 4 (Drosophila) kinase DLG4 discs, large homolog 4 (Drosophila) kinase DLGAP3 discs, large (Drosophila) homolog-associated protein 3 unreported DLGAP4 discs, large (Drosophila) homolog-associated protein 4 unreported DLST dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo- enzyme glutarate complex) DMXL2 Dmx-like 2 unreported DMXL2 Dmx-like 2 unreported DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 unreported DNAJC13 DnaJ (Hsp40) homolog, subfamily C, member 13 unreported 97

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) DNAJC5 DnaJ (Hsp40) homolog, subfamily C, member 5 unreported DNAJC6 DnaJ (Hsp40) homolog, subfamily C, member 6 unreported DNM1 dynamin 1 enzyme DNM1 dynamin 1 enzyme DNM1L dynamin 1-like enzyme DNM1L dynamin 1-like enzyme DNM2 dynamin 2 enzyme DNM3 dynamin 3 enzyme DOCK7 dedicator of cytokinesis 7 unreported Dock9 dedicator of cytokinesis 9 unreported DPP10 dipeptidyl-peptidase 10 (non-functional) peptidase DPP6 dipeptidyl-peptidase 6 peptidase DPYSL2 dihydropyrimidinase-like 2 enzyme DPYSL3 dihydropyrimidinase-like 3 enzyme DPYSL4 dihydropyrimidinase-like 4 enzyme DPYSL4 dihydropyrimidinase-like 4 enzyme DPYSL5 dihydropyrimidinase-like 5 enzyme Dst dystonin unreported DTNA dystrobrevin, alpha unreported DYNC1H1 dynein, cytoplasmic 1, heavy chain 1 peptidase DYNC1I2 dynein, cytoplasmic 1, intermediate chain 2 unreported DYNC1LI1 dynein, cytoplasmic 1, light intermediate chain 1 unreported DYNC1LI2 dynein, cytoplasmic 1, light intermediate chain 2 unreported DYNC2H1 dynein, cytoplasmic 2, heavy chain 1 unreported DZANK1 double zinc ribbon and ankyrin repeat domains 1 unreported ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial enzyme Eci3 enoyl-Coenzyme A delta isomerase 3 enzyme Eef1a1 eukaryotic translation elongation factor 1 alpha 1 translation regulator EEF1B2 eukaryotic translation elongation factor 1 beta 2 translation regulator EEF1D eukaryotic translation elongation factor 1 delta (guanine nucleotide translation exchange protein) regulator EEF1G eukaryotic translation elongation factor 1 gamma translation regulator EEF2 eukaryotic translation elongation factor 2 translation regulator EFHA2 EF-hand domain family, member A2 unreported EFHD2 EF-hand domain family, member D2 unreported EFR3B EFR3 homolog B (S. cerevisiae) unreported EFTUD2 elongation factor Tu GTP binding domain containing 2 enzyme EHD1 EH-domain containing 1 unreported EHD3 EH-domain containing 3 unreported EHD4 EH-domain containing 4 enzyme EIF2S1 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa translation regulator

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) EIF2S2 eukaryotic translation initiation factor 2, subunit 2 beta, 38kDa translation regulator EIF3A eukaryotic translation initiation factor 3, subunit A translation regulator EIF3D eukaryotic translation initiation factor 3, subunit D translation regulator EIF3G eukaryotic translation initiation factor 3, subunit G translation regulator EIF3H eukaryotic translation initiation factor 3, subunit H translation regulator EIF4A1 eukaryotic translation initiation factor 4A1 translation regulator EIF4A2 eukaryotic translation initiation factor 4A2 translation regulator EIF4G3 eukaryotic translation initiation factor 4 gamma, 3 translation regulator ELAVL1 ELAV (embryonic lethal, abnormal vision, Drosophila)-like 1 (Hu unreported antigen R) ENDOD1 endonuclease domain containing 1 enzyme ENO1 enolase 1, (alpha) transcription regulator ENO2 enolase 2 (gamma, neuronal) enzyme EPB41L1 erythrocyte membrane protein band 4.1-like 1 unreported EPB41L1 erythrocyte membrane protein band 4.1-like 1 unreported EPB41L2 erythrocyte membrane protein band 4.1-like 2 unreported EPB41L3 erythrocyte membrane protein band 4.1-like 3 unreported EPB41L3 erythrocyte membrane protein band 4.1-like 3 unreported EPB41L3 erythrocyte membrane protein band 4.1-like 3 unreported EPB42 erythrocyte membrane protein band 4.2 transporter EPDR1 ependymin related protein 1 (zebrafish) unreported ERC1 ELKS/RAB6-interacting/CAST family member 1 unreported ERC2 ELKS/RAB6-interacting/CAST family member 2 unreported ERLIN2 ER lipid raft associated 2 unreported ERMN ermin, ERM-like protein unreported ERP29 endoplasmic reticulum protein 29 transporter ESYT2 extended synaptotagmin-like protein 2 unreported ETF1 eukaryotic translation termination factor 1 translation regulator ETFA electron-transfer-flavoprotein, alpha polypeptide transporter ETFDH electron-transferring-flavoprotein dehydrogenase enzyme EWSR1 Ewing sarcoma breakpoint region 1 unreported EXOC1 exocyst complex component 1 transporter EXOC2 exocyst complex component 2 transporter EXOC3 exocyst complex component 3 transporter EXOC4 exocyst complex component 4 transporter EXOC7 exocyst complex component 7 transporter EXOG endo/exonuclease (5'-3'), endonuclease G-like enzyme EZR ezrin unreported

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) FABP7 fatty acid binding protein 7, brain transporter FAM126B family with sequence similarity 126, member B unreported FAM213A family with sequence similarity 213, member A unreported FAM49B family with sequence similarity 49, member B unreported FAM54B family with sequence similarity 54, member B unreported FAM82A2 family with sequence similarity 82, member A2 unreported FARSB phenylalanyl-tRNA synthetase, beta subunit enzyme FASN fatty acid synthase enzyme FH fumarate hydratase enzyme FKBP4 FK506 binding protein 4, 59kDa enzyme FKBP8 FK506 binding protein 8, 38kDa unreported FLOT1 flotillin 1 unreported FLOT2 flotillin 2 unreported Fmn2 (mouse) formin 2 unreported FMNL2 formin-like 2 unreported FMR1 fragile X mental retardation 1 unreported FSCN1 fascin homolog 1, actin-bundling protein (Strongylocentrotus unreported purpuratus) FUS fused in sarcoma transcription regulator FXR2 fragile X mental retardation, autosomal homolog 2 unreported GABBR1 gamma-aminobutyric acid (GABA) B receptor, 1 G-protein coupled receptor GABBR2 gamma-aminobutyric acid (GABA) B receptor, 2 G-protein coupled receptor GABRA1 gamma-aminobutyric acid (GABA) A receptor, alpha 1 ion channel GABRB1 gamma-aminobutyric acid (GABA) A receptor, beta 1 ion channel GABRB2 gamma-aminobutyric acid (GABA) A receptor, beta 2 ion channel GANAB glucosidase, alpha; neutral AB enzyme GAP43 growth associated protein 43 unreported GAS7 growth arrest-specific 7 transcription regulator GBA glucosidase, beta, acid enzyme GCN1L1 GCN1 general control of amino-acid synthesis 1-like 1 (yeast) translation regulator GDA guanine deaminase enzyme GDAP1 ganglioside induced differentiation associated protein 1 unreported GDI1 GDP dissociation inhibitor 1 unreported GDI2 GDP dissociation inhibitor 2 unreported GFAP glial fibrillary acidic protein unreported GGT7 gamma-glutamyltransferase 7 enzyme GJC3 gap junction protein, gamma 3, 30.2kDa transporter GK glycerol kinase kinase GLG1 (includes golgi glycoprotein 1 unreported EG:20340) GLOD4 glyoxalase domain containing 4 enzyme 100

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) GLS glutaminase enzyme GLUD1 glutamate dehydrogenase 1 enzyme GLUL glutamate-ammonia ligase enzyme Gm10071/Rpl13 ribosomal protein L13 unreported Gm5908 predicted gene 5908 unreported GNA11 guanine nucleotide binding protein (G protein), alpha 11 (Gq class) enzyme GNA13 guanine nucleotide binding protein (G protein), alpha 13 enzyme GNAI2 guanine nucleotide binding protein (G protein), alpha inhibiting enzyme activity polypeptide 2 GNAO1 guanine nucleotide binding protein (G protein), alpha activating enzyme activity polypeptide O GNAO1 guanine nucleotide binding protein (G protein), alpha activating enzyme activity polypeptide O GNAQ guanine nucleotide binding protein (G protein), q polypeptide enzyme Gnas (mouse) GNAS (guanine nucleotide binding protein, alpha stimulating) complex enzyme locus GNAZ guanine nucleotide binding protein (G protein), alpha z polypeptide enzyme GNB1 guanine nucleotide binding protein (G protein), beta polypeptide 1 enzyme GNB2L1 guanine nucleotide binding protein (G protein), beta polypeptide 2-like enzyme 1 GNG12 guanine nucleotide binding protein (G protein), gamma 12 enzyme GNG3 guanine nucleotide binding protein (G protein), gamma 3 enzyme GOT1 glutamic-oxaloacetic transaminase 1, soluble (aspartate enzyme aminotransferase 1) GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate enzyme aminotransferase 2) GPC1 glypican 1 transmembra ne receptor GPD1 glycerol-3-phosphate dehydrogenase 1 (soluble) enzyme GPD2 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) enzyme GPI glucose-6-phosphate isomerase enzyme GPM6A glycoprotein M6A ion channel GPM6B glycoprotein M6B unreported GPR158 G protein-coupled receptor 158 G-protein coupled receptor GPRC5B G protein-coupled receptor, family C, group 5, member B G-protein coupled receptor GPRIN1 G protein regulated inducer of neurite outgrowth 1 unreported GRHPR glyoxylate reductase/hydroxypyruvate reductase enzyme GRIA2 glutamate receptor, ionotropic, AMPA 2 ion channel GRIA3 glutamate receptor, ionotropic, AMPA 3 ion channel GRIN1 glutamate receptor, ionotropic, N-methyl D-aspartate 1 ion channel GRIN2B glutamate receptor, ionotropic, N-methyl D-aspartate 2B ion channel GRM1 glutamate receptor, metabotropic 1 G-protein coupled receptor

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Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) GRM3 glutamate receptor, metabotropic 3 G-protein coupled receptor GRM5 glutamate receptor, metabotropic 5 G-protein coupled receptor GSK3B glycogen synthase kinase 3 beta kinase GSN gelsolin unreported GSTM3 glutathione S-transferase mu 3 (brain) enzyme GSTM5 glutathione S-transferase mu 5 enzyme Gstp1 (includes glutathione S-transferase, pi 1 enzyme unreporteds) GTPBP1 GTP binding protein 1 enzyme H1F0 H1 histone family, member 0 unreported H2afv H2A histone family, member V unreported H2AFY H2A histone family, member Y unreported H2AFY2 H2A histone family, member Y2 unreported HADHA hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA enzyme hydratase (trifunctional protein), alpha subunit HADHB hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA enzyme hydratase (trifunctional protein), beta subunit HBA1/HBA2 hemoglobin, alpha 1 transporter HBB hemoglobin, beta transporter HBD (includes hemoglobin, delta transporter EG:15130) HCN1 hyperpolarization activated cyclic nucleotide-gated potassium channel ion channel 1 HCN2 hyperpolarization activated cyclic nucleotide-gated potassium channel ion channel 2 HDAC11 histone deacetylase 11 transcription regulator HIST1H1E histone cluster 1, H1e unreported Hist1h2bq histone cluster 1, H2bq unreported HK1 hexokinase 1 kinase HK1 hexokinase 1 kinase Hmgb1 high mobility group box 1 transcription (includes regulator EG:100041307) HNRNPA1 heterogeneous nuclear ribonucleoprotein A1 unreported HNRNPA2B1 heterogeneous nuclear ribonucleoprotein A2/B1 unreported HNRNPA3 heterogeneous nuclear ribonucleoprotein A3 unreported HNRNPAB heterogeneous nuclear ribonucleoprotein A/B enzyme HNRNPC heterogeneous nuclear ribonucleoprotein C (C1/C2) unreported HNRNPK heterogeneous nuclear ribonucleoprotein K unreported HNRNPL heterogeneous nuclear ribonucleoprotein L unreported HNRNPM heterogeneous nuclear ribonucleoprotein M unreported HNRNPR heterogeneous nuclear ribonucleoprotein R unreported HNRNPU heterogeneous nuclear ribonucleoprotein U (scaffold attachment factor transporter A) 102

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) HNRNPUL2 heterogeneous nuclear ribonucleoprotein U-like 2 unreported HOMER1 homer homolog 1 (Drosophila) unreported HOMER3 homer homolog 3 (Drosophila) unreported HP1BP3 heterochromatin protein 1, binding protein 3 unreported HPCA hippocalcin unreported HPCAL4 hippocalcin like 4 transporter HRAS v-Ha-ras Harvey rat sarcoma viral oncogene homolog enzyme HRSP12 heat-responsive protein 12 unreported HSD17B4 hydroxysteroid (17-beta) dehydrogenase 4 enzyme HSDL2 hydroxysteroid dehydrogenase like 2 transporter HSP90AA1 heat shock protein 90kDa alpha (cytosolic), class A member 1 enzyme HSP90AB1 heat shock protein 90kDa alpha (cytosolic), class B member 1 enzyme HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 unreported HSPA12A heat shock 70kDa protein 12A unreported HSPA1A/HSPA heat shock 70kDa protein 1A unreported 1B HSPA2 heat shock 70kDa protein 2 unreported HSPA4 heat shock 70kDa protein 4 unreported HSPA4L heat shock 70kDa protein 4-like unreported HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) enzyme HSPA8 heat shock 70kDa protein 8 enzyme HSPA9 heat shock 70kDa protein 9 (mortalin) unreported HSPA9 heat shock 70kDa protein 9 (mortalin) unreported HSPD1 heat shock 60kDa protein 1 (chaperonin) enzyme HSPH1 heat shock 105kDa/110kDa protein 1 unreported HSPH1 heat shock 105kDa/110kDa protein 1 unreported HTT huntingtin transcription regulator HYOU1 hypoxia up-regulated 1 unreported IDH1 isocitrate dehydrogenase 1 (NADP+), soluble enzyme IDH2 isocitrate dehydrogenase 2 (NADP+), mitochondrial enzyme IDH3A isocitrate dehydrogenase 3 (NAD+) alpha enzyme IDH3B isocitrate dehydrogenase 3 (NAD+) beta enzyme IDH3G isocitrate dehydrogenase 3 (NAD+) gamma enzyme IGSF21 immunoglobin superfamily, member 21 unreported IGSF8 immunoglobulin superfamily, member 8 unreported IKBKAP inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase unreported complex-associated protein ILF2 interleukin enhancer binding factor 2, 45kDa transcription regulator ILF3 interleukin enhancer binding factor 3, 90kDa transcription regulator IMMT inner membrane protein, mitochondrial unreported IMPA1 inositol(myo)-1(or 4)-monophosphatase 1 phosphatase IMPDH2 IMP (inosine 5'-monophosphate) dehydrogenase 2 enzyme INA internexin neuronal intermediate filament protein, alpha unreported INPP1 inositol polyphosphate-1-phosphatase phosphatase 103

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) IPO7 importin 7 transporter IQSEC1 IQ motif and Sec7 domain 1 unreported IQSEC2 IQ motif and Sec7 domain 2 unreported ITGB1 integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 transmembra includes MDF2, MSK12) ne receptor ITPR1 inositol 1,4,5-trisphosphate receptor, type 1 ion channel ITPR1 inositol 1,4,5-trisphosphate receptor, type 1 ion channel ITSN1 intersectin 1 (SH3 domain protein) unreported ITSN2 intersectin 2 unreported JUP junction plakoglobin unreported KCNA1 potassium voltage-gated channel, shaker-related subfamily, member 1 ion channel (episodic ataxia with myokymia) KCNA2 potassium voltage-gated channel, shaker-related subfamily, member 2 ion channel KCNA4 potassium voltage-gated channel, shaker-related subfamily, member 4 ion channel KCNAB2 potassium voltage-gated channel, shaker-related subfamily, beta ion channel member 2 KCTD12 potassium channel tetramerisation domain containing 12 ion channel KCTD8 potassium channel tetramerisation domain containing 8 unreported KIAA0090 KIAA0090 unreported KIAA0528 KIAA0528 unreported KIAA1045 KIAA1045 unreported KIAA1244 KIAA1244 unreported KIAA1467 KIAA1467 unreported KIF1A kinesin family member 1A unreported KIF21A kinesin family member 21A unreported KIF2A kinesin heavy chain member 2A unreported KIF5B kinesin family member 5B unreported KIF5C kinesin family member 5C unreported KLC2 kinesin light chain 2 unreported KPNB1 karyopherin (importin) beta 1 transporter KRAS v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog enzyme KTN1 kinectin 1 (kinesin receptor) unreported KXD1 (includes KxDL motif containing 1 unreported EG:39425) L1CAM L1 cell adhesion molecule unreported LAMP1 lysosomal-associated membrane protein 1 unreported LAMTOR1 late endosomal/lysosomal adaptor, MAPK and MTOR activator 1 unreported LANCL2 LanC lantibiotic synthetase component C-like 2 (bacterial) unreported LAP3 leucine aminopeptidase 3 peptidase LDHA lactate dehydrogenase A enzyme LDHA lactate dehydrogenase A enzyme LDHB lactate dehydrogenase B enzyme LETM1 leucine zipper-EF-hand containing transmembrane protein 1 unreported LGI2 leucine-rich repeat LGI family, member 2 unreported LGI3 leucine-rich repeat LGI family, member 3 unreported LINGO1 leucine rich repeat and Ig domain containing 1 unreported

104

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) LLGL1 lethal giant larvae homolog 1 (Drosophila) unreported LMAN2L lectin, mannose-binding 2-like unreported LMNA lamin A/C unreported LMNB1 lamin B1 unreported LMNB2 lamin B2 unreported LNPEP leucyl/cystinyl aminopeptidase peptidase LONP1 lon peptidase 1, mitochondrial peptidase LPCAT4 lysophosphatidylcholine acyltransferase 4 unreported LPPR4 lipid phosphate phosphatase-related protein type 4 phosphatase LRP1 (includes low density lipoprotein receptor-related protein 1 transmembra EG:16971) ne receptor LRPPRC leucine-rich pentatricopeptide repeat containing unreported LRRC47 leucine rich repeat containing 47 unreported LRRC57 leucine rich repeat containing 57 unreported LRRC7 leucine rich repeat containing 7 unreported LSAMP limbic system-associated membrane protein unreported LTA4H leukotriene A4 hydrolase enzyme LXN latexin unreported Macf1 microtubule-actin crosslinking factor 1 enzyme MADD MAP-kinase activating death domain unreported MAG myelin associated glycoprotein unreported MAOA monoamine oxidase A enzyme MAOB monoamine oxidase B enzyme MAP1A microtubule-associated protein 1A unreported MAP1B microtubule-associated protein 1B unreported MAP1S microtubule-associated protein 1S enzyme MAP2 microtubule-associated protein 2 unreported MAP4 microtubule-associated protein 4 unreported MAP6 microtubule-associated protein 6 unreported MAPK1 mitogen-activated protein kinase 1 kinase Mapt microtubule-associated protein tau unreported Mapt microtubule-associated protein tau unreported MARCKS myristoylated alanine-rich protein kinase C substrate unreported MARK1 MAP/microtubule affinity-regulating kinase 1 kinase MARK2 MAP/microtubule affinity-regulating kinase 2 kinase MATR3 matrin 3 unreported MBP myelin basic protein unreported MBP myelin basic protein unreported MCCC1 methylcrotonoyl-CoA carboxylase 1 (alpha) enzyme MCU mitochondrial calcium uniporter ion channel MDH1 malate dehydrogenase 1, NAD (soluble) enzyme MDH2 (includes malate dehydrogenase 2, NAD (mitochondrial) enzyme EG:17448) MECP2 methyl CpG binding protein 2 (Rett syndrome) transcription regulator MFF mitochondrial fission factor unreported 105

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) MFN2 mitofusin 2 enzyme MGLL monoglyceride lipase enzyme MINK1 misshapen-like kinase 1 kinase MLEC malectin unreported MLLT4 myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, unreported Drosophila); translocated to, 4 MOBP myelin-associated oligodendrocyte basic protein unreported MOG myelin oligodendrocyte glycoprotein unreported MOGS mannosyl-oligosaccharide glucosidase enzyme MPP1 membrane protein, palmitoylated 1, 55kDa kinase MPP2 membrane protein, palmitoylated 2 (MAGUK p55 subfamily member kinase 2) MPP6 (includes membrane protein, palmitoylated 6 (MAGUK p55 subfamily member kinase EG:35343) 6) MRPL12 mitochondrial ribosomal protein L12 unreported MSN moesin unreported MTCH1 mitochondrial carrier 1 unreported MTDH metadherin transcription regulator MTHFD1 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, enzyme methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase MTHFD1L methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like enzyme MTOR mechanistic target of rapamycin (serine/threonine kinase) kinase MTX1 metaxin 1 transporter MYCBP2 MYC binding protein 2, E3 ubiquitin protein ligase enzyme MYEF2 myelin expression factor 2 transcription regulator MYH1 myosin, heavy chain 1, skeletal muscle, adult enzyme MYH10 myosin, heavy chain 10, non-muscle unreported MYH14 myosin, heavy chain 14, non-muscle unreported MYH6 myosin, heavy chain 6, cardiac muscle, alpha enzyme MYH9 myosin, heavy chain 9, non-muscle enzyme MYL6 myosin, light chain 6, alkali, smooth muscle and non-muscle unreported MYL9 myosin, light chain 9, regulatory unreported MYO18A myosin XVIIIA unreported MYO1B myosin IB unreported MYO1D myosin ID unreported MYO5A myosin VA (heavy chain 12, myoxin) enzyme MYO6 myosin VI unreported NAP1L1 nucleosome assembly protein 1-like 1 unreported NAPB N-ethylmaleimide-sensitive factor attachment protein, beta transporter NAPG N-ethylmaleimide-sensitive factor attachment protein, gamma transporter NCALD neurocalcin delta unreported NCAM1 neural cell adhesion molecule 1 unreported NCAM1 neural cell adhesion molecule 1 unreported NCAM2 neural cell adhesion molecule 2 unreported

106

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) NCAM2 neural cell adhesion molecule 2 unreported NCAN neurocan unreported NCDN neurochondrin unreported NCEH1 neutral cholesterol ester hydrolase 1 enzyme NCKAP1 NCK-associated protein 1 unreported NCKAP1 NCK-associated protein 1 unreported NCKIPSD NCK interacting protein with SH3 domain unreported Ncl nucleolin unreported NCOA5 nuclear receptor coactivator 5 unreported NCS1 neuronal calcium sensor 1 unreported NCSTN nicastrin peptidase NDEL1 nudE nuclear distribution E homolog (A. nidulans)-like 1 unreported NDRG2 NDRG family member 2 unreported NDRG3 NDRG family member 3 unreported NDUFA10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa enzyme NDUFA12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 enzyme NDUFA13 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 enzyme NDUFA2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa enzyme NDUFA3 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa enzyme NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa enzyme NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa enzyme NDUFA7 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 7, 14.5kDa enzyme NDUFA8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa enzyme NDUFA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa enzyme NDUFB10 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22kDa enzyme NDUFB11 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17.3kDa enzyme NDUFB4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15kDa enzyme NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa enzyme NDUFB6 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6, 17kDa enzyme NDUFB7 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7, 18kDa enzyme NDUFB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa enzyme NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADH- enzyme coenzyme Q reductase) NDUFS2 NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa (NADH- enzyme coenzyme Q reductase) NDUFS3 NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADH- enzyme coenzyme Q reductase) NDUFS4 NADH dehydrogenase (ubiquinone) Fe-S protein 4, 18kDa (NADH- enzyme coenzyme Q reductase) NDUFS7 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH- enzyme coenzyme Q reductase) NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa enzyme NDUFV2 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa enzyme NEFH neurofilament, heavy polypeptide unreported NEFL neurofilament, light polypeptide unreported NEFM neurofilament, medium polypeptide unreported NEGR1 neuronal growth regulator 1 unreported 107

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) NEO1 neogenin 1 transcription regulator NF1 (includes neurofibromin 1 unreported EG:18015) NFASC neurofascin unreported NFASC neurofascin unreported NGEF neuronal guanine nucleotide exchange factor unreported NIPSNAP1 nipsnap homolog 1 (C. elegans) enzyme NME1 (includes non-metastatic cells 1, protein (NM23A) expressed in kinase EG:18102) NME2 non-metastatic cells 2, protein (NM23B) expressed in kinase NNT nicotinamide nucleotide transhydrogenase enzyme NOMO1 NODAL modulator 1 unreported (includes unreporteds) NONO non-POU domain containing, octamer-binding unreported NOP56 NOP56 ribonucleoprotein homolog (yeast) unreported NPEPPS aminopeptidase puromycin sensitive peptidase Nptn neuroplastin unreported Nptn neuroplastin unreported NRAS neuroblastoma RAS viral (v-ras) oncogene homolog enzyme NRCAM neuronal cell adhesion molecule unreported NRXN2 neurexin 2 transporter NRXN3 neurexin 3 transporter NSF N-ethylmaleimide-sensitive factor transporter NSFL1C NSFL1 (p97) cofactor (p47) unreported NTM neurotrimin unreported NUMBL numb homolog (Drosophila)-like unreported NUP210 nucleoporin 210kDa transporter OGDH oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) enzyme OGDHL oxoglutarate dehydrogenase-like enzyme OGT O-linked N-acetylglucosamine (GlcNAc) transferase (UDP-N- enzyme acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) OMG oligodendrocyte myelin glycoprotein G-protein coupled receptor OPA1 optic atrophy 1 (autosomal dominant) enzyme OTUB1 OTU domain, ubiquitin aldehyde binding 1 enzyme OXCT1 3-oxoacid CoA transferase 1 enzyme OXCT1 3-oxoacid CoA transferase 1 enzyme OXR1 (includes oxidation resistance 1 unreported EG:117520) OXR1 (includes oxidation resistance 1 unreported EG:117520) P4HB prolyl 4-hydroxylase, beta polypeptide enzyme PA2G4 proliferation-associated 2G4, 38kDa transcription regulator

108

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) PABPC1 poly(A) binding protein, cytoplasmic 1 translation regulator PACS1 phosphofurin acidic cluster sorting protein 1 unreported PACSIN1 protein kinase C and casein kinase substrate in neurons 1 kinase PACSIN2 protein kinase C and casein kinase substrate in neurons 2 transporter PACSIN3 protein kinase C and casein kinase substrate in neurons 3 unreported PADI2 peptidyl arginine deiminase, type II enzyme PAK1 p21 protein (Cdc42/Rac)-activated kinase 1 kinase PALM paralemmin unreported PALM paralemmin unreported PALM2 paralemmin 2 unreported PC pyruvate carboxylase enzyme PCDH1 protocadherin 1 unreported PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) kinase PCLO piccolo (presynaptic cytomatrix protein) transporter PCMT1 protein-L-isoaspartate (D-aspartate) O-methyltransferase enzyme PCSK1N proprotein convertase subtilisin/kexin type 1 inhibitor unreported PCYOX1 prenylcysteine oxidase 1 enzyme PDCD6IP programmed cell death 6 interacting protein unreported PDDC1 Parkinson disease 7 domain containing 1 unreported PDE10A phosphodiesterase 10A enzyme PDE1B phosphodiesterase 1B, calmodulin-dependent enzyme PDE2A phosphodiesterase 2A, cGMP-stimulated enzyme PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 enzyme PDHB pyruvate dehydrogenase (lipoamide) beta enzyme PDHX pyruvate dehydrogenase complex, component X enzyme PDIA3 protein disulfide isomerase family A, member 3 peptidase PDIA6 protein disulfide isomerase family A, member 6 enzyme PDXK pyridoxal (pyridoxine, vitamin B6) kinase kinase PDXK pyridoxal (pyridoxine, vitamin B6) kinase kinase PDXP pyridoxal (pyridoxine, vitamin B6) phosphatase enzyme PEBP1 phosphatidylethanolamine binding protein 1 unreported PFKM phosphofructokinase, muscle kinase PFKP phosphofructokinase, platelet kinase PFN1 profilin 1 unreported PFN2 profilin 2 unreported PFN2 profilin 2 unreported PGAM1 phosphoglycerate mutase 1 (brain) phosphatase PGD phosphogluconate dehydrogenase enzyme PGK1 phosphoglycerate kinase 1 kinase PGM1 phosphoglucomutase 1 enzyme PGRMC1 progesterone receptor membrane component 1 transmembra ne receptor PGS1 phosphatidylglycerophosphate synthase 1 enzyme PHB prohibitin transcription regulator 109

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) PHB prohibitin transcription regulator PHB2 prohibitin 2 transcription regulator PHGDH phosphoglycerate dehydrogenase enzyme PHLDB1 pleckstrin homology-like domain, family B, member 1 unreported PI4KA phosphatidylinositol 4-kinase, catalytic, alpha kinase PICALM phosphatidylinositol binding clathrin assembly protein unreported PIP4K2A phosphatidylinositol-5-phosphate 4-kinase, type II, alpha kinase PIP4K2B phosphatidylinositol-5-phosphate 4-kinase, type II, beta kinase PIP5K1C phosphatidylinositol-4-phosphate 5-kinase, type I, gamma kinase PITPNM1 phosphatidylinositol transfer protein, membrane-associated 1 transporter PITRM1 pitrilysin metallopeptidase 1 peptidase PKM2 pyruvate kinase, muscle kinase PKM2 pyruvate kinase, muscle kinase PLCB1 phospholipase C, beta 1 (phosphoinositide-specific) enzyme PLCB3 phospholipase C, beta 3 (phosphatidylinositol-specific) enzyme PLCB4 phospholipase C, beta 4 enzyme PLCL1 phospholipase C-like 1 enzyme PLCL2 phospholipase C-like 2 enzyme PLD3 phospholipase D family, member 3 enzyme PLEC plectin unreported PLLP plasmolipin transporter PLP1 (includes proteolipid protein 1 unreported EG:18823) PLXNA1 plexin A1 transmembra ne receptor PPAP2B phosphatidic acid phosphatase type 2B phosphatase Ppfia3 protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), phosphatase interacting protein (liprin), alpha 3 PPIA peptidylprolyl isomerase A (cyclophilin A) enzyme PPIB peptidylprolyl isomerase B (cyclophilin B) enzyme PPM1A protein phosphatase, Mg2+/Mn2+ dependent, 1A phosphatase PPME1 protein phosphatase methylesterase 1 enzyme PPP1CA protein phosphatase 1, catalytic subunit, alpha isozyme phosphatase PPP1R12A protein phosphatase 1, regulatory subunit 12A phosphatase PPP2CA protein phosphatase 2, catalytic subunit, alpha isozyme phosphatase PPP2R1A protein phosphatase 2, regulatory subunit A, alpha phosphatase PPP2R2A protein phosphatase 2, regulatory subunit B, alpha phosphatase PPP2R4 protein phosphatase 2A activator, regulatory subunit 4 phosphatase PPP2R5E protein phosphatase 2, regulatory subunit B', epsilon isoform phosphatase PPP3CA protein phosphatase 3, catalytic subunit, alpha isozyme phosphatase PPP3CB protein phosphatase 3, catalytic subunit, beta isozyme phosphatase PPP3R1 protein phosphatase 3, regulatory subunit B, alpha phosphatase PRDX1 peroxiredoxin 1 enzyme PRDX2 peroxiredoxin 2 enzyme PRDX3 peroxiredoxin 3 enzyme 110

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) PRDX5 peroxiredoxin 5 enzyme PRDX6 peroxiredoxin 6 enzyme PRKACB protein kinase, cAMP-dependent, catalytic, beta kinase PRKAG2 protein kinase, AMP-activated, gamma 2 non-catalytic subunit kinase PRKAR1A protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue kinase specific extinguisher 1) PRKAR2A protein kinase, cAMP-dependent, regulatory, type II, alpha kinase PRKAR2B protein kinase, cAMP-dependent, regulatory, type II, beta kinase PRKCB protein kinase C, beta kinase PRKCE protein kinase C, epsilon kinase PRKCG protein kinase C, gamma kinase PRKCG protein kinase C, gamma kinase PRKCSH protein kinase C substrate 80K-H enzyme PRMT1 protein arginine methyltransferase 1 enzyme PRODH proline dehydrogenase (oxidase) 1 enzyme PRPF8 PRP8 pre-mRNA processing factor 8 homolog (S. cerevisiae) unreported Prrt2 proline-rich transmembrane protein 2 unreported PRRT3 proline-rich transmembrane protein 3 unreported PSAP prosaposin unreported PSD3 pleckstrin and Sec7 domain containing 3 unreported PSMA3 proteasome (prosome, macropain) subunit, alpha type, 3 peptidase PSMA5 proteasome (prosome, macropain) subunit, alpha type, 5 peptidase PSMA7 proteasome (prosome, macropain) subunit, alpha type, 7 peptidase PSMB5 proteasome (prosome, macropain) subunit, beta type, 5 peptidase PSMB6 proteasome (prosome, macropain) subunit, beta type, 6 peptidase PSMC2 proteasome (prosome, macropain) 26S subunit, ATPase, 2 peptidase PTPRA protein tyrosine phosphatase, receptor type, A phosphatase Ptprd protein tyrosine phosphatase, receptor type, D phosphatase Ptprd protein tyrosine phosphatase, receptor type, D phosphatase PTPRF protein tyrosine phosphatase, receptor type, F phosphatase PTPRN2 protein tyrosine phosphatase, receptor type, N polypeptide 2 phosphatase PTPRS protein tyrosine phosphatase, receptor type, S phosphatase PTPRS protein tyrosine phosphatase, receptor type, S phosphatase PTPRZ1 protein tyrosine phosphatase, receptor-type, Z polypeptide 1 phosphatase PVALB parvalbumin unreported (includes EG:19293) PYGB phosphorylase, glycogen; brain enzyme PYGM phosphorylase, glycogen, muscle enzyme QDPR quinoid dihydropteridine reductase enzyme QDPR quinoid dihydropteridine reductase enzyme RAB11B RAB11B, member RAS oncogene family enzyme RAB11FIP5 RAB11 family interacting protein 5 (class I) unreported RAB12 RAB12, member RAS oncogene family enzyme RAB14 RAB14, member RAS oncogene family enzyme RAB18 RAB18, member RAS oncogene family enzyme 111

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) RAB1A RAB1A, member RAS oncogene family enzyme (includes EG:178620) RAB1B RAB1B, member RAS oncogene family enzyme RAB21 RAB21, member RAS oncogene family enzyme RAB27B RAB27B, member RAS oncogene family enzyme RAB2A RAB2A, member RAS oncogene family enzyme RAB35 RAB35, member RAS oncogene family enzyme RAB3A RAB3A, member RAS oncogene family enzyme RAB3B RAB3B, member RAS oncogene family enzyme RAB3C RAB3C, member RAS oncogene family enzyme (includes EG:115827) RAB3GAP1 RAB3 GTPase activating protein subunit 1 (catalytic) unreported RAB3GAP2 RAB3 GTPase activating protein subunit 2 (non-catalytic) enzyme RAB4B RAB4B, member RAS oncogene family enzyme RAB5B RAB5B, member RAS oncogene family enzyme RAB5C RAB5C, member RAS oncogene family enzyme RAB6A RAB6A, member RAS oncogene family enzyme RAB6B RAB6B, member RAS oncogene family enzyme RAB7A RAB7A, member RAS oncogene family enzyme RAB8B RAB8B, member RAS oncogene family enzyme RABGEF1 RAB guanine nucleotide exchange factor (GEF) 1 unreported RALA v-ral simian leukemia viral oncogene homolog A (ras related) enzyme RALB v-ral simian leukemia viral oncogene homolog B (ras related; GTP enzyme binding protein) RALGAPA1 Ral GTPase activating protein, alpha subunit 1 (catalytic) unreported RAP1A RAP1A, member of RAS oncogene family enzyme RAP1B RAP1B, member of RAS oncogene family enzyme RAP1GAP RAP1 GTPase activating protein unreported RAP2B RAP2B, member of RAS oncogene family enzyme RBM39 RNA binding motif protein 39 transcription regulator RBMX RNA binding motif protein, X-linked unreported RCC2 regulator of chromosome condensation 2 unreported RCN2 (includes reticulocalbin 2, EF-hand calcium binding domain unreported EG:26611) RDH11 retinol dehydrogenase 11 (all-trans/9-cis/11-cis) enzyme RDX radixin unreported REEP2 receptor accessory protein 2 unreported RGS6 regulator of G-protein signaling 6 enzyme RGS7 regulator of G-protein signaling 7 enzyme RGS7 regulator of G-protein signaling 7 enzyme RHOA ras homolog family member A enzyme RHOG ras homolog family member G enzyme RHOT1 ras homolog family member T1 enzyme RHOT2 ras homolog family member T2 enzyme 112

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) Rims1 regulating synaptic membrane exocytosis 1 unreported ROCK2 Rho-associated, coiled-coil containing protein kinase 2 kinase RPH3A rabphilin 3A homolog (mouse) transporter RPL10 ribosomal protein L10 unreported Rpl10a ribosomal protein L10A unreported RPL13A ribosomal protein L13a unreported RPL18 ribosomal protein L18 unreported RPL24 ribosomal protein L24 unreported RPL31 ribosomal protein L31 unreported RPL4 ribosomal protein L4 enzyme RPL5 ribosomal protein L5 unreported RPL6 ribosomal protein L6 unreported RPL7 ribosomal protein L7 transcription regulator Rpl8 ribosomal protein L8 unreported Rpl9 ribosomal protein L9 unreported RPLP0 ribosomal protein, large, P0 unreported RPLP2 ribosomal protein, large, P2 unreported RPN1 ribophorin I enzyme RPS14 ribosomal protein S14 translation regulator RPS19 ribosomal protein S19 unreported Rps20 ribosomal protein S20 unreported Rps24 ribosomal protein S24 unreported RPS27 ribosomal protein S27 unreported Rps28 ribosomal protein S28 unreported RPS3 ribosomal protein S3 enzyme RPS3A ribosomal protein S3A unreported RPS6 ribosomal protein S6 unreported RPS8 ribosomal protein S8 unreported RPS9 ribosomal protein S9 translation regulator RRAS2 related RAS viral (r-ras) oncogene homolog 2 enzyme RRBP1 ribosome binding protein 1 homolog 180kDa (dog) transporter RTN1 (includes reticulon 1 unreported EG:104001) RTN1 (includes reticulon 1 unreported EG:104001) RTN3 reticulon 3 unreported RTN4 (includes reticulon 4 unreported EG:57142) RYR2 ryanodine receptor 2 (cardiac) ion channel SACM1L SAC1 suppressor of actin mutations 1-like (yeast) phosphatase SACS spastic ataxia of Charlevoix-Saguenay (sacsin) unreported SAMM50 sorting and assembly machinery component 50 homolog (S. cerevisiae) unreported SAR1A SAR1 homolog A (S. cerevisiae) enzyme SARS seryl-tRNA synthetase enzyme 113

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) SBF1 SET binding factor 1 phosphatase SCAMP1 secretory carrier membrane protein 1 transporter SCAMP3 secretory carrier membrane protein 3 transporter SCN2A sodium channel, voltage-gated, type II, alpha subunit ion channel SCN8A sodium channel, voltage gated, type VIII, alpha subunit ion channel SDHA (includes succinate dehydrogenase complex, subunit A, flavoprotein (Fp) enzyme EG:157074) SDHB succinate dehydrogenase complex, subunit B, iron sulfur (Ip) enzyme SEC23A Sec23 homolog A (S. cerevisiae) transporter SEC24C SEC24 family, member C (S. cerevisiae) transporter 15-Sep 15 kDa selenoprotein enzyme 11-Sep septin 11 unreported 2-Sep septin 2 enzyme 3-Sep septin 3 enzyme 4-Sep septin 4 enzyme 5-Sep septin 5 enzyme 6-Sep septin 6 unreported 7-Sep septin 7 unreported 8-Sep septin 8 unreported 9-Sep septin 9 enzyme Serpina3k serine (or cysteine) peptidase inhibitor, clade A, member 3K unreported (includes unreporteds) SEZ6L2 seizure related 6 homolog (mouse)-like 2 unreported SF3B1 splicing factor 3b, subunit 1, 155kDa unreported SF3B3 splicing factor 3b, subunit 3, 130kDa unreported SFPQ splicing factor proline/glutamine-rich unreported SFXN1 sideroflexin 1 transporter SFXN3 sideroflexin 3 transporter SGIP1 SH3-domain GRB2-like (endophilin) interacting protein 1 unreported SH3GL1 SH3-domain GRB2-like 1 unreported SH3GL2 SH3-domain GRB2-like 2 enzyme SHANK3 SH3 and multiple ankyrin repeat domains 3 transcription regulator SIRPA signal-regulatory protein alpha phosphatase SIRT2 sirtuin 2 transcription regulator SLC12A2 solute carrier family 12 (sodium/potassium/chloride transporters), transporter member 2 SLC12A5 solute carrier family 12 (potassium/chloride transporter), member 5 transporter SLC16A1 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) transporter SLC17A6 solute carrier family 17 (sodium-dependent inorganic phosphate transporter cotransporter), member 6 SLC1A2 solute carrier family 1 (glial high affinity glutamate transporter), transporter member 2 SLC1A2 solute carrier family 1 (glial high affinity glutamate transporter), transporter member 2

114

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) SLC1A3 solute carrier family 1 (glial high affinity glutamate transporter), transporter member 3 SLC25A1 solute carrier family 25 (mitochondrial carrier; citrate transporter), transporter member 1 SLC25A11 solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), transporter member 11 SLC25A12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 transporter SLC25A18 solute carrier family 25 (mitochondrial carrier), member 18 transporter SLC25A23 solute carrier family 25 (mitochondrial carrier; phosphate carrier), transporter member 23 SLC25A3 solute carrier family 25 (mitochondrial carrier; phosphate carrier), transporter member 3 SLC25A4 solute carrier family 25 (mitochondrial carrier; adenine nucleotide transporter translocator), member 4 SLC25A46 solute carrier family 25, member 46 unreported SLC25A6 solute carrier family 25 (mitochondrial carrier; adenine nucleotide transporter translocator), member 6 SLC27A4 solute carrier family 27 (fatty acid transporter), member 4 transporter SLC2A1 solute carrier family 2 (facilitated glucose transporter), member 1 transporter SLC2A3 solute carrier family 2 (facilitated glucose transporter), member 3 transporter SLC32A1 solute carrier family 32 (GABA vesicular transporter), member 1 transporter SLC39A12 solute carrier family 39 (zinc transporter), member 12 transporter SLC3A2 solute carrier family 3 (activators of dibasic and neutral amino acid transporter transport), member 2 SLC4A1 solute carrier family 4, anion exchanger, member 1 (erythrocyte transporter membrane protein band 3, Diego blood group) SLC4A10 solute carrier family 4, sodium bicarbonate transporter, member 10 transporter SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member 4 transporter SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member 4 transporter SLC4A8 solute carrier family 4, sodium bicarbonate cotransporter, member 8 transporter SLC6A1 solute carrier family 6 (neurotransmitter transporter, GABA), member transporter 1 SLC6A11 solute carrier family 6 (neurotransmitter transporter, GABA), member transporter 11 SLC6A15 solute carrier family 6 (neutral amino acid transporter), member 15 transporter SLC6A17 solute carrier family 6, member 17 transporter SLC6A5 solute carrier family 6 (neurotransmitter transporter, glycine), member transporter 5 SLC6A9 solute carrier family 6 (neurotransmitter transporter, glycine), member transporter (includes 9 EG:116509) SLC8A1 solute carrier family 8 (sodium/calcium exchanger), member 1 transporter SLC8A2 solute carrier family 8 (sodium/calcium exchanger), member 2 transporter SMPD3 sphingomyelin phosphodiesterase 3, neutral membrane (neutral enzyme sphingomyelinase II) SNAP25 synaptosomal-associated protein, 25kDa transporter SNAP25 synaptosomal-associated protein, 25kDa transporter SNAP47 synaptosomal-associated protein, 47kDa unreported SNAP91 synaptosomal-associated protein, 91kDa homolog (mouse) unreported SNCA synuclein, alpha (non A4 component of amyloid precursor) unreported 115

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) SND1 staphylococcal nuclease and tudor domain containing 1 enzyme SNRNP200 small nuclear ribonucleoprotein 200kDa (U5) enzyme SNX1 sorting nexin 1 transporter SNX2 sorting nexin 2 transporter SNX3 sorting nexin 3 transporter SOD1 superoxide dismutase 1, soluble enzyme SOGA3 SOGA family member 3 unreported SORCS2 sortilin-related VPS10 domain containing receptor 2 transporter SORL1 sortilin-related receptor, L(DLR class) A repeats containing transporter SPTA1 spectrin, alpha, erythrocytic 1 (elliptocytosis 2) unreported SPTAN1 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) unreported SPTAN1 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) unreported SPTB spectrin, beta, erythrocytic unreported SPTBN1 spectrin, beta, non-erythrocytic 1 unreported SPTBN2 spectrin, beta, non-erythrocytic 2 unreported SPTBN4 spectrin, beta, non-erythrocytic 4 unreported SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) kinase SRCIN1 SRC kinase signaling inhibitor 1 unreported SRGAP3 SLIT-ROBO Rho GTPase activating protein 3 unreported SRPRB signal recognition particle receptor, B subunit unreported SSBP1 single-stranded DNA binding protein 1 unreported SSR3 signal sequence receptor, gamma (translocon-associated protein unreported gamma) ST13 suppression of tumorigenicity 13 (colon carcinoma) (Hsp70 interacting unreported protein) STIP1 stress-induced-phosphoprotein 1 unreported STMN1 stathmin 1 unreported STOML2 stomatin (EPB72)-like 2 unreported STX12 syntaxin 12 unreported STX1A syntaxin 1A (brain) transporter STX1B syntaxin 1B ion channel STXBP1 syntaxin binding protein 1 transporter STXBP3 syntaxin binding protein 3 transporter STXBP5 syntaxin binding protein 5 (tomosyn) unreported STXBP5 syntaxin binding protein 5 (tomosyn) unreported STXBP6 syntaxin binding protein 6 (amisyn) unreported SUCLA2 succinate-CoA ligase, ADP-forming, beta subunit enzyme SUCLG1 succinate-CoA ligase, alpha subunit enzyme SUGP2 SURP and G patch domain containing 2 unreported SV2A synaptic vesicle glycoprotein 2A transporter SV2B synaptic vesicle glycoprotein 2B transporter SVOP SV2 related protein homolog (rat) transporter SYN1 synapsin I transporter SYN2 synapsin II unreported SYNCRIP synaptotagmin binding, cytoplasmic RNA interacting protein unreported SYNE1 spectrin repeat containing, nuclear envelope 1 unreported 116

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) SYNGAP1 synaptic Ras GTPase activating protein 1 unreported SYNGR1 synaptogyrin 1 transporter SYNGR3 synaptogyrin 3 unreported SYNJ1 synaptojanin 1 phosphatase SYNPO synaptopodin unreported SYP synaptophysin transporter SYT1 (includes synaptotagmin I transporter EG:20979) SYT11 synaptotagmin XI transporter SYT12 synaptotagmin XII transporter SYT2 synaptotagmin II transporter SYT3 synaptotagmin III transporter SYT7 synaptotagmin VII transporter TAGLN3 transgelin 3 unreported TANC2 tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 transcription regulator TAOK1 TAO kinase 1 kinase Taok2 (mouse) TAO kinase 2 kinase TARDBP TAR DNA binding protein transcription regulator TARS threonyl-tRNA synthetase enzyme TBC1D10B TBC1 domain family, member 10B enzyme TCERG1 transcription elongation regulator 1 transcription regulator THY1 Thy-1 cell surface antigen unreported TIMM13 translocase of inner mitochondrial membrane 13 homolog (yeast) transporter TIMM50 translocase of inner mitochondrial membrane 50 homolog (S. phosphatase cerevisiae) TJP1 (includes tight junction protein 1 (zona occludens 1) unreported EG:21872) TJP2 tight junction protein 2 (zona occludens 2) kinase TKT transketolase enzyme TLN2 talin 2 unreported TLN2 talin 2 unreported TMED10 transmembrane emp24-like trafficking protein 10 (yeast) transporter TMEM163 transmembrane protein 163 unreported TMEM30A transmembrane protein 30A unreported TMEM33 transmembrane protein 33 unreported TMOD2 tropomodulin 2 (neuronal) unreported TMPO thymopoietin unreported TMX2 thioredoxin-related transmembrane protein 2 enzyme TNIK TRAF2 and NCK interacting kinase kinase TNR tenascin R (restrictin, janusin) unreported TOMM40 translocase of outer mitochondrial membrane 40 homolog (yeast) ion channel TOMM70A translocase of outer mitochondrial membrane 70 homolog A (S. transporter cerevisiae) TPI1 triosephosphate isomerase 1 enzyme

117

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) TPM1 (includes tropomyosin 1 (alpha) unreported EG:22003) TPM3 tropomyosin 3 unreported TPM3 tropomyosin 3 unreported TPP2 tripeptidyl peptidase II peptidase TPPP tubulin polymerization promoting protein unreported TPPP3 tubulin polymerization-promoting protein family member 3 unreported TRIM2 tripartite motif containing 2 enzyme TRIM3 tripartite motif containing 3 unreported TRIO triple functional domain (PTPRF interacting) kinase TSPAN7 tetraspanin 7 unreported TST thiosulfate sulfurtransferase (rhodanese) enzyme TTC7B tetratricopeptide repeat domain 7B unreported TTYH1 tweety homolog 1 (Drosophila) ion channel TUBA1C tubulin, alpha 1c unreported TUBA4A tubulin, alpha 4a unreported TUBB tubulin, beta class I unreported TUBB2A tubulin, beta 2A class IIa unreported TUBB2B tubulin, beta 2B class IIb unreported TUBB3 tubulin, beta 3 class III unreported TUBB4A tubulin, beta 4A class IVa unreported TUBB4B tubulin, beta 4B class IVb unreported TXNL1 thioredoxin-like 1 enzyme TXNRD1 thioredoxin reductase 1 enzyme UBA1 ubiquitin-like modifier activating enzyme 1 enzyme UBAP2L ubiquitin associated protein 2-like unreported UBE2N ubiquitin-conjugating enzyme E2N enzyme UBR4 ubiquitin protein ligase E3 component n-recognin 4 unreported UCHL1 ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) peptidase UFSP2 UFM1-specific peptidase 2 enzyme UGGT1 UDP-glucose glycoprotein glucosyltransferase 1 enzyme UPF1 UPF1 regulator of nonsense transcripts homolog (yeast) enzyme UQCRB ubiquinol-cytochrome c reductase binding protein enzyme UQCRC1 ubiquinol-cytochrome c reductase core protein I enzyme UQCRC2 ubiquinol-cytochrome c reductase core protein II enzyme UQCRFS1 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 enzyme USP5 ubiquitin specific peptidase 5 (isopeptidase T) peptidase VAMP2 vesicle-associated membrane protein 2 (synaptobrevin 2) unreported VAPA VAMP (vesicle-associated membrane protein)-associated protein A, unreported 33kDa VAPB VAMP (vesicle-associated membrane protein)-associated protein B and unreported C VAT1 vesicle amine transport protein 1 homolog (T. californica) transporter VCAN versican unreported VCP valosin containing protein enzyme VDAC1 voltage-dependent anion channel 1 ion channel 118

Table A1. Complete listing of proteins identified in the MAM from mouse brain Symbol Entrez Gene Name Type(s) VDAC2 voltage-dependent anion channel 2 ion channel VDAC3 voltage-dependent anion channel 3 ion channel VIM vimentin unreported VPS13A vacuolar protein sorting 13 homolog A (S. cerevisiae) transporter VPS13C vacuolar protein sorting 13 homolog C (S. cerevisiae) unreported VPS13D vacuolar protein sorting 13 homolog D (S. cerevisiae) unreported VPS18 vacuolar protein sorting 18 homolog (S. cerevisiae) transporter VPS35 vacuolar protein sorting 35 homolog (S. cerevisiae) transporter VSNL1 visinin-like 1 unreported WASF1 WAS protein family, member 1 unreported WDR1 WD repeat domain 1 unreported WDR37 WD repeat domain 37 unreported WDR48 WD repeat domain 48 unreported WDR7 WD repeat domain 7 unreported WFS1 Wolfram syndrome 1 (wolframin) enzyme WWOX WW domain containing oxidoreductase enzyme YARS tyrosyl-tRNA synthetase enzyme YWHAB tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation transcription protein, beta polypeptide regulator YWHAE tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, epsilon polypeptide YWHAG tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, gamma polypeptide YWHAH tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation transcription protein, eta polypeptide regulator Ywhaq tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, theta polypeptide Ywhaq tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, theta polypeptide YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation enzyme protein, zeta polypeptide 1. Gene symbol as it appears in Uniprot database; 2. Gene name references by Entrez; 3. Protein type based on Gene Ontology annotation. “Unreported” protein types have no annotation.

119

Table A2. DRM proteins in Mitochondria and ER Membranes in NG108-15 cells Symbol Entrez Gene Name Type(s) ABCB6 ATP-binding cassette, sub-family B (MDR/TAP), member 6 transporter ACAT1 acetyl-CoA acetyltransferase 1 enzyme ACO2 (includes aconitase 2, mitochondrial enzyme EG:11429) ACTG1 actin, gamma 1 unreported ALDH18A1 aldehyde dehydrogenase 18 family, member A1 kinase AP2A2 adaptor-related protein complex 2, alpha 2 subunit transporter AP2B1 adaptor-related protein complex 2, beta 1 subunit transporter ATAD1 ATPase family, AAA domain containing 1 enzyme ATP1A1 ATPase, Na+/K+ transporting, alpha 1 polypeptide transporter ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 transporter ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha transporter subunit 1, cardiac muscle ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta transporter polypeptide ATP5J2 ATP synthase, H+ transporting, mitochondrial Fo complex, transporter subunit F2 ATP5O ATP synthase, H+ transporting, mitochondrial F1 complex, O transporter subunit CANX calnexin unreported CAPN8 calpain 8 peptidase CCDC47 coiled-coil domain containing 47 unreported CCT4 chaperonin containing TCP1, subunit 4 (delta) unreported CCT5 chaperonin containing TCP1, subunit 5 (epsilon) unreported CKAP4 cytoskeleton-associated protein 4 unreported CLTC clathrin, heavy chain (Hc) unreported COX2 (includes cytochrome c oxidase subunit II enzyme EG:140540) CYB5B cytochrome b5 type B (outer mitochondrial membrane) enzyme CYB5R3 cytochrome b5 reductase 3 enzyme DCAKD dephospho-CoA kinase domain containing unreported DECR1 2,4-dienoyl CoA reductase 1, mitochondrial enzyme DLAT dihydrolipoamide S-acetyltransferase enzyme DLST dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo- enzyme glutarate complex) DYNC1H1 dynein, cytoplasmic 1, heavy chain 1 peptidase Eef1a1 eukaryotic translation elongation factor 1 alpha 1 translation regulator EEF2 eukaryotic translation elongation factor 2 translation regulator FASN fatty acid synthase enzyme GLG1 (includes golgi glycoprotein 1 unreported EG:20340) GLT25D1 glycosyltransferase 25 domain containing 1 unreported GNB1 guanine nucleotide binding protein (G protein), beta polypeptide 1 enzyme

120

Table A2. DRM proteins in Mitochondria and ER Membranes in NG108-15 cells Symbol Entrez Gene Name Type(s) GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate enzyme aminotransferase 2) GRSF1 G-rich RNA sequence binding factor 1 unreported HADHA hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl- enzyme CoA hydratase (trifunctional protein), alpha subunit HNRNPH1 heterogeneous nuclear ribonucleoprotein H1 (H) unreported HNRNPK heterogeneous nuclear ribonucleoprotein K unreported HSD17B12 hydroxysteroid (17-beta) dehydrogenase 12 enzyme HSP90AB1 heat shock protein 90kDa alpha (cytosolic), class B member 1 enzyme HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 unreported HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) enzyme HSPA9 heat shock 70kDa protein 9 (mortalin) unreported HSPD1 heat shock 60kDa protein 1 (chaperonin) enzyme HYOU1 hypoxia up-regulated 1 unreported IMMT inner membrane protein, mitochondrial unreported IQGAP1 IQ motif containing GTPase activating protein 1 unreported KPNB1 karyopherin (importin) beta 1 transporter LETM1 leucine zipper-EF-hand containing transmembrane protein 1 unreported LONP1 lon peptidase 1, mitochondrial peptidase MAP1B microtubule-associated protein 1B unreported MDH2 (includes malate dehydrogenase 2, NAD (mitochondrial) enzyme EG:17448) ME2 malic enzyme 2, NAD(+)-dependent, mitochondrial enzyme MTHFD1L methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1- enzyme like MYH10 myosin, heavy chain 10, non-muscle unreported MYH9 myosin, heavy chain 9, non-muscle enzyme NAP1L1 nucleosome assembly protein 1-like 1 unreported NAPA (includes N-ethylmaleimide-sensitive factor attachment protein, alpha transporter EG:108124) NDUFA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, enzyme 39kDa NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa enzyme (NADH-coenzyme Q reductase) OAT ornithine aminotransferase enzyme OGDH oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) enzyme OGDHL oxoglutarate dehydrogenase-like enzyme PCK2 phosphoenolpyruvate carboxykinase 2 (mitochondrial) kinase PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 enzyme PDHB pyruvate dehydrogenase (lipoamide) beta enzyme PDIA3 protein disulfide isomerase family A, member 3 peptidase PDIA4 protein disulfide isomerase family A, member 4 enzyme PHB prohibitin transcription regulator

121

Table A2. DRM proteins in Mitochondria and ER Membranes in NG108-15 cells Symbol Entrez Gene Name Type(s) PLOD3 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 enzyme PNPT1 polyribonucleotide nucleotidyltransferase 1 enzyme PTCD3 pentatricopeptide repeat domain 3 unreported RAB18 RAB18, member RAS oncogene family enzyme RAB1A (includes RAB1A, member RAS oncogene family enzyme EG:178620) RAB2A RAB2A, member RAS oncogene family enzyme RAB7A RAB7A, member RAS oncogene family enzyme RAP1B RAP1B, member of RAS oncogene family enzyme RARS arginyl-tRNA synthetase enzyme RPL4 ribosomal protein L4 enzyme RPN1 ribophorin I enzyme RPN2 ribophorin II enzyme RRBP1 ribosome binding protein 1 homolog 180kDa (dog) transporter SDHA (includes succinate dehydrogenase complex, subunit A, flavoprotein (Fp) enzyme EG:157074) SFXN1 sideroflexin 1 transporter SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) enzyme SLC1A4 solute carrier family 1 (glutamate/neutral amino acid transporter), transporter member 4 SLC1A5 solute carrier family 1 (neutral amino acid transporter), member 5 transporter SLC25A12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 transporter SLC25A4 solute carrier family 25 (mitochondrial carrier; adenine nucleotide transporter translocator), member 4 SLC3A2 solute carrier family 3 (activators of dibasic and neutral amino acid transporter transport), member 2 SND1 staphylococcal nuclease and tudor domain containing 1 enzyme SPTAN1 spectrin, alpha, non-erythrocytic 1 (alpha-fodrin) unreported STOML2 stomatin (EPB72)-like 2 unreported TIMM50 translocase of inner mitochondrial membrane 50 homolog (S. phosphatase cerevisiae) TUBB tubulin, beta class I unreported TUBB3 tubulin, beta 3 class III unreported UGGT1 UDP-glucose glycoprotein glucosyltransferase 1 enzyme UQCRC1 ubiquinol-cytochrome c reductase core protein I enzyme UQCRC2 ubiquinol-cytochrome c reductase core protein II enzyme VCP valosin containing protein enzyme VDAC1 voltage-dependent anion channel 1 ion channel VDAC2 voltage-dependent anion channel 2 ion channel VDAC3 voltage-dependent anion channel 3 ion channel 1. Gene symbol as it appears in Uniprot database; 2. Gene name references by Entrez; 3. Protein type based on Gene Ontology annotation. “Unreported” protein types have no annotation.

122

Table A3. Proteins Only Identified in the Lipid Raft of MAMs Symbol Entrez Gene Name Type(s) AAAS achalasia, adrenocortical insufficiency, alacrimia unreported ABCB10 ATP-binding cassette, sub-family B (MDR/TAP), member 10 transporter ABCB11 ATP-binding cassette, sub-family B (MDR/TAP), member 11 transporter ABCF3 ATP-binding cassette, sub-family F (GCN20), member 3 transporter ABCG2 ATP-binding cassette, sub-family G (WHITE), member 2 transporter ADO 2-aminoethanethiol (cysteamine) dioxygenase unreported ANKIB1 ankyrin repeat and IBR domain containing 1 transcription regulator ARFGAP1 ADP-ribosylation factor GTPase activating protein 1 transporter ARHGAP21 Rho GTPase activating protein 21 unreported ARL8B ADP-ribosylation factor-like 8B enzyme ASPHD2 aspartate beta-hydroxylase domain containing 2 enzyme ATP5O ATP synthase, H+ transporting, mitochondrial F1 complex, O transporter subunit ATP6V0D1 ATPase, H+ transporting, lysosomal 38kDa, V0 subunit d1 transporter ATP6V1H ATPase, H+ transporting, lysosomal 50/57kDa, V1 subunit H transporter BC003965 cDNA sequence BC003965 unreported BCAN brevican unreported BCL2L13 BCL2-like 13 (apoptosis facilitator) unreported BDH1 (includes 3-hydroxybutyrate dehydrogenase, type 1 enzyme EG:100037356) BRI3BP BRI3 binding protein unreported C12orf57 chromosome 12 open reading frame 57 unreported C1orf95 chromosome 1 open reading frame 95 unreported CACNA2D3 calcium channel, voltage-dependent, alpha 2/delta subunit 3 ion channel CADM4 cell adhesion molecule 4 unreported CAT catalase enzyme CCDC115 coiled-coil domain containing 115 unreported Ccz1 CCZ1 vacuolar protein trafficking and biogenesis associated unreported homolog (S. cerevisiae) CECR6 cat eye syndrome chromosome region, candidate 6 transcription regulator CORO2B coronin, actin binding protein, 2B unreported COX6B1 cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) enzyme COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 (liver) enzyme CYB5B cytochrome b5 type B (outer mitochondrial membrane) enzyme CYB5R1 cytochrome b5 reductase 1 enzyme CYP20A1 cytochrome P450, family 20, subfamily A, polypeptide 1 enzyme DAGLA diacylglycerol lipase, alpha enzyme DDAH2 dimethylarginine dimethylaminohydrolase 2 enzyme DHRS7 dehydrogenase/reductase (SDR family) member 7 enzyme

123

Table A3. Proteins Only Identified in the Lipid Raft of MAMs Symbol Entrez Gene Name Type(s) DSP desmoplakin unreported ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial enzyme EEF1B2 eukaryotic translation elongation factor 1 beta 2 translation regulator ELFN1 extracellular leucine-rich repeat and fibronectin type III unreported domain containing 1 ERLIN2 ER lipid raft associated 2 unreported ESYT2 extended synaptotagmin-like protein 2 unreported FAM162A family with sequence similarity 162, member A unreported FAM54B family with sequence similarity 54, member B unreported FAM81A family with sequence similarity 81, member A unreported FAM82A2 family with sequence similarity 82, member A2 unreported FARP1 FERM, RhoGEF (ARHGEF) and pleckstrin domain protein 1 unreported (chondrocyte-derived) FIS1 (includes fission 1 (mitochondrial outer membrane) homolog (S. unreported EG:288584) cerevisiae) FUT8 fucosyltransferase 8 (alpha (1,6) fucosyltransferase) enzyme FXYD7 FXYD domain containing ion transport regulator 7 ion channel GABBR2 gamma-aminobutyric acid (GABA) B receptor, 2 G-protein coupled receptor GCN1L1 GCN1 general control of amino-acid synthesis 1-like 1 (yeast) translation regulator GIPC1 GIPC PDZ domain containing family, member 1 unreported GLRX5 glutaredoxin 5 unreported GNG4 guanine nucleotide binding protein (G protein), gamma 4 enzyme GOLGA7 golgin A7 unreported GPR56 G protein-coupled receptor 56 G-protein coupled receptor GRID1 glutamate receptor, ionotropic, delta 1 ion channel GRID2 glutamate receptor, ionotropic, delta 2 ion channel GRIK3 glutamate receptor, ionotropic, kainate 3 ion channel GRIN2A glutamate receptor, ionotropic, N-methyl D-aspartate 2A ion channel GRM3 glutamate receptor, metabotropic 3 G-protein coupled receptor HSDL1 hydroxysteroid dehydrogenase like 1 enzyme IGLON5 IgLON family member 5 unreported ILF2 interleukin enhancer binding factor 2, 45kDa transcription regulator IQGAP2 IQ motif containing GTPase activating protein 2 unreported JAGN1 jagunal homolog 1 (Drosophila) unreported KCNA6 potassium voltage-gated channel, shaker-related subfamily, ion channel member 6 KCND2 potassium voltage-gated channel, Shal-related subfamily, ion channel

member 2

124

Table A3. Proteins Only Identified in the Lipid Raft of MAMs Symbol Entrez Gene Name Type(s) KCNQ3 potassium voltage-gated channel, KQT-like subfamily, ion channel member 3 KIAA0090 KIAA0090 unreported KPNB1 karyopherin (importin) beta 1 transporter KRT2 keratin 2 unreported KRT80 keratin 80 unreported KRTCAP2 keratinocyte associated protein 2 enzyme LEMD2 LEM domain containing 2 unreported LGI1 leucine-rich, glioma inactivated 1 unreported MANEAL mannosidase, endo-alpha-like unreported MCU mitochondrial calcium uniporter ion channel MTOR mechanistic target of rapamycin (serine/threonine kinase) kinase MTX3 metaxin 3 unreported NAPA (includes N-ethylmaleimide-sensitive factor attachment protein, alpha transporter EG:108124) NCAN neurocan unreported Ncl nucleolin unreported NDUFA12 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 12 enzyme NDUFA5 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 5, enzyme 13kDa NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, enzyme 14kDa NDUFA8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, enzyme 19kDa NDUFA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, enzyme 39kDa NLGN2 neuroligin 2 enzyme OPA1 optic atrophy 1 (autosomal dominant) enzyme OPA1 optic atrophy 1 (autosomal dominant) enzyme OSBPL8 oxysterol binding protein-like 8 unreported OSTC oligosaccharyltransferase complex subunit enzyme PACS1 phosphofurin acidic cluster sorting protein 1 unreported PCYOX1 prenylcysteine oxidase 1 enzyme PEX10 peroxisomal biogenesis factor 10 unreported PEX5L peroxisomal biogenesis factor 5-like ion channel PGAP1 post-GPI attachment to proteins 1 enzyme PHB prohibitin transcription regulator PITPNM1 phosphatidylinositol transfer protein, membrane-associated 1 transporter PLEKHA5 pleckstrin homology domain containing, family A member 5 unreported PLLP plasmolipin transporter Prss3 (includes protease, serine, 3 peptidase unreporteds) PURB purine-rich element binding protein B transcription regulator 125

Table A3. Proteins Only Identified in the Lipid Raft of MAMs Symbol Entrez Gene Name Type(s) RANBP2 RAN binding protein 2 enzyme RCN2 (includes reticulocalbin 2, EF-hand calcium binding domain unreported EG:26611) RDH14 retinol dehydrogenase 14 (all-trans/9-cis/11-cis) enzyme RFTN2 raftlin family member 2 unreported RGS7BP regulator of G-protein signaling 7 binding protein unreported RMND1 required for meiotic nuclear division 1 homolog (S. unreported cerevisiae) RYR2 ryanodine receptor 2 (cardiac) ion channel S1PR1 sphingosine-1-phosphate receptor 1 G-protein coupled receptor SACM1L SAC1 suppressor of actin mutations 1-like (yeast) phosphatase SCYL1 SCY1-like 1 (S. cerevisiae) kinase SHANK3 SH3 and multiple ankyrin repeat domains 3 transcription regulator SLC1A1 solute carrier family 1 (neuronal/epithelial high affinity transporter glutamate transporter, system Xag), member 1 SLC2A1 solute carrier family 2 (facilitated glucose transporter), transporter member 1 SLC2A13 solute carrier family 2 (facilitated glucose transporter), transporter member 13 SMC1A structural maintenance of chromosomes 1A transporter SNX4 sorting nexin 4 transporter SPCS2 signal peptidase complex subunit 2 homolog (S. cerevisiae) unreported SPECC1L sperm antigen with calponin homology and coiled-coil unreported domains 1-like Svip small VCP/p97-interacting protein unreported SYNE1 spectrin repeat containing, nuclear envelope 1 unreported SYNE1 spectrin repeat containing, nuclear envelope 1 unreported SYNGAP1 synaptic Ras GTPase activating protein 1 unreported SYT11 synaptotagmin XI transporter SYT2 synaptotagmin II transporter TAMM41 TAM41, mitochondrial translocator assembly and unreported maintenance protein, homolog (S. cerevisiae) TAPT1 transmembrane anterior posterior transformation 1 G-protein coupled receptor TBC1D10B TBC1 domain family, member 10B enzyme TDRKH tudor and KH domain containing unreported TIMM13 translocase of inner mitochondrial membrane 13 homolog transporter (yeast) TIMM50 translocase of inner mitochondrial membrane 50 homolog (S. phosphatase cerevisiae) TIMM9 translocase of inner mitochondrial membrane 9 homolog transporter (yeast) TM9SF4 transmembrane 9 superfamily protein member 4 transporter TMEM111 transmembrane protein 111 unreported TMEM85 transmembrane protein 85 unreported 126

Table A3. Proteins Only Identified in the Lipid Raft of MAMs Symbol Entrez Gene Name Type(s) TMX3 thioredoxin-related transmembrane protein 3 enzyme TNKS1BP1 tankyrase 1 binding protein 1, 182kDa unreported TOMM22 translocase of outer mitochondrial membrane 22 homolog transporter (yeast) TOMM40 translocase of outer mitochondrial membrane 40 homolog ion channel (yeast) TRHDE thyrotropin-releasing hormone degrading enzyme peptidase TRPV2 transient receptor potential cation channel, subfamily V, ion channel member 2 TTC9B tetratricopeptide repeat domain 9B unreported UQCRC1 ubiquinol-cytochrome c reductase core protein I enzyme VPS45 vacuolar protein sorting 45 homolog (S. cerevisiae) transporter WASF1 WAS protein family, member 1 unreported 1. Gene symbol as it appears in Uniprot database; 2. Gene name references by Entrez; 3. Protein type based on Gene Ontology annotation. “Unreported” protein types have no annotation.

127

Table A4. Lipid Raft-Enriched Proteins in the MAM* ID Entrez Gene Name Type(s) Q921H8 acetyl-CoA acyltransferase 1 enzyme Q9JI91 actinin, alpha 2 transcription regulator Q3ULT2 actinin, alpha 4 unreported Q8VHH5 ArfGAP with GTPase domain, ankyrin repeat and PH domain 3 transcription regulator Q9ESW4 acylglycerol kinase kinase B1AU25 apoptosis-inducing factor, mitochondrion-associated, 1 enzyme E9Q3Q6 activated leukocyte cell adhesion molecule unreported O54774 adaptor-related protein complex 3, delta 1 subunit transporter Q8VDN2 ATPase, Na+/K+ transporting, alpha 1 polypeptide transporter Q3UHK5 ATPase, Na+/K+ transporting, alpha 2 polypeptide transporter Q6PIC6 ATPase, Na+/K+ transporting, alpha 3 polypeptide transporter Q03265 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha transporter subunit 1, cardiac muscle P56480 ATP synthase, H+ transporting, mitochondrial F1 complex, beta transporter polypeptide Q8C2Q8 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma transporter polypeptide 1 Q9DCX2 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d enzyme P56135 ATP synthase, H+ transporting, mitochondrial Fo complex, subunit F2 transporter P50516 ATPase, H+ transporting, lysosomal 70kDa, V1 subunit A transporter P62814 ATPase, H+ transporting, lysosomal 56/58kDa, V1 subunit B2 transporter Q9Z1G3 ATPase, H+ transporting, lysosomal 42kDa, V1 subunit C1 transporter Q54A87 ATPase, H+ transporting, lysosomal 13kDa, V1 subunit G2 transporter Q91XV3 brain abundant, membrane attached signal protein 1 transcription regulator F7BNZ5 breast carcinoma amplified sequence 1 unreported Q5RJI5 BR serine/threonine kinase 1 kinase Q80TN1 calcium/calmodulin-dependent protein kinase II alpha kinase Q5SVJ0 calcium/calmodulin-dependent protein kinase II beta kinase Q91WS0 CDGSH iron sulfur domain 1 unreported P12960 contactin 1 enzyme Q61330 contactin 2 (axonal) unreported P12787 cytochrome c oxidase subunit Va enzyme O54734 dolichyl-diphosphooligosaccharide--protein glycosyltransferase enzyme Q52KF7 discs, large homolog 3 (Drosophila) kinase Q62108 discs, large homolog 4 (Drosophila) kinase Q7TNF0 double C2-like domains, alpha transporter O35465 FK506 binding protein 8, 38kDa unreported Q3UPA1 guanine nucleotide binding protein (G protein), alpha 11 (Gq class) enzyme Q3TQ70 guanine nucleotide binding protein (G protein), beta polypeptide 1 Enzyme Q9DAS9 guanine nucleotide binding protein (G protein), gamma 12 enzyme

128

Table A4. Lipid Raft Enriched Proteins in the MAM* ID Entrez Gene Name Type(s)

P63216 guanine nucleotide binding protein (G protein), gamma 3 enzyme A2AQR0 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) enzyme P23819-4 glutamate receptor, ionotropic, AMPA 2 ion channel Q3TU85 heat shock 70kDa protein 1A unreported Q6WVG3 potassium channel tetramerisation domain containing 12 ion channel Q7TQ95 KIAA1715 unreported F6RJV6 LanC lantibiotic synthetase component C-like 2 (bacterial) unreported A9DA50 leucine rich repeat and Ig domain containing 1 unreported B3RH23 lamin A/C unreported P14733 lamin B1 unreported E9Q6L9 leucine rich repeat containing 7 unreported E9PY01 myelin associated glycoprotein unreported Q64133 monoamine oxidase A enzyme Q8BW75 monoamine oxidase B enzyme Q7TSJ2 microtubule-associated protein 6 unreported Q9QYG0 NDRG family member 2 unreported Q9ERS2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 13 enzyme Q9DCS9 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22kDa enzyme B1AV40 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17.3kDa enzyme Q91VD9 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADH- enzyme coenzyme Q reductase) Q91WD5 NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa (NADH- enzyme coenzyme Q reductase) Q9DCT2 NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADH- enzyme coenzyme Q reductase) Q9DC70 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH- enzyme coenzyme Q reductase) D3YUM1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa enzyme Q9D6J6 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa enzyme Q61941 nicotinamide nucleotide transhydrogenase enzyme Q60597 oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) enzyme Q9Z0P4 paralemmin unreported Q5PR72 phosphodiesterase 2A, cGMP-stimulated enzyme Q9D051 pyruvate dehydrogenase (lipoamide) beta enzyme P47857 phosphofructokinase, muscle kinase Q8C605 phosphofructokinase, platelet kinase Q3V235 prohibitin 2 transcription regulator E9PYJ7 phosphatidylinositol transfer protein, membrane-associated 2 enzyme B8QI35 protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), phosphatase interacting protein (liprin), alpha 3 Q61171 peroxiredoxin 2 enzyme Q5SXR0 peptidyl-tRNA hydrolase 2 enzyme

129

Table A4. Lipid Raft Enriched Proteins in the MAM* ID Entrez Gene Name Type(s) Q0PD66 RAB1B, member RAS oncogene family enzyme Q4FJQ0 RAB7A, member RAS oncogene family enzyme A2CG14 v-ral simian leukemia viral oncogene homolog A (ras related) enzyme Q3UDZ1 ras homolog family member G enzyme Q3U505 ribophorin II enzyme P62071 related RAS viral (r-ras) oncogene homolog 2 enzyme Q8BGH2 sorting and assembly machinery component 50 homolog (S. unreported cerevisiae) Q6PDS3 sterile alpha and TIR motif containing 1 transmembra ne receptor O08547 SEC22 vesicle trafficking protein homolog B (S. cerevisiae) unreported (gene/pseudogene) P28661 septin 4 enzyme B1AQZ0 septin 8 unreported Q99JR1 sideroflexin 1 transporter Q91V61 sideroflexin 3 transporter A2AIZ0 SH3-domain GRB2-like (endophilin) interacting protein 1 unreported Q5SX53 solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), transporter member 11 Q8BH59 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 transporter P48962 solute carrier family 25 (mitochondrial carrier; adenine nucleotide transporter translocator), member 4 A2ARC5 solute carrier family 27 (fatty acid transporter), member 4 transporter Q3TPL8 solute carrier family 2 (facilitated glucose transporter), member 3 transporter Q5FWH7 solute carrier family 39 (zinc transporter), member 12 transporter P10852 solute carrier family 3 (activators of dibasic and neutral amino acid transporter transport), member 2 Q53ZN9 solute carrier family 4, anion exchanger, member 1 (erythrocyte transporter membrane protein band 3, Diego blood group) E9PX29 spectrin, beta, non-erythrocytic 4 unreported A2AG40 stomatin (EPB72)-like 2 unreported Q497P1 syntaxin 1A (brain) transporter Q9JIS5 synaptic vesicle glycoprotein 2A transporter E9PZA8 synaptotagmin VII transporter Q8C996 transmembrane protein 163 unreported Q8BYI9 tenascin R (restrictin, janusin) unreported P68368 tubulin, alpha 4a unreported P99024 tubulin, beta class I unreported Q9ERD7 tubulin, beta 3 class III unreported Q9D6F9 tubulin, beta 4A class IVa unreported Q9DB77 ubiquinol-cytochrome c reductase core protein II enzyme D3YTU0 vesicle-associated membrane protein 1 (synaptobrevin 1) transporter Q60932 voltage-dependent anion channel 1 ion channel Q60930 voltage-dependent anion channel 2 ion channel 130

Table A4. Lipid Raft Enriched Proteins in the MAM* ID Entrez Gene Name Type(s) Q3TX38 voltage-dependent anion channel 3 ion channel Q5SS40 tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, epsilon polypeptide E9Q070 Unreported A8DUK4 Unreported *Lipid raft –enriched proteins have a 2-fold increase in spectral counts. 1. Gene symbol as it appears in Uniprot database; 2. Gene name references by Entrez; 3. Protein type based on Gene Ontology annotation. “Unreported” protein types have no annotation.

131

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) 1300017J02Rik RIKEN cDNA 1300017J02 gene unreported 1700019G17Ri camello-like 2 unreported k/Cml2 2810007J24Rik RIKEN cDNA 2810007J24 gene enzyme AADAC arylacetamide deacetylase (esterase) enzyme ABAT 4-aminobutyrate aminotransferase enzyme ABCA6 ATP-binding cassette, sub-family A (ABC1), member 6 transporter ABCB10 ATP-binding cassette, sub-family B (MDR/TAP), member 10 transporter ABCB11 ATP-binding cassette, sub-family B (MDR/TAP), member 11 transporter ABCB4 ATP-binding cassette, sub-family B (MDR/TAP), member 4 transporter ABCB6 ATP-binding cassette, sub-family B (MDR/TAP), member 6 transporter ABCC2 ATP-binding cassette, sub-family C (CFTR/MRP), member 2 transporter ABCC6 ATP-binding cassette, sub-family C (CFTR/MRP), member 6 transporter ABCD3 ATP-binding cassette, sub-family D (ALD), member 3 transporter ABCG2 ATP-binding cassette, sub-family G (WHITE), member 2 transporter ABHD12 abhydrolase domain containing 12 unreported ABHD14B abhydrolase domain containing 14B enzyme ABHD6 abhydrolase domain containing 6 enzyme ACAA1 acetyl-CoA acyltransferase 1 enzyme Acaa1b acetyl-Coenzyme A acyltransferase 1B enzyme ACAA2 acetyl-CoA acyltransferase 2 enzyme ACAD11 acyl-CoA dehydrogenase family, member 11 enzyme ACAD9 acyl-CoA dehydrogenase family, member 9 enzyme ACADL acyl-CoA dehydrogenase, long chain enzyme ACADM acyl-CoA dehydrogenase, C-4 to C-12 straight chain enzyme ACADS acyl-CoA dehydrogenase, C-2 to C-3 short chain enzyme ACADVL acyl-CoA dehydrogenase, very long chain enzyme ACAT1 acetyl-CoA acetyltransferase 1 enzyme ACAT2 acetyl-CoA acetyltransferase 2 enzyme ACBD5 acyl-CoA binding domain containing 5 unreported Acnat1/Acnat2 acyl-coenzyme A amino acid N-acyltransferase 1 enzyme ACO1 aconitase 1, soluble enzyme ACO2 aconitase 2, mitochondrial enzyme (includes EG:11429) ACOX1 acyl-CoA oxidase 1, palmitoyl enzyme ACOX2 acyl-CoA oxidase 2, branched chain enzyme ACP5 acid phosphatase 5, tartrate resistant phosphatase ACSF2 acyl-CoA synthetase family member 2 enzyme ACSL1 acyl-CoA synthetase long-chain family member 1 enzyme ACSL5 acyl-CoA synthetase long-chain family member 5 enzyme ACSM1 acyl-CoA synthetase medium-chain family member 1 enzyme 132

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) ACTC1 actin, alpha, cardiac muscle 1 enzyme ACTG1 actin, gamma 1 unreported ACTN4 actinin, alpha 4 unreported ACTR2 ARP2 actin-related protein 2 homolog (yeast) unreported ACTR3 ARP3 actin-related protein 3 homolog (yeast) unreported ADH1C alcohol dehydrogenase 1C (class I), gamma polypeptide enzyme ADH5 alcohol dehydrogenase 5 (class III), chi polypeptide enzyme (includes EG:11532) ADHFE1 alcohol dehydrogenase, iron containing, 1 enzyme ADK adenosine kinase kinase AGL amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase enzyme AGL amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase enzyme AGMAT agmatine ureohydrolase (agmatinase) enzyme Agmo alkylglycerol monooxygenase enzyme AGPAT2 1-acylglycerol-3-phosphate O-acyltransferase 2 (lysophosphatidic enzyme acid acyltransferase, beta) AGPAT3 1-acylglycerol-3-phosphate O-acyltransferase 3 enzyme AGXT alanine-glyoxylate aminotransferase enzyme AGXT2 alanine--glyoxylate aminotransferase 2 enzyme AHCY adenosylhomocysteinase enzyme AHSA1 AHA1, activator of heat shock 90kDa protein ATPase homolog 1 unreported (yeast) AHSG alpha-2-HS-glycoprotein unreported AIFM1 apoptosis-inducing factor, mitochondrion-associated, 1 enzyme AK2 adenylate kinase 2 kinase AK3 adenylate kinase 3 kinase AKR1B1 aldo-keto reductase family 1, member B1 (aldose reductase) enzyme Akr1c14 aldo-keto reductase family 1, member C14 enzyme ALB albumin transporter ALCAM activated leukocyte cell adhesion molecule unreported ALDH1A1 aldehyde dehydrogenase 1 family, member A1 enzyme ALDH1B1 aldehyde dehydrogenase 1 family, member B1 enzyme ALDH1L1 aldehyde dehydrogenase 1 family, member L1 enzyme ALDH2 aldehyde dehydrogenase 2 family (mitochondrial) enzyme ALDH3A2 aldehyde dehydrogenase 3 family, member A2 enzyme ALDH4A1 aldehyde dehydrogenase 4 family, member A1 enzyme ALDH5A1 aldehyde dehydrogenase 5 family, member A1 enzyme ALDH6A1 aldehyde dehydrogenase 6 family, member A1 enzyme ALDH7A1 aldehyde dehydrogenase 7 family, member A1 enzyme ALDH8A1 aldehyde dehydrogenase 8 family, member A1 enzyme ALDOB aldolase B, fructose-bisphosphate enzyme

133

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) AMACR alpha-methylacyl-CoA racemase enzyme ANPEP alanyl (membrane) aminopeptidase peptidase ANXA11 annexin A11 unreported ANXA2 annexin A2 unreported ANXA4 annexin A4 unreported ANXA5 annexin A5 unreported ANXA6 annexin A6 unreported ANXA7 annexin A7 ion channel AP2A2 adaptor-related protein complex 2, alpha 2 subunit transporter AP2B1 adaptor-related protein complex 2, beta 1 subunit transporter AP2M1 adaptor-related protein complex 2, mu 1 subunit transporter APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1 enzyme APOA1 apolipoprotein A-I transporter APOB apolipoprotein B (including Ag(x) antigen) transporter APOC3 apolipoprotein C-III transporter APOE apolipoprotein E transporter APOH apolipoprotein H (beta-2-glycoprotein I) transporter APOM apolipoprotein M transporter APOO apolipoprotein O unreported APOOL apolipoprotein O-like unreported ARF6 ADP-ribosylation factor 6 transporter ARG1 arginase, liver enzyme ARPC3 actin related protein 2/3 complex, subunit 3, 21kDa unreported ARPC5L actin related protein 2/3 complex, subunit 5-like unreported ASGR1 asialoglycoprotein receptor 1 transmembrane receptor ASPH (includes aspartate beta-hydroxylase enzyme EG:312981) ASS1 argininosuccinate synthase 1 enzyme ATAD1 ATPase family, AAA domain containing 1 enzyme ATAD3A/ATA ATPase family, AAA domain containing 3A unreported D3B ATL2 atlastin GTPase 2 unreported ATL3 atlastin GTPase 3 unreported ATP11C ATPase, class VI, type 11C transporter ATP13A1 ATPase type 13A1 transporter ATP1A1 ATPase, Na+/K+ transporting, alpha 1 polypeptide transporter ATP1B1 ATPase, Na+/K+ transporting, beta 1 polypeptide transporter ATP1B3 ATPase, Na+/K+ transporting, beta 3 polypeptide transporter ATP2A2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 transporter ATP2B4 ATPase, Ca++ transporting, plasma membrane 4 transporter ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha transporter

134

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) subunit 1, cardiac muscle ATP5B ATP synthase, H+ transporting, mitochondrial F1 complex, beta transporter polypeptide ATP5C1 ATP synthase, H+ transporting, mitochondrial F1 complex, gamma transporter polypeptide 1 ATP5F1 ATP synthase, H+ transporting, mitochondrial Fo complex, subunit transporter B1 Atp5h (includes ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d enzyme EG:100039281) ATP5J ATP synthase, H+ transporting, mitochondrial Fo complex, subunit transporter F6 ATP5J2 ATP synthase, H+ transporting, mitochondrial Fo complex, subunit transporter F2 ATP5L ATP synthase, H+ transporting, mitochondrial Fo complex, subunit transporter G ATP5O ATP synthase, H+ transporting, mitochondrial F1 complex, O transporter subunit AUP1 ancient ubiquitous protein 1 unreported BAAT bile acid CoA: amino acid N-acyltransferase (glycine N- enzyme choloyltransferase) BAIAP2 BAI1-associated protein 2 kinase BC021614 cDNA sequence BC021614 unreported BCAP31 B-cell receptor-associated protein 31 transporter BCKDHA branched chain keto acid dehydrogenase E1, alpha polypeptide enzyme BDH1 3-hydroxybutyrate dehydrogenase, type 1 enzyme (includes EG:100037356) BLVRB biliverdin reductase B (flavin reductase (NADPH)) enzyme BPHL biphenyl hydrolase-like (serine hydrolase) enzyme BRI3BP BRI3 binding protein unreported BRP44 brain protein 44 unreported BSG (includes basigin (Ok blood group) transporter EG:12215) C19orf10 chromosome 19 open reading frame 10 cytokine C21orf33 chromosome 21 open reading frame 33 unreported C2orf72 chromosome 2 open reading frame 72 unreported C4B (includes complement component 4B (Chido blood group) unreported unreporteds) C8B complement component 8, beta polypeptide unreported C8G complement component 8, gamma polypeptide transporter CALR calreticulin transcription regulator CANX calnexin unreported CAT catalase enzyme CCBL1 cysteine conjugate-beta lyase, cytoplasmic enzyme CCDC47 coiled-coil domain containing 47 unreported CCT2 chaperonin containing TCP1, subunit 2 (beta) kinase

135

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) CCT3 chaperonin containing TCP1, subunit 3 (gamma) unreported CCT4 chaperonin containing TCP1, subunit 4 (delta) unreported CCT7 chaperonin containing TCP1, subunit 7 (eta) unreported CD38 CD38 molecule enzyme CD47 CD47 molecule unreported CD81 CD81 molecule unreported Cdc42 cell division cycle 42 homolog (S. cerevisiae) enzyme CDH2 cadherin 2, type 1, N-cadherin (neuronal) unreported CDK5RAP3 CDK5 regulatory subunit associated protein 3 unreported CDS2 CDP-diacylglycerol synthase (phosphatidate cytidylyltransferase) 2 enzyme Ceacam1 carcinoembryonic antigen-related cell adhesion molecule 1 transmembrane receptor CEP57L1 centrosomal protein 57kDa-like 1 unreported CERS2 ceramide synthase 2 transcription regulator Ces1e carboxylesterase 1E enzyme Ces1f (includes carboxylesterase 1F enzyme EG:234564) Ces1g carboxylesterase 1G enzyme

Ces2a carboxylesterase 2A enzyme Ces2b/Ces2c carboxylesterase 2C enzyme Ces2e carboxylesterase 2E enzyme Ces2g carboxylesterase 2G enzyme CFL1 cofilin 1 (non-muscle) unreported CHCHD3 coiled-coil-helix-coiled-coil-helix domain containing 3 unreported CHDH choline dehydrogenase enzyme CHP calcium binding protein P22 transporter CISD1 CDGSH iron sulfur domain 1 unreported CISD2 CDGSH iron sulfur domain 2 unreported CLDN1 claudin 1 unreported CLDN3 claudin 3 transmembrane receptor CLIC4 chloride intracellular channel 4 ion channel CLPTM1 cleft lip and palate associated transmembrane protein 1 unreported CLTC clathrin, heavy chain (Hc) unreported CLU clusterin unreported Cml1 camello-like 1 unreported CNPY2 canopy 2 homolog (zebrafish) unreported CNPY3 canopy 3 homolog (zebrafish) unreported COBLL1 COBL-like 1 unreported COMT catechol-O-methyltransferase enzyme COX4I1 cytochrome c oxidase subunit IV isoform 1 enzyme 136

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) COX5A cytochrome c oxidase subunit Va enzyme (includes EG:12858) Cox5b cytochrome c oxidase, subunit Vb enzyme COX6A1 cytochrome c oxidase subunit VIa polypeptide 1 enzyme COX7A2 cytochrome c oxidase subunit VIIa polypeptide 2 (liver) enzyme CPOX coproporphyrinogen oxidase enzyme CPS1 carbamoyl-phosphate synthase 1, mitochondrial enzyme CPT1A carnitine palmitoyltransferase 1A (liver) enzyme CPT2 carnitine palmitoyltransferase 2 enzyme CRYZ crystallin, zeta (quinone reductase) enzyme CS citrate synthase enzyme CTAGE5 CTAGE family, member 5 enzyme CTH cystathionase (cystathionine gamma-lyase) enzyme CTNNA1 catenin (cadherin-associated protein), alpha 1, 102kDa unreported CTNNB1 catenin (cadherin-associated protein), beta 1, 88kDa transcription regulator CTNND1 catenin (cadherin-associated protein), delta 1 unreported CTSB cathepsin B peptidase CTSD cathepsin D peptidase CTTN cortactin unreported CYB5A cytochrome b5 type A (microsomal) enzyme (includes EG:109672) CYB5B cytochrome b5 type B (outer mitochondrial membrane) enzyme CYB5R3 cytochrome b5 reductase 3 enzyme CYC1 cytochrome c-1 enzyme Cycs cytochrome c, somatic transporter CYP1A2 cytochrome P450, family 1, subfamily A, polypeptide 2 enzyme CYP27A1 cytochrome P450, family 27, subfamily A, polypeptide 1 enzyme Cyp2a12/Cyp2 cytochrome P450, family 2, subfamily a, polypeptide 12 enzyme a22 CYP2A6 cytochrome P450, family 2, subfamily A, polypeptide 6 enzyme (includes unreporteds) CYP2C19 cytochrome P450, family 2, subfamily C, polypeptide 19 enzyme CYP2C19 cytochrome P450, family 2, subfamily C, polypeptide 19 enzyme Cyp2c29 cytochrome P450, family 2, subfamily c, polypeptide 29 enzyme (includes unreporteds) Cyp2c29 cytochrome P450, family 2, subfamily c, polypeptide 29 enzyme (includes unreporteds) Cyp2c40 cytochrome P450, family 2, subfamily c, polypeptide 40 enzyme (includes unreporteds) 137

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) Cyp2c40 cytochrome P450, family 2, subfamily c, polypeptide 40 enzyme (includes unreporteds) Cyp2c44 cytochrome P450, family 2, subfamily c, polypeptide 44 enzyme Cyp2c70 cytochrome P450, family 2, subfamily c, polypeptide 70 enzyme Cyp2d26 cytochrome P450, family 2, subfamily d, polypeptide 26 enzyme CYP2D6 cytochrome P450, family 2, subfamily D, polypeptide 6 enzyme Cyp2d9 cytochrome P450, family 2, subfamily d, polypeptide 9 enzyme (includes unreporteds) Cyp2d9 cytochrome P450, family 2, subfamily d, polypeptide 9 enzyme (includes unreporteds) CYP2E1 cytochrome P450, family 2, subfamily E, polypeptide 1 enzyme CYP2F1 cytochrome P450, family 2, subfamily F, polypeptide 1 enzyme Cyp2j5 cytochrome P450, family 2, subfamily j, polypeptide 5 enzyme CYP3A4 cytochrome P450, family 3, subfamily A, polypeptide 4 enzyme CYP3A43 cytochrome P450, family 3, subfamily A, polypeptide 43 enzyme CYP3A7 cytochrome P450, family 3, subfamily A, polypeptide 7 enzyme CYP4A22 cytochrome P450, family 4, subfamily A, polypeptide 22 enzyme CYP4F12 cytochrome P450, family 4, subfamily F, polypeptide 12 enzyme CYP4V2 cytochrome P450, family 4, subfamily V, polypeptide 2 enzyme CYP7B1 cytochrome P450, family 7, subfamily B, polypeptide 1 enzyme D2HGDH D-2-hydroxyglutarate dehydrogenase unreported DAD1 defender against cell death 1 enzyme (includes EG:13135) DAK dihydroxyacetone kinase 2 homolog (S. cerevisiae) unreported DBI diazepam binding inhibitor (GABA receptor modulator, acyl-CoA unreported binding protein) DBT dihydrolipoamide branched chain transacylase E2 enzyme DCAKD dephospho-CoA kinase domain containing unreported DDOST dolichyl-diphosphooligosaccharide--protein glycosyltransferase enzyme DDRGK1 DDRGK domain containing 1 unreported DDT D-dopachrome tautomerase enzyme DDX1 DEAD (Asp-Glu-Ala-Asp) box helicase 1 enzyme DECR1 2,4-dienoyl CoA reductase 1, mitochondrial enzyme DECR2 2,4-dienoyl CoA reductase 2, peroxisomal enzyme DERL1 Der1-like domain family, member 1 unreported DGAT1 diacylglycerol O-acyltransferase 1 enzyme DHCR24 24-dehydrocholesterol reductase enzyme DHCR7 7-dehydrocholesterol reductase enzyme DHRS1 dehydrogenase/reductase (SDR family) member 1 enzyme DHRS4 dehydrogenase/reductase (SDR family) member 4 enzyme

138

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) DHRS7B dehydrogenase/reductase (SDR family) member 7B unreported DHTKD1 dehydrogenase E1 and transketolase domain containing 1 enzyme DLAT dihydrolipoamide S-acetyltransferase enzyme DLD dihydrolipoamide dehydrogenase enzyme DLST dihydrolipoamide S-succinyltransferase (E2 component of 2-oxo- enzyme glutarate complex) DMGDH dimethylglycine dehydrogenase enzyme DNAJA3 DnaJ (Hsp40) homolog, subfamily A, member 3 unreported DNAJB11 DnaJ (Hsp40) homolog, subfamily B, member 11 unreported DNAJC3 DnaJ (Hsp40) homolog, subfamily C, member 3 unreported DPM1 dolichyl-phosphate mannosyltransferase polypeptide 1, catalytic enzyme (includes subunit EG:13480) DPP4 dipeptidyl-peptidase 4 peptidase DPYD dihydropyrimidine dehydrogenase enzyme DPYS dihydropyrimidinase enzyme EBP emopamil binding protein (sterol isomerase) enzyme ECH1 enoyl CoA hydratase 1, peroxisomal enzyme ECHDC2 enoyl CoA hydratase domain containing 2 unreported ECHS1 enoyl CoA hydratase, short chain, 1, mitochondrial enzyme ECI1 enoyl-CoA delta isomerase 1 enzyme ECI2 enoyl-CoA delta isomerase 2 enzyme Eef1a1 eukaryotic translation elongation factor 1 alpha 1 translation regulator EEF2 eukaryotic translation elongation factor 2 translation regulator EGFR epidermal growth factor receptor kinase ELOVL2 ELOVL fatty acid elongase 2 enzyme ENO1 enolase 1, (alpha) transcription regulator ENPEP glutamyl aminopeptidase (aminopeptidase A) peptidase ENTPD5 ectonucleoside triphosphate diphosphohydrolase 5 enzyme EPHX1 epoxide hydrolase 1, microsomal (xenobiotic) peptidase EPHX2 epoxide hydrolase 2, cytoplasmic enzyme EPS8L2 EPS8-like 2 unreported ERAP1 endoplasmic reticulum aminopeptidase 1 peptidase ERLEC1 endoplasmic reticulum lectin 1 unreported ERLIN2 ER lipid raft associated 2 unreported ERO1L ERO1-like (S. cerevisiae) enzyme ERP29 endoplasmic reticulum protein 29 transporter ERP44 endoplasmic reticulum protein 44 enzyme ESD esterase D enzyme ETFA electron-transfer-flavoprotein, alpha polypeptide transporter

139

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) ETFB electron-transfer-flavoprotein, beta polypeptide transporter ETFDH electron-transferring-flavoprotein dehydrogenase enzyme FAAH fatty acid amide hydrolase enzyme FABP1 fatty acid binding protein 1, liver transporter FADS2 fatty acid desaturase 2 enzyme FAF2 Fas associated factor family member 2 unreported FAH fumarylacetoacetate hydrolase (fumarylacetoacetase) enzyme FAM162A family with sequence similarity 162, member A unreported FAM213A family with sequence similarity 213, member A unreported FAM82A1 family with sequence similarity 82, member A1 unreported FARSB phenylalanyl-tRNA synthetase, beta subunit enzyme FASN fatty acid synthase enzyme FAU Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) unreported ubiquitously expressed FBP1 fructose-1,6-bisphosphatase 1 phosphatase FDFT1 farnesyl-diphosphate farnesyltransferase 1 enzyme FDPS farnesyl diphosphate synthase enzyme FECH ferrochelatase enzyme FGA fibrinogen alpha chain unreported FGB (includes fibrinogen beta chain unreported EG:110135) FGG fibrinogen gamma chain unreported FIS1 (includes fission 1 (mitochondrial outer membrane) homolog (S. cerevisiae) unreported EG:288584) FKBP11 FK506 binding protein 11, 19 kDa enzyme FKBP2 FK506 binding protein 2, 13kDa enzyme FKBP8 FK506 binding protein 8, 38kDa unreported FMO1 flavin containing monooxygenase 1 enzyme (includes EG:14261) FMO5 flavin containing monooxygenase 5 enzyme FTCD formiminotransferase cyclodeaminase enzyme FTL ferritin, light polypeptide enzyme GANAB glucosidase, alpha; neutral AB enzyme GBA glucosidase, beta, acid enzyme GC group-specific component (vitamin D binding protein) transporter GCAT glycine C-acetyltransferase enzyme GCDH glutaryl-CoA dehydrogenase enzyme GGCX gamma-glutamyl carboxylase enzyme GLT25D1 glycosyltransferase 25 domain containing 1 unreported GLTPD2 glycolipid transfer protein domain containing 2 unreported GLUD1 glutamate dehydrogenase 1 enzyme GLUL glutamate-ammonia ligase enzyme 140

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) GLYCTK glycerate kinase kinase Gm10071/Rpl1 ribosomal protein L13 unreported 3 Gm5093 predicted gene 5093 unreported Gm5409/Try10 predicted pseudogene 5409 peptidase Gm6139 predicted gene 6139 unreported GNAI2 guanine nucleotide binding protein (G protein), alpha inhibiting enzyme activity polypeptide 2 Gnas (mouse) GNAS (guanine nucleotide binding protein, alpha stimulating) enzyme complex locus GNB2 guanine nucleotide binding protein (G protein), beta polypeptide 2 enzyme GNB2L1 guanine nucleotide binding protein (G protein), beta polypeptide 2- enzyme like 1 GNG12 guanine nucleotide binding protein (G protein), gamma 12 enzyme

GNMT glycine N-methyltransferase enzyme

GOT1 glutamic-oxaloacetic transaminase 1, soluble (aspartate enzyme aminotransferase 1) GOT2 glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate enzyme aminotransferase 2) GPAM glycerol-3-phosphate acyltransferase, mitochondrial enzyme GPD2 glycerol-3-phosphate dehydrogenase 2 (mitochondrial) enzyme GPT glutamic-pyruvate transaminase (alanine aminotransferase) enzyme GPT2 (includes glutamic pyruvate transaminase (alanine aminotransferase) 2 enzyme EG:108682) GPX1 (includes glutathione peroxidase 1 enzyme EG:14775) GRHPR glyoxylate reductase/hydroxypyruvate reductase enzyme GRPEL1 GrpE-like 1, mitochondrial (E. coli) unreported GSTA3 glutathione S-transferase alpha 3 enzyme GSTK1 glutathione S-transferase kappa 1 enzyme Gstm3 glutathione S-transferase, mu 3 enzyme GSTM5 glutathione S-transferase mu 5 enzyme Gstp1 (includes glutathione S-transferase, pi 1 enzyme unreporteds) GSTZ1 glutathione transferase zeta 1 enzyme Gulo gulonolactone (L-) oxidase enzyme H2-L histocompatibility 2, D region locus L unreported H6PD hexose-6-phosphate dehydrogenase (glucose 1-dehydrogenase) enzyme HAAO 3-hydroxyanthranilate 3,4-dioxygenase enzyme HACL1 2-hydroxyacyl-CoA lyase 1 enzyme HADHA hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl- enzyme CoA hydratase (trifunctional protein), alpha subunit HADHB hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl- enzyme CoA hydratase (trifunctional protein), beta subunit 141

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) HAO1 hydroxyacid oxidase (glycolate oxidase) 1 enzyme HBA1/HBA2 hemoglobin, alpha 1 transporter HBB hemoglobin, beta transporter HBD (includes hemoglobin, delta transporter EG:15130) HDHD2 haloacid dehalogenase-like hydrolase domain containing 2 unreported HGD homogentisate 1,2-dioxygenase enzyme HIBADH 3-hydroxyisobutyrate dehydrogenase enzyme HIBCH 3-hydroxyisobutyryl-CoA hydrolase enzyme HINT2 histidine triad nucleotide binding protein 2 unreported HIST1H2AB/H histone cluster 1, H2ae unreported IST1H2AE HLA-C major histocompatibility complex, class I, C transmembrane receptor HM13 histocompatibility (minor) 13 peptidase HMGCL 3-hydroxymethyl-3-methylglutaryl-CoA lyase enzyme HMGCS2 3-hydroxy-3-methylglutaryl-CoA synthase 2 (mitochondrial) enzyme HOGA1 4-hydroxy-2-oxoglutarate aldolase 1 enzyme HPD 4-hydroxyphenylpyruvate dioxygenase enzyme HPN hepsin peptidase HPX hemopexin transporter HRSP12 heat-responsive protein 12 unreported HSD11B1 hydroxysteroid (11-beta) dehydrogenase 1 enzyme HSD17B10 hydroxysteroid (17-beta) dehydrogenase 10 enzyme HSD17B11 hydroxysteroid (17-beta) dehydrogenase 11 enzyme HSD17B12 hydroxysteroid (17-beta) dehydrogenase 12 enzyme HSD17B2 hydroxysteroid (17-beta) dehydrogenase 2 enzyme HSD17B4 hydroxysteroid (17-beta) dehydrogenase 4 enzyme HSD17B6 hydroxysteroid (17-beta) dehydrogenase 6 homolog (mouse) enzyme HSD17B7 hydroxysteroid (17-beta) dehydrogenase 7 enzyme Hsd3b3 hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta- enzyme isomerase 3 Hsd3b4 hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta- enzyme (includes isomerase 4 unreporteds) HSD3B7 hydroxy-delta-5-steroid dehydrogenase, 3 beta-and steroid delta- enzyme isomerase 7 HSDL2 hydroxysteroid dehydrogenase like 2 transporter HSP90AB1 heat shock protein 90kDa alpha (cytosolic), class B member 1 enzyme HSP90B1 heat shock protein 90kDa beta (Grp94), member 1 unreported HSPA1L heat shock 70kDa protein 1-like unreported HSPA5 heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) enzyme HSPA8 heat shock 70kDa protein 8 enzyme HSPA9 heat shock 70kDa protein 9 (mortalin) unreported 142

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) HSPD1 heat shock 60kDa protein 1 (chaperonin) enzyme HSPE1 heat shock 10kDa protein 1 (chaperonin 10) enzyme HYOU1 hypoxia up-regulated 1 unreported ICAM1 intercellular adhesion molecule 1 transmembrane receptor IDH1 isocitrate dehydrogenase 1 (NADP+), soluble enzyme Igtp interferon gamma induced GTPase enzyme IQGAP2 IQ motif containing GTPase activating protein 2 unreported ISOC2 isochorismatase domain containing 2 enzyme IVD isovaleryl-CoA dehydrogenase enzyme Keg1 kidney expressed gene 1 unreported KHK ketohexokinase (fructokinase) kinase KMO kynurenine 3-monooxygenase (kynurenine 3-hydroxylase) enzyme KRT8 keratin 8 unreported KRTCAP2 keratinocyte associated protein 2 enzyme KYNU kynureninase enzyme LACTB2 lactamase, beta 2 unreported LAMP1 lysosomal-associated membrane protein 1 unreported LAP3 leucine aminopeptidase 3 peptidase LDHA lactate dehydrogenase A enzyme LDHD lactate dehydrogenase D enzyme LETM1 leucine zipper-EF-hand containing transmembrane protein 1 unreported LGALS9B lectin, galactoside-binding, soluble, 9B unreported LIMA1 LIM domain and actin binding 1 unreported LMAN1 lectin, mannose-binding, 1 unreported LMF1 lipase maturation factor 1 unreported LMF2 lipase maturation factor 2 unreported LONP1 lon peptidase 1, mitochondrial peptidase LONP2 lon peptidase 2, peroxisomal peptidase LPCAT3 lysophosphatidylcholine acyltransferase 3 unreported LRP1 (includes low density lipoprotein receptor-related protein 1 transmembrane EG:16971) receptor LRPAP1 low density lipoprotein receptor-related protein associated protein 1 transmembrane receptor LRRC59 leucine rich repeat containing 59 unreported MAL2 mal, T-cell differentiation protein 2 (gene/pseudogene) transporter MAN1A2 mannosidase, alpha, class 1A, member 2 enzyme MAN2A1 mannosidase, alpha, class 2A, member 1 enzyme MANF mesencephalic astrocyte-derived neurotrophic factor unreported MAOB monoamine oxidase B enzyme MAR1 mitochondrial amidoxime reducing component 1 enzyme MAR2 mitochondrial amidoxime reducing component 2 enzyme

143

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) MAT1A methionine adenosyltransferase I, alpha enzyme MAVS mitochondrial antiviral signaling protein unreported MBL2 mannose-binding lectin (protein C) 2, soluble unreported MDH1 malate dehydrogenase 1, NAD (soluble) enzyme MDH2 malate dehydrogenase 2, NAD (mitochondrial) enzyme (includes EG:17448) ME1 malic enzyme 1, NADP(+)-dependent, cytosolic enzyme METTL7A methyltransferase like 7A unreported METTL7B methyltransferase like 7B enzyme MGST1 microsomal glutathione S-transferase 1 enzyme MIA3 melanoma inhibitory activity family, member 3 unreported MLEC malectin unreported MLYCD malonyl-CoA decarboxylase enzyme MOGS mannosyl-oligosaccharide glucosidase enzyme MPDU1 mannose-P-dolichol utilization defect 1 unreported MPST mercaptopyruvate sulfurtransferase enzyme MSN moesin unreported MTCH2 mitochondrial carrier 2 unreported MTDH metadherin transcription regulator MTHFD1 methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1, enzyme methenyltetrahydrofolate cyclohydrolase, formyltetrahydrofolate synthetase MTTP microsomal triglyceride transfer protein transporter MTX1 metaxin 1 transporter Mug1/Mug2 murinoglobulin 1 transporter Mup1 (includes major urinary protein 1 unreported unreporteds) Mup1 (includes major urinary protein 1 unreported unreporteds) Mup1 (includes major urinary protein 1 unreported unreporteds) MVP major vault protein unreported MYH9 myosin, heavy chain 9, non-muscle enzyme MYO1B myosin IB unreported MYO1C myosin IC unreported MYO6 myosin VI unreported Naca nascent polypeptide-associated complex alpha polypeptide transcription regulator NCLN nicalin peptidase NDRG2 NDRG family member 2 unreported NDUFA10 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 10, 42kDa enzyme NDUFA11 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 11, enzyme 14.7kDa 144

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) NDUFA2 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2, 8kDa enzyme NDUFA3 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3, 9kDa enzyme NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9kDa enzyme NDUFA6 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 6, 14kDa enzyme NDUFA8 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 8, 19kDa enzyme NDUFA9 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39kDa enzyme NDUFB10 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 10, 22kDa enzyme NDUFB11 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 11, 17.3kDa enzyme NDUFB4 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 4, 15kDa enzyme NDUFB5 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5, 16kDa enzyme NDUFB7 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 7, 18kDa enzyme NDUFB8 NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 8, 19kDa enzyme NDUFS1 NADH dehydrogenase (ubiquinone) Fe-S protein 1, 75kDa (NADH- enzyme coenzyme Q reductase) NDUFS2 NADH dehydrogenase (ubiquinone) Fe-S protein 2, 49kDa (NADH- enzyme coenzyme Q reductase) NDUFS3 NADH dehydrogenase (ubiquinone) Fe-S protein 3, 30kDa (NADH- enzyme coenzyme Q reductase) NDUFS5 NADH dehydrogenase (ubiquinone) Fe-S protein 5, 15kDa (NADH- enzyme coenzyme Q reductase) NDUFS6 NADH dehydrogenase (ubiquinone) Fe-S protein 6, 13kDa (NADH- enzyme coenzyme Q reductase) NDUFS7 NADH dehydrogenase (ubiquinone) Fe-S protein 7, 20kDa (NADH- enzyme coenzyme Q reductase) NDUFS8 NADH dehydrogenase (ubiquinone) Fe-S protein 8, 23kDa (NADH- enzyme coenzyme Q reductase) NDUFV1 NADH dehydrogenase (ubiquinone) flavoprotein 1, 51kDa enzyme NDUFV2 NADH dehydrogenase (ubiquinone) flavoprotein 2, 24kDa enzyme NIPSNAP1 nipsnap homolog 1 (C. elegans) enzyme NME2 non-metastatic cells 2, protein (NM23B) expressed in kinase NNT nicotinamide nucleotide transhydrogenase enzyme NOMO1 NODAL modulator 1 unreported (includes unreporteds) NPC1 Niemann-Pick disease, type C1 transporter NSDHL NAD(P) dependent steroid dehydrogenase-like enzyme NUCB1 nucleobindin 1 unreported NUDT7 nudix (nucleoside diphosphate linked moiety X)-type motif 7 enzyme OAT ornithine aminotransferase enzyme OGDH oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) enzyme OSTC oligosaccharyltransferase complex subunit enzyme OTC ornithine carbamoyltransferase enzyme P4HB prolyl 4-hydroxylase, beta polypeptide enzyme PA2G4 proliferation-associated 2G4, 38kDa transcription regulator 145

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) PACSIN3 protein kinase C and casein kinase substrate in neurons 3 unreported PAH phenylalanine hydroxylase enzyme PAPSS2 3'-phosphoadenosine 5'-phosphosulfate synthase 2 enzyme PARK7 parkinson protein 7 enzyme PARP9 poly (ADP-ribose) polymerase family, member 9 unreported PBLD phenazine biosynthesis-like protein domain containing enzyme PC pyruvate carboxylase enzyme PCCA propionyl CoA carboxylase, alpha polypeptide enzyme PCCB propionyl CoA carboxylase, beta polypeptide enzyme PCYOX1 prenylcysteine oxidase 1 enzyme PDHA1 pyruvate dehydrogenase (lipoamide) alpha 1 enzyme PDHB pyruvate dehydrogenase (lipoamide) beta enzyme PDIA3 protein disulfide isomerase family A, member 3 peptidase PDIA4 protein disulfide isomerase family A, member 4 enzyme PDIA5 protein disulfide isomerase family A, member 5 enzyme PDIA6 protein disulfide isomerase family A, member 6 enzyme PDZK1 PDZ domain containing 1 transporter PECR peroxisomal trans-2-enoyl-CoA reductase enzyme PEX11A peroxisomal biogenesis factor 11 alpha unreported PEX16 peroxisomal biogenesis factor 16 unreported PFN1 profilin 1 unreported PGAM1 phosphoglycerate mutase 1 (brain) phosphatase PGD phosphogluconate dehydrogenase enzyme PGK1 phosphoglycerate kinase 1 kinase PGM2 phosphoglucomutase 2 enzyme PGRMC1 progesterone receptor membrane component 1 transmembrane receptor PGRMC2 progesterone receptor membrane component 2 ligand- dependent nuclear receptor PHB prohibitin transcription regulator PHB2 prohibitin 2 transcription regulator PHYH phytanoyl-CoA 2-hydroxylase enzyme PICALM phosphatidylinositol binding clathrin assembly protein unreported PIPOX pipecolic acid oxidase enzyme PLA2G12B phospholipase A2, group XIIB enzyme PLG plasminogen peptidase PLXNB2 plexin B2 transmembrane receptor PM20D1 peptidase M20 domain containing 1 peptidase PON1 paraoxonase 1 phosphatase

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Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) PON2 paraoxonase 2 enzyme PON3 paraoxonase 3 enzyme POR P450 (cytochrome) oxidoreductase enzyme PPAP2B phosphatidic acid phosphatase type 2B phosphatase PPIA peptidylprolyl isomerase A (cyclophilin A) enzyme PPIB peptidylprolyl isomerase B (cyclophilin B) enzyme PPT1 palmitoyl-protein thioesterase 1 enzyme PRDX1 peroxiredoxin 1 enzyme PRDX2 peroxiredoxin 2 enzyme PRDX3 peroxiredoxin 3 enzyme PRDX4 peroxiredoxin 4 enzyme PRDX5 peroxiredoxin 5 enzyme PREB prolactin regulatory element binding transcription regulator PRKCSH protein kinase C substrate 80K-H enzyme PRODH proline dehydrogenase (oxidase) 1 enzyme PRODH2 proline dehydrogenase (oxidase) 2 enzyme PTGES2 prostaglandin E synthase 2 transcription regulator PTRH2 peptidyl-tRNA hydrolase 2 enzyme PXMP2 peroxisomal membrane protein 2, 22kDa unreported Pzp pregnancy zone protein unreported QDPR quinoid dihydropteridine reductase enzyme RAB10 RAB10, member RAS oncogene family enzyme RAB14 RAB14, member RAS oncogene family enzyme RAB18 RAB18, member RAS oncogene family enzyme RAB1A RAB1A, member RAS oncogene family enzyme (includes EG:178620) RAB1B RAB1B, member RAS oncogene family enzyme RAB2A RAB2A, member RAS oncogene family enzyme RAB7A RAB7A, member RAS oncogene family enzyme RAB8A RAB8A, member RAS oncogene family unreported RBP4 retinol binding protein 4, plasma transporter RDH11 retinol dehydrogenase 11 (all-trans/9-cis/11-cis) enzyme RDH16 retinol dehydrogenase 16 (all-trans) enzyme Rdh7 retinol dehydrogenase 7 enzyme RDX radixin unreported REEP6 receptor accessory protein 6 unreported RER1 RER1 retention in endoplasmic reticulum 1 homolog (S. cerevisiae) unreported RETSAT retinol saturase (all-trans-retinol 13,14-reductase) enzyme RGN regucalcin (senescence marker protein-30) enzyme

147

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) RPL10 ribosomal protein L10 unreported Rpl10a ribosomal protein L10A unreported Rpl12 ribosomal protein L12 unreported RPL13A ribosomal protein L13a unreported RPL14 ribosomal protein L14 unreported Rpl15 ribosomal protein L15 unreported RPL17 ribosomal protein L17 unreported RPL18 ribosomal protein L18 unreported RPL18A ribosomal protein L18a unreported RPL19 ribosomal protein L19 unreported Rpl21 (includes ribosomal protein L21 unreported EG:100039601) RPL22 ribosomal protein L22 unreported RPL23 ribosomal protein L23 unreported RPL23A ribosomal protein L23a unreported RPL24 ribosomal protein L24 unreported RPL27 ribosomal protein L27 unreported RPL27A ribosomal protein L27a unreported RPL3 ribosomal protein L3 unreported RPL31 ribosomal protein L31 unreported RPL32 ribosomal protein L32 unreported RPL35 ribosomal protein L35 unreported Rpl36 ribosomal protein L36 unreported RPL37A ribosomal protein L37a unreported Rpl38 ribosomal protein L38 unreported RPL39 ribosomal protein L39 unreported (includes EG:25347) RPL4 ribosomal protein L4 enzyme RPL5 ribosomal protein L5 unreported RPL6 ribosomal protein L6 unreported RPL7 ribosomal protein L7 transcription regulator RPL7A ribosomal protein L7a unreported Rpl8 ribosomal protein L8 unreported Rpl9 ribosomal protein L9 unreported RPLP0 ribosomal protein, large, P0 unreported RPLP1 ribosomal protein, large, P1 unreported RPLP2 ribosomal protein, large, P2 unreported RPN1 ribophorin I enzyme RPN2 ribophorin II enzyme RPS10 ribosomal protein S10 unreported

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Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) RPS11 ribosomal protein S11 unreported Rps12 ribosomal protein S12 unreported Rps13 ribosomal protein S13 unreported RPS14 ribosomal protein S14 translation regulator Rps15 ribosomal protein S15 unreported RPS16 ribosomal protein S16 unreported Rps17 ribosomal protein S17 unreported RPS18 ribosomal protein S18 unreported RPS19 ribosomal protein S19 unreported Rps20 ribosomal protein S20 unreported RPS23 ribosomal protein S23 translation regulator Rps24 ribosomal protein S24 unreported RPS25 ribosomal protein S25 unreported RPS26 ribosomal protein S26 unreported Rps27a ribosomal protein S27A unreported RPS27L ribosomal protein S27-like unreported Rps28 ribosomal protein S28 unreported RPS3 ribosomal protein S3 enzyme RPS3A ribosomal protein S3A unreported RPS4X ribosomal protein S4, X-linked unreported RPS5 ribosomal protein S5 unreported RPS6 ribosomal protein S6 unreported RPS7 ribosomal protein S7 unreported RPS9 ribosomal protein S9 translation regulator RRBP1 ribosome binding protein 1 homolog 180kDa (dog) transporter SARDH sarcosine dehydrogenase enzyme SCARB1 scavenger receptor class B, member 1 transporter SCARB2 scavenger receptor class B, member 2 unreported SCP2 sterol carrier protein 2 transporter SDC4 syndecan 4 unreported SDF2L1 stromal cell-derived factor 2-like 1 unreported SDHA succinate dehydrogenase complex, subunit A, flavoprotein (Fp) enzyme (includes EG:157074) SDHB succinate dehydrogenase complex, subunit B, iron sulfur (Ip) enzyme SDHC succinate dehydrogenase complex, subunit C, integral membrane enzyme protein, 15kDa SEC11A SEC11 homolog A (S. cerevisiae) peptidase SEC14L4 SEC14-like 4 (S. cerevisiae) transporter SEC22B SEC22 vesicle trafficking protein homolog B (S. cerevisiae) unreported (gene/pseudogene) 149

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) SEC61A1 Sec61 alpha 1 subunit (S. cerevisiae) transporter SEC61G Sec61 gamma subunit transporter SEC63 SEC63 homolog (S. cerevisiae) transporter (includes EG:11231) SELENBP1 selenium binding protein 1 unreported SERBP1 SERPINE1 mRNA binding protein 1 unreported SERPINA1 serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, unreported antitrypsin), member 1 Serpina3k serine (or cysteine) peptidase inhibitor, clade A, member 3K unreported (includes unreporteds) SERPINC1 serpin peptidase inhibitor, clade C (antithrombin), member 1 unreported SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 unreported SFXN1 sideroflexin 1 transporter SGPL1 sphingosine-1-phosphate lyase 1 enzyme SHMT2 serine hydroxymethyltransferase 2 (mitochondrial) enzyme SIGMAR1 sigma non-opioid intracellular receptor 1 G-protein coupled receptor SLC22A1 solute carrier family 22 (organic cation transporter), member 1 transporter SLC22A18 solute carrier family 22, member 18 transporter SLC25A1 solute carrier family 25 (mitochondrial carrier; citrate transporter), transporter member 1 SLC25A10 solute carrier family 25 (mitochondrial carrier; dicarboxylate transporter transporter), member 10 SLC25A11 solute carrier family 25 (mitochondrial carrier; oxoglutarate carrier), transporter member 11 SLC25A12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12 transporter SLC25A13 solute carrier family 25, member 13 (citrin) transporter SLC25A15 solute carrier family 25 (mitochondrial carrier; ornithine transporter) transporter member 15 SLC25A20 solute carrier family 25 (carnitine/acylcarnitine translocase), member transporter 20 SLC25A22 solute carrier family 25 (mitochondrial carrier: glutamate), member transporter 22 SLC25A3 solute carrier family 25 (mitochondrial carrier; phosphate carrier), transporter member 3 SLC26A1 solute carrier family 26 (sulfate transporter), member 1 transporter SLC27A2 solute carrier family 27 (fatty acid transporter), member 2 transporter SLC27A5 solute carrier family 27 (fatty acid transporter), member 5 transporter SLC29A1 solute carrier family 29 (nucleoside transporters), member 1 transporter SLC2A2 solute carrier family 2 (facilitated glucose transporter), member 2 transporter SLC2A9 solute carrier family 2 (facilitated glucose transporter), member 9 transporter SLC31A1 solute carrier family 31 (copper transporters), member 1 transporter SLC35A3 solute carrier family 35 (UDP-N-acetylglucosamine (UDP-GlcNAc) transporter transporter), member A3 150

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) SLC35A3 solute carrier family 35 (UDP-N-acetylglucosamine (UDP-GlcNAc) transporter transporter), member A3 SLC37A4 solute carrier family 37 (glucose-6-phosphate transporter), member 4 transporter SLC38A3 solute carrier family 38, member 3 transporter SLC39A11 solute carrier family 39 (metal ion transporter), member 11 transporter SLC3A2 solute carrier family 3 (activators of dibasic and neutral amino acid transporter transport), member 2 SLC6A12 solute carrier family 6 (neurotransmitter transporter, betaine/GABA), transporter member 12 SLC6A13 solute carrier family 6 (neurotransmitter transporter, GABA), transporter member 13 Slco1a1 solute carrier organic anion transporter family, member 1a1 transporter SLCO1B3 solute carrier organic anion transporter family, member 1B3 transporter SLCO2B1 solute carrier organic anion transporter family, member 2B1 transporter SND1 staphylococcal nuclease and tudor domain containing 1 enzyme SNTB1 syntrophin, beta 1 (dystrophin-associated protein A1, 59kDa, basic unreported component 1) SOD1 superoxide dismutase 1, soluble enzyme SOD2 superoxide dismutase 2, mitochondrial enzyme SORD sorbitol dehydrogenase enzyme SPCS2 signal peptidase complex subunit 2 homolog (S. cerevisiae) unreported SPR (includes sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase) enzyme EG:20751) SQRDL sulfide quinone reductase-like (yeast) enzyme SRPR signal recognition particle receptor (docking protein) unreported SRPRB signal recognition particle receptor, B subunit unreported SRSF7 serine/arginine-rich splicing factor 7 unreported SSBP1 single-stranded DNA binding protein 1 unreported SSR1 signal sequence receptor, alpha unreported SSR3 signal sequence receptor, gamma (translocon-associated protein unreported gamma) SSR4 signal sequence receptor, delta unreported STAB2 stabilin 2 transmembrane receptor STOML2 stomatin (EPB72)-like 2 unreported STS steroid sulfatase (microsomal), isozyme S enzyme STT3A STT3, subunit of the oligosaccharyltransferase complex, homolog A enzyme (S. cerevisiae) STT3B STT3, subunit of the oligosaccharyltransferase complex, homolog B enzyme (S. cerevisiae) STX11 syntaxin 11 transporter SUCLG1 succinate-CoA ligase, alpha subunit enzyme SUCLG2 succinate-CoA ligase, GDP-forming, beta subunit enzyme SULT1A1 sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1 enzyme SUOX sulfite oxidase enzyme

151

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) SURF4 surfeit 4 unreported TAPBP TAP binding protein (tapasin) transporter TBL2 transducin (beta)-like 2 unreported TCIRG1 T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 enzyme subunit A3 TCP1 t-complex 1 unreported TDO2 tryptophan 2,3-dioxygenase enzyme TECR trans-2,3-enoyl-CoA reductase enzyme TEX264 testis expressed 264 unreported TFR2 transferrin receptor 2 transporter TGM1 transglutaminase 1 (K polypeptide epidermal type I, protein- enzyme glutamine-gamma-glutamyltransferase) TIMM50 translocase of inner mitochondrial membrane 50 homolog (S. phosphatase cerevisiae) TIMM8A translocase of inner mitochondrial membrane 8 homolog A (yeast) transporter TKT transketolase enzyme TLN1 talin 1 unreported TM7SF2 transmembrane 7 superfamily member 2 enzyme TM9SF2 transmembrane 9 superfamily member 2 transporter TM9SF4 transmembrane 9 superfamily protein member 4 transporter TMBIM6 transmembrane BAX inhibitor motif containing 6 unreported TMED10 transmembrane emp24-like trafficking protein 10 (yeast) transporter TMED2 transmembrane emp24 domain trafficking protein 2 transporter TMED4 transmembrane emp24 protein transport domain containing 4 transporter TMED5 transmembrane emp24 protein transport domain containing 5 unreported TMED7 transmembrane emp24 protein transport domain containing 7 transporter TMEM109 transmembrane protein 109 unreported TMEM111 transmembrane protein 111 unreported TMEM135 transmembrane protein 135 unreported TMEM14C transmembrane protein 14C unreported TMEM150A transmembrane protein 150A unreported TMEM19 transmembrane protein 19 unreported TMEM205 transmembrane protein 205 unreported TMEM214 transmembrane protein 214 unreported TMEM30A transmembrane protein 30A unreported TMEM33 transmembrane protein 33 unreported TMEM38B transmembrane protein 38B ion channel TMEM56 transmembrane protein 56 unreported TMEM97 transmembrane protein 97 unreported TMX1 thioredoxin-related transmembrane protein 1 enzyme TMX2 thioredoxin-related transmembrane protein 2 enzyme TOMM22 translocase of outer mitochondrial membrane 22 homolog (yeast) transporter

152

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) TPI1 triosephosphate isomerase 1 enzyme TPM1 (includes tropomyosin 1 (alpha) unreported EG:22003) TRABD TraB domain containing unreported TRAM1 translocation associated membrane protein 1 unreported TST thiosulfate sulfurtransferase (rhodanese) enzyme TTC35 tetratricopeptide repeat domain 35 unreported TTPA tocopherol (alpha) transfer protein transporter TUBA1B tubulin, alpha 1b unreported TUBA4A tubulin, alpha 4a unreported TUBB3 tubulin, beta 3 class III unreported TXN (includes thioredoxin enzyme EG:116484) TXNDC5 thioredoxin domain containing 5 (endoplasmic reticulum) enzyme UBA1 ubiquitin-like modifier activating enzyme 1 enzyme UFSP2 UFM1-specific peptidase 2 enzyme UGGT1 UDP-glucose glycoprotein glucosyltransferase 1 enzyme UGP2 UDP-glucose pyrophosphorylase 2 enzyme UGT1A1 UDP glucuronosyltransferase 1 family, polypeptide A1 enzyme UGT1A6 UDP glucuronosyltransferase 1 family, polypeptide A6 enzyme UGT1A9 UDP glucuronosyltransferase 1 family, polypeptide A9 enzyme (includes unreporteds) UGT2A3 UDP glucuronosyltransferase 2 family, polypeptide A3 enzyme UGT2B10 UDP glucuronosyltransferase 2 family, polypeptide B10 enzyme UGT2B15 UDP glucuronosyltransferase 2 family, polypeptide B15 enzyme UGT2B15 UDP glucuronosyltransferase 2 family, polypeptide B15 enzyme UGT2B17 UDP glucuronosyltransferase 2 family, polypeptide B17 enzyme UGT2B4 UDP glucuronosyltransferase 2 family, polypeptide B4 enzyme UGT3A2 UDP glycosyltransferase 3 family, polypeptide A2 unreported UGT3A2 UDP glycosyltransferase 3 family, polypeptide A2 unreported UOX urate oxidase, pseudogene enzyme UQCR10 ubiquinol-cytochrome c reductase, complex III subunit X enzyme UQCRB ubiquinol-cytochrome c reductase binding protein enzyme UQCRC1 ubiquinol-cytochrome c reductase core protein I enzyme UQCRC2 ubiquinol-cytochrome c reductase core protein II enzyme UQCRFS1 ubiquinol-cytochrome c reductase, Rieske iron-sulfur polypeptide 1 enzyme UQCRHL ubiquinol-cytochrome c reductase hinge protein-like enzyme UROC1 urocanase domain containing 1 enzyme VAPA VAMP (vesicle-associated membrane protein)-associated protein A, unreported 33kDa VCP valosin containing protein enzyme VDAC1 voltage-dependent anion channel 1 ion channel 153

Table A5. MAM Proteins Identified in Mouse Liver Symbol Entrez Gene Name Type(s) VDAC2 voltage-dependent anion channel 2 ion channel VDAC3 voltage-dependent anion channel 3 ion channel VKORC1 vitamin K epoxide reductase complex, subunit 1 enzyme VTN vitronectin unreported YWHAE tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, epsilon polypeptide YWHAG tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation unreported protein, gamma polypeptide ZADH2 zinc binding alcohol dehydrogenase domain containing 2 enzyme 1. Gene symbol as it appears in Uniprot database; 2. Gene name references by Entrez; 3. Protein type based on Gene Ontology annotation. “Unreported” protein types have no annotation.

154

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