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Title A small molecule dissociates the PAM motor from the TIM23 channel

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Author Cheung, Tsz Mei Jesmine

Publication Date 2017

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A small molecule dissociates the PAM motor from the TIM23 channel

A dissertation submitted in partial satisfaction of the

requirements for the degree Doctor of Philosophy

in Biochemistry and Molecular Biology

by

Tsz Mei Jesmine Cheung

2017

© Copyright by

Tsz Mei Jesmine Cheung

2017

ABSTRACT OF THE DISSERTATION

A small molecule dissociates the PAM motor from the TIM23 channel

by

Tsz Mei Jesmine Cheung

Doctor of Philosophy in Biochemistry and Molecular Biology

University of California, Los Angeles, 2017

Professor Carla Marie Koehler, Chair

Mitochondrial dysfunction is a contributing factor in various neural and muscular degenerative diseases. Modulation of mitochondrial protein pathways can have regulatory effects on mitochondrial function. Conventional methods using yeast genetics and RNAi approaches are powerful, but limited because RNAi requires several days and during this time, cells can alter metabolism. Therefore, we have developed several approaches to identify small molecule modulators for mitochondrial protein translocation.

This study utilizes a high-throughput small molecule screening approach to identify modulators of the TIM23 import pathway, which transports proteins across the inner membrane and into the matrix. This strategy will be useful in generating a toolbox of small molecule modulators for mitochondrial translocation to understand how defects in mitochondrial assembly contribute to disease.

ii To this end, we have identified and characterized a small molecule probe MitoBloCK-20

(MB-20) that binds specifically to Tim17 in the TIM23 complex. MB-20 inhibits the import of substrates that use the TIM23 translocon, but not the TIM22 translocon. MB-20 seems to target

Tim17, because Tim17p, but not Tim23p and the PAM complex, was resistant to protease in the presence of MB-20. In pull-down experiments with His-tagged components, the interaction between Tim17 and Tim23 was stabilized but Tim44, Pam18 and Pam16 of the motor precipitated, suggesting the interaction between Tim17/Tim23 and the PAM complex was disrupted. Based upon biochemical analysis, MB-20 seems to target Tim17 and suggests that

Tim17 plays a critical role in coordinating activities between the channel and the import motor.

This modulator also showed a critical role for mitochondria in development of the zebrafish neural system.

iii The dissertation of Tsz Mei Jesmine Cheung is approved.

Catherine F. Clarke

Alexander M. Van der Bliek

Carla Marie Koehler, Committee Chair

University of California, Los Angeles

2017

iv TABLE OF CONTENTS

List of Figures Acknowledgements Vita

Chapter 1. Introduction – High-throughput screening methods to develop small molecule probes targeting mitochondria ...... 1 Chapter 2. Discovery of MB-20, a small molecule modulator of the Tim23 channel ...... 39 Chapter 3. MB-20 reveals the role of Tim17 in stabilizing the TIM23/PAM complex ...... 69

v LIST OF FIGURES

Chapter 1. Figure 1-1. The diverse roles of mitochondria ...... 8 Figure 1-2. Integration of different organelles in cells ...... 9 Figure 1-3. Main components of the mitochondrial import system ...... 10 Figure 1-4. The TIM23 and TIM22 import pathways ...... 11 Figure 1-5. Design of the small molecule screen targeting the TIM23 pathway ...... 12 Figure 1-6. Hits from the screen grouped by similar scaffold on the structure ...... 13

Chapter 2 Figure 2-1. Respiration assay of the hits ...... 48 Figure 2-2. Disc3(5) assay of the hits...... 49 Figure 2-3. Membrane permeability assay of the hits ...... 50 Figure 2-4. Illustration of the rationale of using in vitro import to identify the target complex of the small molecules ...... 51 Figure 2-5. Examples of the results obtained from the in vitro import assay with different small molecules ...... 52 Figure 2-6. In vitro import assay with mitochondria and mitoplast ...... 53 Figure 2-7. Results obtained from the in vitro import assay of Cytb2(167)-DHFR and Cytb2(167)A63P-DHFR with EN30, A01 and LS59 ...... 54

Chapter 3. Figure 3-1. Structure and MIC50 of MB-20 and MB-20.1 ...... 99 Figure 3-2. MB-20 targets the TIM23 complex ...... 101 Figure 3-3. MB-20 targets Tim17 specifically ...... 102 Figure 3-4. MB-20 disrupts the Tim44-Tim17 interaction and destabilizes the PAM motor ...... 103 Figure 3-5. MB-20 does not induce mitophagy ...... 105 Figure 3-6. MB-20 treatment induces LC3 lipidation and LC3 tubules formation ...... 108

Figure 3-7. MB-20 treatment in zebrafish showed aberrant accumulation of neuronal phenotype ...... 110 Figure S3-1 MB-20 does not impair general mitochondrial functions ...... 112 Figure S3-2 MB-20 does not affect mitochondrial membrane potential in HeLa cells .... 114 Figure S3-3 Mulan KO cells exhibit the same phenotype as WT ...... 115 Figure S3-4 LC3 does not localize to the mitochondria or to the ER ...... 117 Figure S3-5 LC3 tubules formation is also observed in MEF cells and MEF cells stably expressing Parkin-flag ...... 118 Figure S3-6 MB-20 does not affect mitophagy ...... 119

vi LIST OF TABLES

Chapter 1 Table 1-1. Small molecules purchased for secondary screen ...... 31

Chapter 2 Table 2-1. Summary of the results from the respiration assay ...... 54 Table 2-2. List of candidates remained after the secondary screen ...... 55 Table 2-3. Summary of the in vitro import assay of the hits ...... 56

vii ACKNOWLEDGEMENTS

It was truly an amazing and challenging journey. Not only did I receive an incredible

scientific training at a renowned university, I have also made a lot of friendship that would last a

lifetime. First, I would like to thank my advisor, Dr. Carla Koehler, for the opportunity to work

in a lab. Carla was a great mentor and I am very grateful for her support for all these years. I

would also like to extend my gratitude to my committee members, Dr. Alex van der Bliek, Dr.

Michael A Teitell, Dr. Steven G Clarke, and Dr. Catherine Clarke for their advice and guidance

over the years.

Thank you past and present members of the Koehler lab: Non Miyata, Meghan Johnson,

Colin Douglas, Eric Torres, Eriko Shimada, Vivian Zhang, Jennifer Ngo, Gary Schmorgan,

Deepa Dabir, Jisoo Han, Christina Jayson, Thao Vi, Michael Conti, Matthew Maland and Janos

Steffen. Thank you all for the scientific discussions, random discussions, whining, teasing, happy hours, basically everything that happened for the last couple years. Graduate school would not

have been the same without any of you.

Last but not least, I would not have survived my graduate career without my family. Aunt

Anna and Uncle Steve, without your generous support, I would have been able to come to the

States for my education, and I would not be able to achieve what I achieved today. There is no

word that can describe my gratitude. Mom and Dad, thank you so much for everything you have

done for me. I am not very good at expressing my feelings, but I love both of you more than I

could ever tell you and I hope you are proud of me. My little brother Jeff, thank you for taking

care of mom and dad when I can’t and I am very grateful that I can do what I wanted to do

because I know you are there taking care of them. Finally, I would like to thank my husband (a

viii recent upgrade), Ramsay Macdonald, for being there with me through this journey. Thank you for putting up with me all these years and more!

ix Chapter 1 describes the logistic of the high-throughput screening for the identification of

small molecule modulators in the TIM23 protein import pathway.

Chapter 2 highlights the characterization of the screening hits. From the 64 potential hits,

I narrowed the list down using different approaches and focused on one small molecule, named

MitoBloCK-20 (MB-20).

Chapter 3 is a version of a manuscript in preparation to be authored by myself, Christina

Jayson, Thao Vi, and Koehler CM. Small molecule modulator MB-20 reveals the role of Tim17

in stabilizing the complex PAM complex formation.

x VITA

2006-2008 De Anza College Cupertino, California

2010 B.S. in Chemical Biology University of California, Berkeley

2010-2011 Research Assistant Division of Biological Sciences University of California, San Diego

2011-2012 Staff Research Associate Department of Biological Chemistry University of California, Los Angeles

2012-2017 Graduate Student Researcher Department of Chemistry and Biochemistry University of California, Los Angeles

2012-2017 Teaching Assistant Department of Chemistry and Biochemistry University of California, Los Angeles

PRESENTATIONS AND PUBLICATIONS

Shen QF, Yamano KJ, Head B, Kawajiri SH, Cheung TM Jesmine, Wang CX, Cho JH, Hattori NT, Youle RJ, van der Bliek A (2014). Mutations in Fis1 disrupt orderly disposal of defective mitochondria. Mol. Biol. Cell 25:145-159.

TM Jesmine Cheung, Christina Jayson, Thao Vi and Carla Koehler (2016). A small molecules disrupts Tim44 interactions with the TIM channel in mitochondrial protein import. Presented at Gordon Research Conference: Protein Translocation Across Membranes, Galveston, Texas.

TM Jesmine Cheung, Christina Jayson, Thao Vi and Carla Koehler (2017). A small molecule dissociates the PAM motor from the TIM23 channel. Presented at Keystone Symposia: Mitochondria Communication. Taos, New Mexico.

HONORS AND AWARDS

2015 Hanson-Dow Teaching Assistant Award Department of Chemistry and Biochemistry University of California, Los Angeles

xi

2006-2008 Dean’s Honors List De Anza College

xii Chapter 1: Introduction – High-throughput screening methods to develop small molecule probes targeting mitochondria

Introduction

Mitochondria are known as the power house of the cells, generating ATP to support the

vast range of activities in cells that are essential for life. As scientists explore these fascinating,

yet complicated organelles, we found that they are much more than we once thought1.

Mitochondria also perform numerous vital functions in cells, including activation of apoptosis2,3, maintenance of calcium homeostasis4,5 and management of oxidative stress6,7 (Figure 1-1).

Besides the heterogeneity of mitochondrial functions, mitochondria are found to be

involved and connected with other components of the cell8 (Figure 1-2). These organelles can be

considered as an integral part of cellular signaling and cell communication and survival9–13. The

mitochondrial outer membrane plays a crucial role in communicating with other cellular

compartments and signaling under different cellular conditions14–16. The emerging network of

mitochondria-organelle contacts revealed pivotal mitochondrial functions for proper cell

functions and survival. The best-studied mitochondrial organelle contact site, the ER- mitochondria encounter structure (ERMES) has been linked to many vital processes including mitochondrial fission, inheritance, mitophagy and phospholipid transport17–19. All these

regulations and processes are detrimental in maintaining the quality of mitochondria and in

restoration of proper mitochondrial functions. For instance, bidirectional signaling between the

nucleus and mitochondria regulates the nuclear transcriptional response triggered by

accumulation of misfolded proteins within the mitochondria20–22 (UPRmt). If misfolded proteins

1 accumulate to a level that exceeds the capacity of the UPRmt, autophagy of mitochondria

(mitophagy) follows to mitigate the proteotoxic stress23–26.

As highlighted above, mitochondria are intimately linked to a wide range of processes

that are important to the cells. Dysfunctions of these processes contribute to various muscular

and neurodegenerative diseases27. Thus, enormous endeavor from the scientific communities has

been put to better understand the mechanism of the different functions of mitochondria, with the

goal to gain insight for developing effective mitochondria-targeted therapies.

The mitochondrial protein import system

Mitochondria retain features of their bacterial ancestors, including a genome28,29.

However, 99% of the mitochondrial proteins are synthesized in the cytosol containing different

targeting and sorting information and must be imported across one or both mitochondrial

membranes and transported to their functional destination30–33. Mitochondria are complex

organelles that contain different compartments, the outer membrane (OM), the inner membrane

(IM), the intermembrane space (IMS) between the OM and IM, and the mitochondrial matrix

inside the IM. Mitochondria have developed a sophisticated translocation system for the import

of nuclear-coded proteins (Figure 1-3). The outer membrane contains the TOM (translocase of

the outer membrane) complex for translocation across the outer membrane34,35 and the SAM

(sorting and assembly machinery) complex for assembly of outer membrane proteins36–39. At the

inner membrane, the TIM23 (translocase of the inner membrane) complex mediates the import of

precursor proteins with a typical N-terminal targeting sequence40,41, and the TIM22 complex

mediates the import of carrier proteins42–44.

Almost all mitochondrial proteins are imported through the TOM complex45 (Figure 1-4).

The central component of TOM is Tom40p, which has a β-barrel structure that forms the main 2 channel for protein to cross the outer membrane35,46. In the complex, Tom20p, Tom22p, Tom70p

and Tom5p are the receptors, recognizing the precursor targeting sequence, guiding the

precursors to the TOM channel34,47. For proteins with an N-terminal targeting presequence,

import is then mediated by the TIM23 complex41,48 (Figure 1-4), which contains Tim17p,

Tim23p, Tim50p and Tim21p, and it is associated with the PAM (protein-associated translocation motor) complex. The translocation motor consists of Tim44p, mitochondrial Hsp70

(mHsp70), the nucleotide exchange factor Mge1p, and J-proteins Pam16p and Pam18p49–51. The

TIM23 import pathway is membrane potential (∆ψ) dependent. For the matrix-targeted protein,

targeting sequence is cleaved by the matrix processing protease (MPP) once it is translocated

into the matrix40,52. For inner-membrane-sorted pre-proteins, they are cleaved twice, first by

MPP, then by the inner-membrane peptidase53 (IMP).

The carrier proteins and import components utilize the TIM22 import pathway (Figure 1-

4). Most carrier proteins are located in the inner membrane and are synthesized without a cleavable presequence. This pathway is specific for the import of the mitochondrial carrier family, including the ADP/ATP carrier (AAC), phosphate carrier (PiC), and the dicarboxylate

carrier (DiC) and also the inner membrane translocation components Tim17p, Tim22p and

Tim23p54–56. Components of the TIM22 translocation system include the small Tim proteins

(Tim8p, Tim9p, Tim10p, Tim12p and Tim13p)57–59 and the membrane components58,60 (Tim18p,

Tim22p and Tim54p). The small Tim proteins function as chaperones. Tom40 of the TOM

complex recruits small TIM chaperones of the intermembrane space to the channel exit,

escorting the substrates to the insertion complex, which are then inserted into the inner

membrane of the mitochondria46,61.

3 Mitochondrial diseases associated with defective import system

Dysfunctions in mitochondrial import machineries are found to be associated with

numerous muscular and neurodegenerative diseases. The human deafness dystonia syndrome

(MTS/DFN-1) is a recessive, X-linked neurodegenerative disorder characterized by progressive

sensorineural deafness, cortical blindness, dystonia, dysphagia, and paranoia62. It is caused by

truncation or deletion of DDP1/Tim8, which is involved in the import of mitochondrial carrier

proteins63. Similarly, mutations in other mitochondrial import components directly cause

mitochondrial diseases including chaperone Hsp60 in hereditary spastic paraplegia64,65 and

atypical mitochondria disease involving multisystem failure and DNAJC19 in dilated

cardiomyopathy with ataxia66–68. Moreover, the TOM and TIM translocons have recently been associated with Parkinson disease24,69 and Alzheimer’s disease70–74, and alteration of import

pathways may prevent disease progression. Although there exist numerous mitochondrial

diseases affecting a lot of people, due to inadequate tools and approaches, it has been difficult to

make further progress in studying mitochondrial protein machineries and import process.

High-throughput screening approaches to discover and develop small molecules probes

As most of the transport proteins are essential for viability75, it has been challenging to

study how defects in mitochondrial protein translocation contribute to diseases using

conventional genetic and biochemical approaches. Genetic knockout of essential proteins cannot

be generated. Temperature-sensitive mutants is an alternative approach to study essential

proteins, however, this method in studying the molecular mechanisms are very limited and often

have secondary effect or inefficient. Therefore, there is a critical need to develop small molecule

4 modulators as tools to dissect how mitochondrial dysfunction contributes to the characterization

of diseases.

The overall research goal is to develop verified chemical probes that alter mitochondrial

assembly and use these agents in model systems to shed lights on the mechanistic detail on the

mitochondrial import system. In our lab, we have successfully identify and characterize small

molecules that targets different part of the import machineries, including MitoBloCK-1 (MB-1), which is a Tim9-Tim10 complex inhibitor76 and MitoBloCK-6 (MB-6), which is an Erv1

inhibitor77.

As part of this goal, this project focuses on small molecules inhibitors that modulate the

TIM23 import pathway, using saccharomyces cerevisiae (budding yeast) as a model. As proof of

principle, three small molecules, MitoBloCK-10 (MB-10), MitoBloCK-11 (MB-11), DECA have been shown to inhibit protein translocation in yeast, and results show that MB-10 targets Tim44 of the TIM23 translocon78 and MB-11 potentially blocks Tom70 of the TOM translocon.

Design of small molecule screen targeting the TIM23 pathway

The mitochondrial import pathways are highly conserved between yeast and mammalian

cells, and their overall structures are very similar with 25% to 30% sequence homology79.

Therefore, we used yeast as our model organism for the high-throughput small molecule screen

as it is an easier organism to manipulate.

Saccharomyces cerevisiae contains several drug pumps, which are not ideal in small molecule screening because of small molecule efflux. Therefore, two drug pumps were knocked out to increase the intracellular concentration of the small molecule (Figure 1-5 A). Su9-Ura3 that is imported to the matrix was integrated into this yeast strain (GA74 ∆PDR5 ∆SNQ2). High-

5 throughput screening of import inhibition was performed by a growth assay. Ura3 is an enzyme

that is involved in the uracil biosynthetic pathway and normally functions in the cytosol so that

yeast can grow in the absence of uracil. In this strain, the fusion protein Su9-Ura3 was

genetically integrated into the yeast genome, therefore Ura3 is targeted to the mitochondrial

matrix by the N-terminal mitochondrial targeting sequence (Su9), and the strain failed to grow in

media lacking uracil (Figure 1-5 B). However, growth was restored by the subset of small

molecules that impaired protein import using the TIM23 pathway (Figure 1-5 C).

Characterization of hits obtained from the high-throughput screens

Results showed a total of 64 hits from the small molecule libraries we screened.

Unfortunately, it would be impossible to further investigate all 64 compounds. Therefore, based

on similar scaffold on the structure, the hits were grouped into 11 different groups (Figure 1-6).

Small molecules can be quite expensive, and sometimes unavailable due to difficulties regarding

their synthesis. Therefore, I picked a few molecules from each group depending on the price,

availability and the growth rate from the high-throughput screening. A total of 25 small

molecules were selected and 24 were purchased (no molecule was selected from group 4 due to price and unavailability) for secondary screening (Table 1-1).

Concluding remarks

Mitochondria dysfunction has been implicated in increasing number of diseases and

cellular processes. As scientists constantly uncover new and exciting functions of mitochondria,

we anticipate that the field of mitochondria studies will continue to expand. In addition, as

technology advances, including imaging techniques, mass spectrometer analysis, interactome

6 studies, these can hopefully help scientists to get a better understanding of these fascinating, yet complicated organelles.

Mitochondria are mysterious, there are still a lot of unknowns about these organelles, and using the high-throughput screening approach, we will be able to develop a toolbox consisting different small molecule probes to study mitochondrial functions. Hopefully, that can shed some lights into the mechanisms and can lead to drug discoveries for mitochondrial diseases.

7 Figures

Figure 1-180. The diverse roles of mitochondria. (A) Function of mitochondria under normal cell conditions. (B) Mitochondria response during different stress conditions.

8

Figure 1-28. Integration of different organelles in cells. Organelles in cell are organized through a network of contact sites. ER-mitochondria-peroxisome contact is shown in box 1.

Nucleus-vacuole contact is shown in box 2. Mitochondria-vacuole contact is shown in box 3.

9

Figure 1-3. Main components of the mitochondrial import system. There are five main

complexes that are important for protein translocation across mitochondrial membranes.

Translocase of Outer Membrane (TOM) and Sorting and Assembly Machinery (SAM) are

located in the outer membrane of the mitochondria. Translocase of the Inner Membrane (TIM22

and TIM23) and Oxidase Assembly Translocase (OXA) are located in the inner membrane of the mitochondria.

10 Figure 1-481. The TIM23 and TIM22 import pathways. Mitochondrial proteins are synthesized as precursor proteins in the cytosol. N-terminal targeting sequence of the precursor proteins is recognized by the Translocase of Outer Membrane (TOM) complex and then to the matrix through the Translocase of Inner Membrane (TIM23) translocon. The targeting sequence was then cleaved by the mitochondrial processing peptidase (MPP) in the matrix. Carrier proteins also pass through the TOM complex and then transferred to the TIM22 complex by the

Tim9-Tim10 complex in the intermembrane space. The protein is then inserted into the inner membrane of the mitochondria.

11

Figure 1-5. Design of the small molecule screen targeting the TIM23 pathway. (A) Two drug pumps were knocked out from the yeast strain to maintain intracellular concentration of the small molecule. (B) Su9, an N-terminal targeting sequence was fused with the gene URA3 and integrated into the yeast genome. (C) By inhibiting mitochondrial import, Su9-URA3 can stay in the cytosol.

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Figure 1-6. Hits from the screen grouped by similar scaffold on the structure. The hits results from the screen described in Figure 1-5 were grouped according to their structure scaffolding.

30 Group Name Survival Rate Short Name Group 1 AEM 11113437 35.89% AN17 Group 1 AEM 11113384 30.70% AN14 Group 1 ASN 04428878 49.37% AN18 Group 2 ASN 03775847 43.61% B02 Group 2 ASN 04188696 35.96% A05 Group 2 ASN 05303721 38.49% A06 Group 3 6639260 72.63% EN30 Group 3 5145909 43.17% CB39 Group 3 Gossypol 37.40% ------Group 4 Syrosingopine CDD-46925 * 37.26% ------Group 5 F2536-1579 23.16% LS59 Group 5 F3226-0462 25.26% LS52 Group 6 SYN 15590170 37.76% AN60 Group 7 F2624-0032 97.89% LS72 Group 7 SYN 15446633 34.67% AN73 Group 7 T5577396 46.29% EN76 Group 7 T5591571 39.58% EN71 Group 8 Benzethonium chloride 50.20% ------Group 9 F3007-0019 24.21% LS99 Group 10 T6008280 39.36% EN108 Group 11 ASN 03886845 32.47% AN114 Group 11 ASN 04363508 32.11% A01 Group 11 SYN 15633705 38.34% AN110 Group 11 T5882433 34.39% EN113 Group 11 T5932896 42.80% EN116

Table 1-1. Small Molecules purchased for secondary screen. A total of 25 small molecules were selected based on survival rates and structural scaffold.

*This is a natural compound, and we did not order it because it was too expensive.

31 References

1. McBride, H. M., Neuspiel, M. & Wasiak, S. Mitochondria: More Than Just a Powerhouse. Curr. Biol. 16, 551–560 (2006). 2. Kroemer, G. & Reed, J. C. Mitochondrial control of cell death. Nat Med 6, 513–519 (2000). 3. Newmeyer, D. D. & Ferguson-Miller, S. Mitochondria: Releasing power for life and unleashing the machineries of death. Cell 112, 481–490 (2003). 4. Contreras, L., Drago, I., Zampese, E. & Pozzan, T. Mitochondria: The calcium connection. Biochim. Biophys. Acta - Bioenerg. 1797, 607–618 (2010). 5. Rizzuto, R., De Stefani, D., Raffaello, A. & Mammucari, C. Mitochondria as sensors and regulators of calcium signalling. Nat. Rev. Mol. Cell Biol. 13, 566–578 (2012). 6. Ott, M., Gogvadze, V., Orrenius, S. & Zhivotovsky, B. Mitochondria, oxidative stress and cell death. Apoptosis 12, 913–922 (2007). 7. Lenaz, G. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta - Bioenerg. 1366, 53–67 (1998). 8. Murley, A. & Nunnari, J. The Emerging Network of Mitochondria-Organelle Contacts. Mol. Cell 61, 648–653 (2016). 9. Karbowski, M., Norris, K. L., Cleland, M. M., Jeong, S.-Y. & Youle, R. J. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006). 10. Kuznetsov, A. V., Janakiraman, M., Margreiter, R. & Troppmair, J. Regulating cell survival by controlling cellular energy production: Novel functions for ancient signaling pathways? FEBS Lett. 577, 1–4 (2004). 11. Kuznetsov, A. V et al. Survival signaling by C-RAF: mitochondrial reactive oxygen species and Ca2+ are critical targets. Mol. Cell. Biol. 28, 2304–2313 (2008). 12. Riedl, S. J. & Salvesen, G. S. The apoptosome: signalling platform of cell death. Nat. Rev. Mol. Cell Biol. 8, 405–413 (2007). 13. Youle, R. J. CELL BIOLOGY: Cellular Demolition and the Rules of Engagement. Science (80-. ). 315, 776–777 (2007). 14. Kotiadis, V. N., Duchen, M. R. & Osellame, L. D. Mitochondrial quality control and communications with the nucleus are important in maintaining mitochondrial function and cell health. Biochim. Biophys. Acta - Gen. Subj. 1840, 1254–1265 (2014). 15. van Vliet, A. R., Verfaillie, T. & Agostinis, P. New functions of mitochondria associated membranes in cellular signaling. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 2253– 2262 (2014). 16. Meisinger, C. et al. Contents, Ed. Board + Forthc. articles. Trends Biochem. Sci. 30, i (2005). 17. Csordás, G. et al. Structural and functional features and significance of the physical

32 linkage between ER and mitochondria. J. Cell Biol. 174, 915–921 (2006). 18. AhYoung, A. P. et al. Conserved SMP domains of the ERMES complex bind phospholipids and mediate tether assembly. Proc. Natl. Acad. Sci. 112, E3179–E3188 (2015). 19. Murley, A. et al. ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast. Elife 2013, 1–16 (2013). 20. Sun, N., Youle, R. J. & Finkel, T. The Mitochondrial Basis of Aging. Mol. Cell 61, 654– 666 (2016). 21. Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411–4419 (2002). 22. Martinus, R. D. et al. Selective Induction of Mitochondrial Chaperones in Response to Loss of the Mitochondrial Genome. Eur. J. Biochem. 240, 98–103 (1996). 23. Eiyama, A. & Okamoto, K. PINK1/Parkin-mediated mitophagy in mammalian cells. Curr. Opin. Cell Biol. 33, 95–101 (2015). 24. Pickrell, A. M. & Youle, R. J. The roles of PINK1, Parkin, and mitochondrial fidelity in parkinson’s disease. Neuron 85, 257–273 (2015). 25. Jin, S. M. & Youle, R. J. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria. Autophagy 9, 1750–1757 (2013). 26. Pellegrino, M. W. & Haynes, C. M. Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial infection. BMC Biol. 13, 22 (2015). 27. Nunnari, J. & Suomalainen, A. Mitochondria: In sickness and in health. Cell 148, 1145– 1159 (2012). 28. Dyall, S. D. Ancient Invasions: From Endosymbionts to Organelles. Science (80-. ). 304, 253–257 (2004). 29. Gray, M. W., Gray, M. W., Burger, G. & Lang, B. F. Mitochondrial Evolution. 1476, 1– 16 (2008). 30. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing Mitochondrial Proteins: Machineries and Mechanisms. Cell 138, 628–644 (2009). 31. Koehler, C. M. New Developments in Mitochondrial Assembly. Annu. Rev. Cell Dev. Biol. 20, 309–335 (2004). 32. Sickmann, A. et al. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. 100, 13207–13212 (2003). 33. Taylor, S. W. et al. Characterization of the human heart mitochondrial proteome. J. Proteome Res. 21, 281–286 (2003). 34. Mokranjac, D. & Neupert, W. Cell biology: Architecture of a protein entry gate. Nature 528, 201–202 (2015). 35. Hill, K. et al. Tom40 forms the hydrophilic channel of the mitochondrial import pore for preproteins. Nature 395, 516–521 (1998). 36. Gentle, I., Gabriel, K., Beech, P., Waller, R. & Lithgow, T. The Omp85 family of proteins 33 is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol. 164, 19–24 (2004). 37. Kozjak, V. et al. An essential role of Sam50 in the protein sorting and assembly machinery of the mitochondrial outer membrane. J. Biol. Chem. 278, 48520–48523 (2003). 38. Paschen, S. a. et al. Evolutionary conservation of biogenesis of b -barrel membrane proteins. Nature 426, 862–866 (2003). 39. Wiedemann, N. & Pfanner, N. Mitochondrial Machineries for Protein Import and Assembly. Annu. Rev. Biochem. 86, annurev-biochem-060815-014352 (2017). 40. Hawlitschek, G. et al. Mitochondrial protein import: Identification of processing peptidase and of PEP, a processing enhancing protein. Cell 53, 795–806 (1988). 41. Chacinska, A. et al. Mitochondrial presequence translocase: Switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817–829 (2005). 42. Koehler, C. M., Merchant, S. & Schatz, G. How membrane proteins travel across the mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428–432 (1999). 43. Truscott, K. N., Brandner, K. & Pfanner, N. Mechanisms of protein import into mitochondria. Curr. Biol. 13, 326–337 (2003). 44. Pfanner, N. Mitochondrial import: crossing the aqueous intermembrane space. Curr. Biol. 8, R262-5 (1998). 45. Ahting, U. et al. The Tom Core Complex. J. Cell Biol. 147, 959–968 (1999). 46. Shiota, T. et al. Molecular architecture of the active mitochondrial protein gate. Science (80-. ). 349, 1544–1548 (2015). 47. Abe, Y. {[Structural} basis of presequence recognition by the mitochondrial protein import receptor Tom20]. Fukuoka Igaku Zasshi 92, 266–271 (2001). 48. Roise, D., Horvath, S. J., Tomich, J. M., Richards, J. H. & Schatz, G. A chemically synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic helix and perturb natural and artificial phospholipid bilayers. EMBO J. 5, 1327–34 (1986). 49. Popov-Čeleketić, D., Mapa, K., Neupert, W. & Mokranjac, D. Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria. EMBO J. 27, 1469–1480 (2008). 50. Ting, S. Y., Schilke, B. A., Hayashi, M. & Craig, E. A. Architecture of the TIM23 inner mitochondrial translocon and interactions with the matrix import motor. J. Biol. Chem. 289, 28689–28696 (2014). 51. Kang, P. J. et al. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348, 137–143 (1990). 52. Vögtle, F. N. et al. Global Analysis of the Mitochondrial N-Proteome Identifies a Processing Peptidase Critical for Protein Stability. Cell 139, 428–439 (2009). 53. Mossmann, D., Meisinger, C. & Vögtle, F. N. Processing of mitochondrial presequences. Biochim. Biophys. Acta - Gene Regul. Mech. 1819, 1098–1106 (2012). 54. Davis, a J., Ryan, K. R. & Jensen, R. E. Tim23p contains separate and distinct signals for

34 targeting to mitochondria and insertion into the inner membrane. Mol. Biol. Cell 9, 2577– 2593 (1998). 55. Endres, M., Neupert, W. & Brunner, M. Transport of the ADP / ATP carrier of mitochondria from the TOM complex to the TIM22 · 54 complex. 18, 3214–3221 (1999). 56. Leuenberger, D., Bally, N. A., Schatz, G. & Koehler, C. M. Different import pathways through the mitochondrial intermembrane space for inner membrane proteins. EMBO J. 18, 4816–4822 (1999). 57. Webb, C. T., Gorman, M. A., Lazarou, M., Ryan, M. T. & Gulbis, J. M. Crystal structure of the mitochondrial chaperone TIM9???10 reveals a six-bladed ??-propeller. Mol. Cell 21, 123–133 (2006). 58. Gebert, N. et al. Assembly of the three small Tim proteins precedes docking to the mitochondrial carrier translocase. EMBO Rep. 9, 548–554 (2008). 59. Lionaki, E. et al. The essential function of Tim12 in vivo is ensured by the assembly interactions of its C-terminal domain. J. Biol. Chem. 283, 15747–15753 (2008). 60. Paschen, S. A. The role of the TIM8-13 complex in the import of Tim23 into mitochondria. EMBO J. 19, 6392–6400 (2000). 61. Murphy, M. P., Leuenberger, D., Curran, S. P., Oppliger, W. & Koehler, C. M. The Essential Function of the Small Tim Proteins in the TIM22 Import Pathway Does Not Depend on Formation of the Soluble 70-Kilodalton Complex. Mol. Cell. Biol. 21, 6132– 6138 (2001). 62. Tranebjaerg, L. et al. A new X linked recessive deafness syndrome with blindness, dystonia, fractures, and mental deficiency is linked to Xq22. J. Med. Genet. 32, 257–263 (1995). 63. Koehler, C. M., Leuenberger, D., Merchant, S., Renold, A. & Junne, T. Human deafness dystonia syndrome is a mitochondrial disease. Cell Biol. 96, 2141–2146 (1999). 64. Magen, D. et al. Mitochondrial Hsp60 Chaperonopathy Causes an Autosomal-Recessive Neurodegenerative Disorder Linked to Brain Hypomyelination and Leukodystrophy. Am. J. Hum. Genet. 83, 30–42 (2008). 65. Christensen, J. H. et al. Inactivation of the hereditary spastic paraplegia-associated Hspd1 gene encoding the Hsp60 chaperone results in early embryonic lethality in mice. Cell Stress Chaperones 15, 851–863 (2010). 66. Richter-Dennerlein, R. et al. DNAJC19, a mitochondrial cochaperone associated with cardiomyopathy, forms a complex with prohibitins to regulate cardiolipin remodeling. Cell Metab. 20, 158–171 (2014). 67. Al Teneiji, A., Siriwardena, K., George, K., Mital, S. & Mercimek-Mahmutoglu, S. Progressive Cerebellar Atrophy and a Novel Homozygous Pathogenic DNAJC19 Variant as a Cause of Dilated Cardiomyopathy Ataxia Syndrome. Pediatr. Neurol. 62, 58–61 (2016). 68. Ojala, T. et al. New mutation of mitochondrial DNAJC19 causing dilated and noncompaction cardiomyopathy, anemia, ataxia, and male genital anomalies. Pediatr. Res. 72, 432–437 (2012).

35 69. Lazarou, M., Jin, S. M., Kane, L. A. & Youle, R. J. Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment and Activation of the E3 Ligase Parkin. Dev. Cell 22, 320–333 (2012). 70. Manczak, M. et al. Mitochondria are a direct site of A beta accumulation in Alzheimer’s disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum. Mol. Genet. 15, 1437–49 (2006). 71. Mossmann, D. et al. Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition of Preprotein Maturation. Cell Metab. (2014). doi:10.1016/j.cmet.2014.07.024 72. Gottschalk, W. K. et al. HHS Public Access. 1, 1–25 (2015). 73. Devi, L. Accumulation of Amyloid Precursor Protein in the Mitochondrial Import Channels of Human Alzheimer’s Disease Brain Is Associated with Mitochondrial Dysfunction. J. Neurosci. 26, 9057–9068 (2006). 74. Pagani, L. & Eckert, A. Amyloid-Beta Interaction with Mitochondria. Int. J. Alzheimers. Dis. 2011, 1–12 (2011). 75. Baker, K. P. & Schatz, G. Mitochondrial proteins essential for viability mediate protein import into yeast mitochondria. Nature 349, 205–8 (1991). 76. Hasson, S. a et al. Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation. Proc. Natl. Acad. Sci. U. S. A. 107, 9578–9583 (2010). 77. Dabir, D. V et al. A small molecule inhibitor of redox-regulated protein translocation into mitochondria. Dev. Cell 25, 81–92 (2013). 78. Miyata, N. et al. Adaptation of a genetic screen reveals an inhibitor for mitochondrial protein import component Tim44. J. Biol. Chem. 292, 5429–5442 (2017). 79. Hoogenraad, N. J., Ward, L. A. & Ryan, M. T. Import and assembly of proteins into mitochondria of mammalian cells. Biochim. Biophys. Acta 1592, 97–105 (2002). 80. Kuznetsov, A. V. & Margreiter, R. Heterogeneity of mitochondria and mitochondrial function within cells as another level of mitochondrial complexity. Int. J. Mol. Sci. 10, 1911–1929 (2009). 81. Bolender, N., Sickmann, A., Wagner, R., Meisinger, C. & Pfanner, N. Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep. 9, 42–49 (2008). 82. van der Laan, M., Schrempp, S. G. & Pfanner, N. Voltage-coupled conformational dynamics of mitochondrial protein-import channel. Nat. Struct. Mol. Biol. 20, 915–917 (2013). 83. Schulz, C., Schendzielorz, A. & Rehling, P. Unlocking the presequence import pathway. Trends Cell Biol. 25, 265–275 (2015). 84. Meinecke, M. et al. Inner Membrane. 653, 2003–2006 (2006). 85. Lytovchenko, O. et al. Signal recognition initiates reorganization of the presequence translocase during protein import. EMBO J. 32, 886–898 (2013). 86. Schulz, C. & Rehling, P. Remodelling of the active presequence translocase drives motor- dependent mitochondrial protein translocation. Nat. Commun. 5, 1–9 (2014).

36 87. Wrobel, L., Sokol, A. M., Chojnacka, M. & Chacinska, A. The presence of disulfide bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein translocase assembly. Sci. Rep. 6, 27484 (2016). 88. Martinez-Caballero, S., Grigoriev, S. M., Herrmann, J. M., Campo, M. L. & Kinnally, K. W. Tim17p regulates the twin pore structure and voltage gating of the mitochondrial protein import complex TIM23. J. Biol. Chem. 282, 3584–3593 (2007). 89. Ramesh, A. et al. A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import. J. Cell Biol. 214, 417–431 (2016). 90. Rainbolt, T. K., Atanassova, N., Genereux, J. C. & Wiseman, R. L. Stress-regulated translational attenuation adapts mitochondrial protein import through Tim17A degradation. Cell Metab. 18, 908–919 (2013). 91. Ieva, R. et al. Mgr2 functions as lateral gatekeeper for preprotein sorting in the mitochondrial inner membrane. Mol. Cell 56, 641–652 (2014). 92. Horst, M. et al. 1842.Full. 16, 1842–1849 (1997). 93. Kang, P. J. et al. Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins. Nature 348, 137–143 (1990). 94. Banerjee, R., Gladkova, C., Mapa, K., Witte, G. & Mokranjac, D. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein. Elife 4, 1–17 (2015). 95. Liu, Q. Regulated Cycling of Mitochondrial Hsp70 at the Protein Import Channel. Science (80-. ). 300, 139–141 (2003). 96. Slutsky-Leiderman, O. et al. The interplay between components of the mitochondrial protein translocation motor studied using purified components. J. Biol. Chem. 282, 33935–33942 (2007). 97. Hasson, S. a. et al. Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation. Proc. Natl. Acad. Sci. 107, 9578–9583 (2010). 98. Hurt, E. C. & van Loon, A. P. G. M. How proteins find mitochondria and intramitochondrial compartments. Trends Biochem. Sci. 11, 204–207 (1986). 99. Hartl, F. U., Ostermann, J., Guiard, B. & Neupert, W. Successive translocation into and out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a bipartite signal peptide. Cell 51, 1027–1037 (1987). 100. Glick, B. S. et al. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast mitochondria by a stop-transfer mechanism. Cell 69, 809–822 (1992). 101. Koll, H. et al. Antifolding activity of hsp60 couples protein import into the mitochondrial matrix with export to the intermembrane space. Cell 68, 1163–1175 (1992). 102. Gartner, F., Bomer, U., Guiard, B. & Pfanner, N. The sorting signal of cytochrome b2 promotes early divergence from the general mitochondrial import pathway and restricts the unfoldase activity of matrix Hsp70. Embo J. 14, 6043–6057 (1995). 103. Gruhler, a, Ono, H., Guiard, B., Neupert, W. & Stuart, R. a. A novel intermediate on the import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting. 37 EMBO J. 14, 1349–1359 (1995). 104. Pratje, E. & Guiard, B. One nuclear. 5, 1313–1317 (1986). 105. Schneider, A., Behrens, M. & Scherer, P. Inner membrane protease I, an enzyme mediating intramitochondrial protein sorting in yeast. EMBO … 10, 247–254 (1991). 106. Geissler, A. et al. Membrane potential-driven protein import into mitochondria. The sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation of the matrix-targeting sequence. Mol. Biol. Cell 11, 3977–91 (2000). 107. Claypool, S. M., Boontheung, P., McCaffery, J. M., Loo, J. A. & Koehler, C. M. The Cardiolipin Transacylase, Tafazzin, Associates with Two Distinct Respiratory Components Providing Insight into Barth Syndrome. Mol. Biol. Cell 19, 5143–5155 (2008). 108. Koehler, C. M. New Developments in Mitochondrial Assembly. Annu. Rev. Cell Dev. Biol. 20, 309–335 (2004). 109. Alder, N. N., Jensen, R. E. & Johnson, A. E. Fluorescence Mapping of Mitochondrial TIM23 Complex Reveals a Water-Facing, Substrate-Interacting Helix Surface. Cell 134, 439–450 (2008). 110. van der Laan, M. et al. Motor-free mitochondrial presequence translocase drives membrane integration of preproteins. Nat. Cell Biol. 9, 1152–1159 (2007). 111. Neupert, W. & Herrmann, J. M. Translocation of Proteins into Mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007). 112. D’Silva, P. R., Schilke, B., Hayashi, M. & Craig, E. A. Interaction of the J-Protein Heterodimer Pam18/Pam16 of the Mitochondrial Import Motor with the Translocon of the Inner Membrane. Mol. Biol. Cell 19, 424–432 (2008). 113. Demishtein-Zohary, K. & Azem, A. The TIM23 mitochondrial protein import complex: function and dysfunction. Cell Tissue Res. 367, 33–41 (2017). 114. Roesch, K., Curran, S. P., Tranebjaerg, L. & Koehler, C. M. Human deafness dystonia syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex. Hum. Mol. Genet. 11, 477–486 (2002). 115. Rothbauer, U. et al. Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human Tim23 into the Inner Membrane of Mitochondria. J. Biol. Chem. 276, 37327–37334 (2001). 116. Iacovino, M. et al. The conserved translocase Tim17 prevents mitochondrial DNA loss. Hum. Mol. Genet. 18, 65–74 (2008). 117. Salhab, M., Patani, N., Jiang, W. & Mokbel, K. High TIMM17A expression is associated with adverse pathological and clinical outcomes in human breast cancer. Breast Cancer 19, 153–160 (2012). 118. Xu, X. et al. Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer. Proteomics 10, 1374–1390 (2010). 119. Yang, X. et al. The impact of timm17a on aggressiveness of human breast cancer cells. Anticancer Res. 36, 1237–1242 (2016). 38 120. Gao, S.-P. et al. Loss of TIM50 suppresses proliferation and induces apoptosis in breast cancer. Tumor Biol. 37, 1279–1287 (2016). 121. Dabir, D. V. et al. A Small Molecule Inhibitor of Redox-Regulated Protein Translocation into Mitochondria. Dev. Cell 25, 81–92 (2013). 122. Voos, W., Gambill, B. D., Guiard, B., Pfanner, N. & Craig, E. A. Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. J. Cell Biol. 123, 119–126 (1993). 123. Beasley, E. M., Müller, S. & Schatz, G. The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions. EMBO J. 12, 2303–2311 (1993). 124. Pai, M. Y. et al. in 287–298 (2015). doi:10.1007/978-1-4939-2269-7_22 125. Waldmann, H. & Janning, P. Chemical Biology. 46, 0–340 (2009). 126. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998). 127. van der Laan, M. et al. A Role for Tim21 in Membrane-Potential-Dependent Preprotein Sorting in Mitochondria. Curr. Biol. 16, 2271–2276 (2006). 128. de Rijk, M. C. et al. Prevalence of Parkinson’s disease in the elderly: the Rotterdam Study. Neurology 45, 2143–6 (1995). 129. Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211– 221 (2010). 130. Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, (2010). 131. Vives-Bauza, C. et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc. Natl. Acad. Sci. U. S. A. 107, 378–383 (2010). 132. Greene, A. W. et al. Mitochondrial processing peptidase regulates PINK1 processing, import and Parkin recruitment. EMBO Rep. 13, 378–85 (2012). 133. Jin, S. M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010). 134. Meissner, C., Lorenz, H., Weihofen, A., Selkoe, D. J. & Lemberg, M. K. The mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1 trafficking. J. Neurochem. 117, 856–867 (2011). 135. Yamano, K. & Youle, R. J. PINK1 is degraded through the N-end rule pathway. Autophagy 9, 1758–1769 (2013). 136. Okatsu, K. et al. A dimeric pink1-containing complex on depolarized mitochondria stimulates parkin recruitment. J. Biol. Chem. 288, 36372–36384 (2013). 137. Tanida, I., Ueno, T. & Kominami, E. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 2503–2518 (2004). 138. Tanida, I. Autophagosome Formation and Molecular Mechanism of Autophagy. 14, (2011).

39 139. Hammerling, B. C. & Gustafsson, Å. B. Mitochondrial quality control in the myocardium: Cooperation between protein degradation and mitophagy. J. Mol. Cell. Cardiol. 75, 122– 130 (2014). 140. Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415–24 (2014). 141. Attaix, D. & Taillandier, D. The missing link: Mul1 signals mitophagy and muscle wasting. Cell Metab. 16, 551–552 (2012). 142. Cilenti, L. et al. Inactivation of Omi/HtrA2 protease leads to the deregulation of mitochondrial Mulan E3 ubiquitin ligase and increased mitophagy. Biochim. Biophys. Acta 1843, 1295–1307 (2014). 143. Lokireddy, S. et al. The ubiquitin ligase Mul1 induces mitophagy in skeletal muscle in response to muscle-wasting stimuli. Cell Metab. 16, 613–624 (2012). 144. Levesque, M. P., Krauss, J., Koehler, C., Boden, C. & Harris, M. P. New tools for the identification of developmentally regulated enhancer regions in embryonic and adult zebrafish. Zebrafish 10, 21–29 (2013). 145. Schiller, D., Cheng, Y. C., Liu, Q., Walter, W. & Craig, E. A. Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering. Mol. Cell. Biol. 28, 4424–33 (2008). 146. Demishtein-Zohary, K. et al. Role of Tim17 in coupling the import motor to the translocation channel of the mitochondrial presequence translocase. Elife 6, 1–11 (2017). 147. Glick, B. S. & Pon, L. A. Isolation of highly purified mitochondria from Saccharomyces cerevisiae. Methods Enzymol. 260, 213–23 (1995).

40 Chapter 2: Discovery of MB-20, a small molecule modulator of the Tim23 channel

Introduction

Most mitochondrial matrix proteins contained a characteristic N-terminal presequence, upon folding, it forms a positively charged amphiphathic α-helix. Once the proteins are synthesized in the cytosol, the presequence directs the proteins to the mitochondria and transport through both the outer and inner mitochondrial membranes into the matrix where the presequence is usually cleaved by the mitochondrial processing peptidase (MPP).

Upon translocation through the TOM, presequence-carrying proteins are then headed

towards the TIM23 complex82,83. The TIM23 complex can recognize the preproteins and

transport the proteins into two directions, into the inner membrane or into the matrix. The

binding of Tim50 to Tim23 in the absence of a substrate keeps the Tim23 channel in a closed

state84. The subunit Tim50 of the TIM23 complex functions as a receptor that binds the

presequence, and upon binding, the channel is activated and opened for the preprotein to be

translocated across the inner membrane. Tim50 cooperates with the regulatory subunit Tim21 to recruit Pam17, and subsequently association of the PAM complex for matrix translocation85,86.

Tim17 is a central, membrane-embedded subunit of the TIM23 machinery, but the exact

molecular function has not been reported. However, it is shown to be involved in regulation of

the Tim23 channel and in preprotein sorting at the inner membrane87–90. The most recently

identified subunit Mgr2 has shown to be in close proximity to the inner membrane sorting signal,

providing quality control for the release of the preproteins into the inner membrane91.

41 Protein translocation into the matrix cannot be complete without the import motor PAM.

The ATP-driven chaperone mitochondrial heat-shock protein 70 (mtHsp70) forms the core of the

motor92,93. The N-terminal domain of Tim44 interacts with the import motor, whereas its C-

terminal domain interacts with the translocation channel94, serving as a middle-man between the

two complexes and help transfer the preprotein to mtHsp70 from the channel. The two J-proteins

Pam18 and Pam16 are associated with the ATP-hydrolyzing activity of mtHsp70, and the

nucleotide exchange factor Mge1 regulates the cycling of mtHsp70 through the release of ADP

for the new reaction cycle95,96.

Results

Secondary screens of the small molecule hits

Previous screens in the lab have shown small molecules that affect the quality of

mitochondria, thus before further investigating any of the small molecules, several assays need to be performed to ensure the small molecules do not affect the quality of mitochondria. There are two classes of hits that are found to be false-positive from previous screens: general uncouplers

of the membrane potential and compounds that affect mitochondrial membrane integrity, causing

nonspecific release of mitochondrial proteins. Therefore, the small molecules were further

selected using 3 secondary screening assays to remove these false-positives97. There are two

ways to determine if the small molecules uncouple mitochondria: using a spectrofluorimetric

assay based on quenching of a fluorescent dye, DiSC3(5), in energized mitochondria and testing

whether energized mitochondria become uncoupled in an oxygen electrode. The oxygen

electrode is advantageous if the small molecule interferes with the fluorescent dye. To determine

42 if the small molecule nonselectively permeabilize mitochondrial membrane, isolated

mitochondria were treated with the small molecule and both the pellet and supernatant fractions

were collected to determine whether mitochondrial proteins are released. Small molecules that

did not pass all the tests were removed from the candidate lists for further study.

Respiration Assay

The oxygen consumption rates from isolated mitochondria can be measured using the

Clarke-type oxygen electrode. It is a useful technique to evaluate the small molecules’ effect to mitochondrial respiration since ADP-dependent oxygen consumption directly reflects coupled respiration or oxidative phosphorylation (OXPHOS). Respiration was initiated by addition of

Nicotinamide adenine dinucleotide (NADH), and the rate of oxygen consumption was monitored

using the oxygen electrode. DMSO or small molecule was then added, the rate of oxygen

consumption was continuously monitored during the process, and carbonyl cyanide m-

chlorophenyl hydrazine (CCCP), a known uncoupler was added at the end to ensure the

mitochondria are behaving as expected. Figure 2-1 showed different examples of the results

obtained from the oxygen electrode and DMSO is used as the vehicle control (Figure 2-1 A).

Table 2-1 gave a summary of the results obtained from all the small molecules. Small molecules that showed inhibition of respiration or uncouple of mitochondria were removed from the candidate lists for further study.

DiSC3(5) Assay

3,3’-Dipropylthiadicarbocyanine Iodide or DiSC3(5) is a cationic dye that accumulates on

hyperpolarized membranes and is translocated into the lipid bilayer. Mitochondrial membrane

potential is critical for maintaining the physiological function of the respiratory chain for ATP production. When mitochondria are coupled, DiSC3(5) accumulates in the membranes of the

43 mitochondria and the fluorescence is quenched. However, in the presence of depolarized mitochondria, DiSC3(5) is released to the supernatant and high fluorescence was observed. Small molecules were incubated with isolated mitochondria and DiSC3(5) and fluorescence was measured (Figure 2-2). DMSO was used as a negative control and CCCP was used as a positive control for comparison. Small molecules that showed high fluorescence measurement (higher or close to the reading for CCCP) were removed from the candidate lists for further study.

Permeability Assay

To ensure the integrity of the mitochondria, small molecules were assessed for the non- specific permeabilization of mitochondrial membranes. The small molecules were incubated with isolated mitochondria, and the supernatant and pellet fractions were collected separately.

The samples were then separated using SDS-PAGE and analyzed by Coomassie staining (Figure

2-3). Most of the proteins should be in the pellet fraction if the mitochondria were intact.

However, in the case of MB-4, a known small molecule that permeabilize mitochondrial membrane, most proteins were found in the supernatant fraction, indicating that the mitochondria were no longer intact. Small molecules that showed permeabilization of mitochondrial membranes were removed from the candidate lists for further study.

Identify the target of the small molecules

There are different components of the TIM23 pathways, including the TOM complex, the

TIM23 complex and the PAM motor, where each complex contains multiple subunits. The small molecules can be inhibiting the pathway by targeting any of these components. Ideally, the small molecule is specifically targeting one component of the pathway, such that it can used to study

44 that particular protein. Various biochemical assays were used on the remaining candidates after

the secondary screen (Table 2-2) to further investigate their potential target.

In vitro import assay

In order to identify the target, different precursors that are known to be imported through

different pathways were used in an in vitro import assay (Figure 2-4). The protein Su9-DHFR

was generated by the fusion of protein subunit 9 (Su9) and dihydrofolate reductase (DHFR). This

protein consists of the 69-residue presequence of ATPase Su9, and it is translocated into the

matrix, where the presequence is cleaved. ADP/ATP carrier (AAC) is typical substrates of the

TIM22 complex that is commonly used. If a small molecule inhibits the import of both Su9-

DHFR and AAC, it is very likely that the small molecule is targeting the TOM complex or it can

also be non-specific (Figure 2-5 B). On the other hand, if the small molecule only inhibits Su9-

DHFR import but not AAC, which would suggest the small molecule could be targeting the

TIM23/PAM complex (Figure 2-5 C). Some small molecules showed no inhibition towards any

of the precursors (Figure 2-5 A).

For small molecules that inhibit both Su9-DHFR and AAC import, it suggests that the small molecule could be targeting the TOM complex. In order to verify that, import of Su9-

DHFR into mitochondria and mitoplasts were performed (Figure 2-6 A). Mitoplasts are mitochondria post-hypotonic treatment. After treatment, the outer membrane of mitochondria was burst open, and only inner membrane is intact. Therefore, if the small molecule is targeting the TOM complex, it should not exhibit import inhibition with mitoplasts (Figure 2-6 B).

The TIM23 complex can also transport preproteins with an additional sorting sequence

98–103 into the intermembrane space, and cytochrome (Cyt) b2 represents a typical example . Its

preprotein carries a second cleavable segment, which arrests the protein in the inner membrane

45 and prevents it from crossing the membrane completely. The sequence is then cleaved by the

104,105 inner membrane peptidase I . A single mutation of Cytb2, a substitution of alanine 63 by

proline generates a preprotein that is targeted to the matrix space106. Using these two different precursors, if a small molecule inhibits import of both preproteins, the target would likely to be the TIM23 complex, and if a small molecule only inhibits the Cytb2(167)A63P-DHFR, it would

suggest the target to be the PAM complex. Results obtained suggest that EN30 and A01 are

targeting the PAM motor while LS59 targets the TIM23 complex (Figure 2-7). In our lab, there

is no small molecule modulator that targets the TIM23 complex, therefore I focused on

characterizing LS59, named MitoBloCK-20 (MB-20) during the remaining of my graduate study.

Concluding Remarks

The goal of the screen was to identify and create a collection of small molecule

modulators for different components in the import pathways. From the Ura3 screen, we have identified a total of 6 molecules (MB-10, MB-11, DECA, EN30, A01 and LS59) that targets different components of the Tim23 pathway. These small molecules can be used to investigate mechanisms that were previously too challenging due to inadequate tools, and hopefully expand our knowledge of how import dysfunction contributes to various degenerative diseases.

46 Materials and Methods

Monitoring the rate of oxygen consumption using Clarke-type oxygen electrode. Oxygen

consumption of isolated mitochondria were performed as described previously107. Briefly, 100

μg of mitochondria isolated from yeast were incubated in 1 ml of respiration buffer with gentle

stirring. Respiration was initiated with addition of 2 mM NADH. Once a steady-state level was

reached, small molecule or DMSO (vehicle control) is added. As a control, mitochondria were

uncoupled by addition of 20µM Carbonyl cyanide 4-(trifluoromethoxy) phenlhydrazone (CCCP)

at the end of the experiment. Slope indicates the rates of oxygen consumption.

Membrane potential measurements using DiSC3(5). Membrane potential measurements of

purified mitochondria were performed with fluorescent 3,3’-Dipropylthiadicarbocyanine Iodide

dye DiSC3(5). 1% DMSO, CCCP or small molecules were added to mitochondria in import

buffer and incubated for 10 minutes, 0.2 μM of dye was then added. 5 minutes later, fluorescence

was measured at excitation and emission length of 620 nm and 670 nm respectively.

Mitochondria integrity assay. 25 μg of yeast isolated mitochondria were incubated in import buffer (0.6 M sorbitol, 2 mM KH2PO4, 60 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 2.5

mM EDTA,5 mM L-methionine, pH 7.1) the presence of small molecules at 25ºC for 30

minutes. Mitochondria were then pelleted by centrifugation at 8,000 x g for 10 minutes at 4ºC.

Supernatant containing released proteins from mitochondria were TCA precipitated on ice for 30

minutes. Precipitated proteins were recovered by centrifugation at maximum speed for 15

minutes at 4ºC. Both mitochondrial pellet and released proteins were resuspended in 5x-Laemmli

47 sample buffer (0.25 M Tris-Cl pH 6.8, 10% SDS, 30% glycerol, 0.02% bromophenol blue) with

5% β-mercaptoethanol (βME) and analyzed on SDS-PAGE. Resulting gel were stained with

coomassie blue for 30 minutes and destained in destaining solution (20% methanol, 10% acetic

acid) overnight in order to visualize the proteins.

Import of radiolabeled proteins into isolated yeast mitochondria. Mitochondria were purified

from yeast grown in ethanol-glycerol media. 35S-labeled precursors were synthesized in vitro

using TNT Quick Coupled Transcription/Translation kits (Promega) from purified plasmid. 15

µg mitochondria were used per import reaction. The mitochondria were then incubated with

small molecules or DMSO vehicle control for 15 minutes at 25 ºC in import buffer (0.6 M

sorbitol, 2 mM KH2PO4, 60 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 2.5 mM EDTA,5

mM L-methionine, pH 7.1) supplemented with 2 mM NADH. Import reactions were initiated by

precursor addition. Import was stopped by adding 50 µg/mL trypsin in cold buffer, and the samples were transferred to ice for 15 minutes. 250 µg/mL soybean trypsin inhibitor was then added. Mitochondria were isolated by centrifugation at 8,000 x g at 4ºC for 10 minutes. Final

mitochondria pellet were dissolved in 5x Laemlli sample buffer. Samples were resolved on SDS-

PAGE. Gels were dried prior to exposure to film.

Mitoplasting. 100 µg of mitochondria were incubated in 1 ml of 20mM HEPES pH 7.4 for 30 minutes on ice. Treated mitochondria (mitoplasts) were then recovered by centrifugation at maximum speed for 15 minutes. The mitoplasts were then resuspended in import buffer (0.6 M sorbitol, 2 mM KH2PO4, 60 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 2.5 mM EDTA,5

mM L-methionine, pH 7.1) for in vitro import assay.

48

Miscellaneous. Mitochondrial proteins were analyzed by SDS-PAGE using a 12 or 15% polyacrylamide gel and a Tricine-based running buffer. Proteins were detected by immunoblotting using PVDF membranes.

49 Figures and tables

Figure 2-1. Respiration assay of the hits. (A) DMSO was used as a control of the assay. (B) An example of a small molecule that slows down respiration. (C) An example of a small molecule that potentially uncouples mitochondria. (D) An example of a small molecule that does not affect respiration.

50

Figure 2-2. DiSC3(5) assay of the hits. Isolated mitochondria were incubated with DMSO/small molecules and DiSC3 (5) and the fluorescence was measured. DMSO is used as a negative control whereas CCCP is used as a positive control.

51

Figure 2-3. Membrane permeability assay of the hits. The assay was conducted using 50μM of the small molecules. Supernatant (S) and pellet (P) fractions were collected separately.

Samples were then separated by SDS-PAGE and analyzed by Coomassie staining. DMSO is

used as a negative control and MB-4, a known compound that permeabilizes mitochondrial membrane, is used as a positive control.

52

Figure 2-4. Illustration of the rationale of using in vitro import to identify the target complex of the small molecules. By using different precursors that are known to be imported using different pathways, we can get

53

Figure 2-5. Examples of the results obtained from the in vitro import assay of Su9-DHFR and AAC with different small molecules. (A) An example of a small molecule that does not inhibit either Su9-DHFR or AAC. (B) An example of a small molecule that inhibits both Su9-

DHFR and AAC. (C) An example of a small molecule that only inhibits Su9-DHFR but not

AAC.

54

Figure 2-6. In vitro import assay with mitochondria and mitoplast. (A) Generation of mitoplasts using hypotonic treatment from mitochondria. (B) An example of a small molecule that inhibits Su9-DHFR import with mitochondria and mitoplasts. MB-11, a known TOM complex inhibitor was used as a positive control.

55

Figure 2-7. Results obtained from the in vitro import assay of Cytb2(167)-DHFR and

Cytb2(167)A63P-DHFR with EN30, A01 and LS59. EN30 and A01 showed inhibition for only

Cytb2(167)A63P-DHFR and LS59 showed inhibition for both Cytb2(167)-DHFR and

Cytb2(167)A63P-DHFR.

56 Name Respiration effect AN17 No change AN14 No change AN18 No change B02 No change A05 slow down respiration A06 No change EN30 slow down respiration CB39 slow down CCCP oxygen level Gossypol increased LS59 No change LS52 oxygen level increased AN60 No change LS72 possible uncoupler AN73 No change EN76 No change EN71 slow down respiration oxygen level Benzethonium chloride increased LS99 oxygen level increased EN108 slow down respiration A01 No change AN110 No change EN113 No change EN116 slow down CCCP A04 No change

Table 2-1. Summary of the results from the respiration assay.

57 Name AN17 AN14 AN18 B02 A05 A06 EN30 CB39 LS59 AN60 AN73 EN76 EN71 EN108 A01 AN110 EN113 A04

Table 2-2. List of candidates remained after the secondary screen.

58 Su9-DHFR AAC import Import into Cytb2(167)- Cytb2(167)A63P- Name import mitoplasts DHFR import DHFR import AN17 No inhibition No inhibition AN14 No inhibition No inhibition AN18 No inhibition No inhibition B02 No inhibition No inhibition A05 + No inhibition A06 ++ ++ ++ EN30 ++ No inhibition No inhibition ++ CB39 +++ +++ +++ LS59 +++ No inhibition +++ ++ AN60 + No inhibition AN73 No inhibition No inhibition EN76 No inhibition No inhibition EN71 ++ ++ ++ EN108 ++ + ++ A01 ++ No inhibition No inhibition ++ AN110 ++ ++ + EN113 + A04 ++ + +++

Very slight inhibition, therefore no further investigation Nonspecific inhibition, therefore no further investigation No inhibition, therefore no further investigation No data Small molecule of interest, named MB-20

Table 2-3. Summary of the small molecules on the import assays.

59 References

1. McBride, H. M., Neuspiel, M. & Wasiak, S. Mitochondria: More Than Just a Powerhouse.

Curr. Biol. 16, 551–560 (2006).

2. Kroemer, G. & Reed, J. C. Mitochondrial control of cell death. Nat Med 6, 513–519

(2000).

3. Newmeyer, D. D. & Ferguson-Miller, S. Mitochondria: Releasing power for life and

unleashing the machineries of death. Cell 112, 481–490 (2003).

4. Contreras, L., Drago, I., Zampese, E. & Pozzan, T. Mitochondria: The calcium

connection. Biochim. Biophys. Acta - Bioenerg. 1797, 607–618 (2010).

5. Rizzuto, R., De Stefani, D., Raffaello, A. & Mammucari, C. Mitochondria as sensors and

regulators of calcium signalling. Nat. Rev. Mol. Cell Biol. 13, 566–578 (2012).

6. Ott, M., Gogvadze, V., Orrenius, S. & Zhivotovsky, B. Mitochondria, oxidative stress and

cell death. Apoptosis 12, 913–922 (2007).

7. Lenaz, G. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta -

Bioenerg. 1366, 53–67 (1998).

8. Murley, A. & Nunnari, J. The Emerging Network of Mitochondria-Organelle Contacts.

Mol. Cell 61, 648–653 (2016).

9. Karbowski, M., Norris, K. L., Cleland, M. M., Jeong, S.-Y. & Youle, R. J. Role of Bax

and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006).

10. Kuznetsov, A. V., Janakiraman, M., Margreiter, R. & Troppmair, J. Regulating cell

survival by controlling cellular energy production: Novel functions for ancient signaling

pathways? FEBS Lett. 577, 1–4 (2004).

60 11. Kuznetsov, A. V et al. Survival signaling by C-RAF: mitochondrial reactive oxygen

species and Ca2+ are critical targets. Mol. Cell. Biol. 28, 2304–2313 (2008).

12. Riedl, S. J. & Salvesen, G. S. The apoptosome: signalling platform of cell death. Nat. Rev.

Mol. Cell Biol. 8, 405–413 (2007).

13. Youle, R. J. CELL BIOLOGY: Cellular Demolition and the Rules of Engagement.

Science (80-. ). 315, 776–777 (2007).

14. Kotiadis, V. N., Duchen, M. R. & Osellame, L. D. Mitochondrial quality control and

communications with the nucleus are important in maintaining mitochondrial function and

cell health. Biochim. Biophys. Acta - Gen. Subj. 1840, 1254–1265 (2014).

15. van Vliet, A. R., Verfaillie, T. & Agostinis, P. New functions of mitochondria associated

membranes in cellular signaling. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 2253–

2262 (2014).

16. Meisinger, C. et al. Contents, Ed. Board + Forthc. articles. Trends Biochem. Sci. 30, i

(2005).

17. Csordás, G. et al. Structural and functional features and significance of the physical

linkage between ER and mitochondria. J. Cell Biol. 174, 915–921 (2006).

18. AhYoung, A. P. et al. Conserved SMP domains of the ERMES complex bind

phospholipids and mediate tether assembly. Proc. Natl. Acad. Sci. 112, E3179–E3188

(2015).

19. Murley, A. et al. ER-associated mitochondrial division links the distribution of

mitochondria and mitochondrial DNA in yeast. Elife 2013, 1–16 (2013).

20. Sun, N., Youle, R. J. & Finkel, T. The Mitochondrial Basis of Aging. Mol. Cell 61, 654–

666 (2016).

61 21. Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21,

4411–4419 (2002).

22. Martinus, R. D. et al. Selective Induction of Mitochondrial Chaperones in Response to

Loss of the Mitochondrial Genome. Eur. J. Biochem. 240, 98–103 (1996).

23. Eiyama, A. & Okamoto, K. PINK1/Parkin-mediated mitophagy in mammalian cells. Curr.

Opin. Cell Biol. 33, 95–101 (2015).

24. Pickrell, A. M. & Youle, R. J. The roles of PINK1, Parkin, and mitochondrial fidelity in

parkinson’s disease. Neuron 85, 257–273 (2015).

25. Jin, S. M. & Youle, R. J. The accumulation of misfolded proteins in the mitochondrial

matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized

mitochondria. Autophagy 9, 1750–1757 (2013).

26. Pellegrino, M. W. & Haynes, C. M. Mitophagy and the mitochondrial unfolded protein

response in neurodegeneration and bacterial infection. BMC Biol. 13, 22 (2015).

27. Nunnari, J. & Suomalainen, A. Mitochondria: In sickness and in health. Cell 148, 1145–

1159 (2012).

28. Dyall, S. D. Ancient Invasions: From Endosymbionts to Organelles. Science (80-. ). 304,

253–257 (2004).

29. Gray, M. W., Gray, M. W., Burger, G. & Lang, B. F. Mitochondrial Evolution. 1476, 1–

16 (2008).

30. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing

Mitochondrial Proteins: Machineries and Mechanisms. Cell 138, 628–644 (2009).

31. Koehler, C. M. New Developments in Mitochondrial Assembly. Annu. Rev. Cell Dev.

Biol. 20, 309–335 (2004).

62 32. Sickmann, A. et al. The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl.

Acad. Sci. 100, 13207–13212 (2003).

33. Taylor, S. W. et al. Characterization of the human heart mitochondrial proteome. J.

Proteome Res. 21, 281–286 (2003).

34. Mokranjac, D. & Neupert, W. Cell biology: Architecture of a protein entry gate. Nature

528, 201–202 (2015).

35. Hill, K. et al. Tom40 forms the hydrophilic channel of the mitochondrial import pore for

preproteins. Nature 395, 516–521 (1998).

36. Gentle, I., Gabriel, K., Beech, P., Waller, R. & Lithgow, T. The Omp85 family of proteins

is essential for outer membrane biogenesis in mitochondria and bacteria. J. Cell Biol. 164,

19–24 (2004).

37. Kozjak, V. et al. An essential role of Sam50 in the protein sorting and assembly

machinery of the mitochondrial outer membrane. J. Biol. Chem. 278, 48520–48523

(2003).

38. Paschen, S. a. et al. Evolutionary conservation of biogenesis of b -barrel membrane

proteins. Nature 426, 862–866 (2003).

39. Wiedemann, N. & Pfanner, N. Mitochondrial Machineries for Protein Import and

Assembly. Annu. Rev. Biochem. 86, annurev-biochem-060815-014352 (2017).

40. Hawlitschek, G. et al. Mitochondrial protein import: Identification of processing peptidase

and of PEP, a processing enhancing protein. Cell 53, 795–806 (1988).

41. Chacinska, A. et al. Mitochondrial presequence translocase: Switching between TOM

tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817–829 (2005).

42. Koehler, C. M., Merchant, S. & Schatz, G. How membrane proteins travel across the

63 mitochondrial intermembrane space. Trends Biochem. Sci. 24, 428–432 (1999).

43. Truscott, K. N., Brandner, K. & Pfanner, N. Mechanisms of protein import into

mitochondria. Curr. Biol. 13, 326–337 (2003).

44. Pfanner, N. Mitochondrial import: crossing the aqueous intermembrane space. Curr. Biol.

8, R262-5 (1998).

45. Ahting, U. et al. The Tom Core Complex. J. Cell Biol. 147, 959–968 (1999).

46. Shiota, T. et al. Molecular architecture of the active mitochondrial protein gate. Science

(80-. ). 349, 1544–1548 (2015).

47. Abe, Y. {[Structural} basis of presequence recognition by the mitochondrial protein

import receptor Tom20]. Fukuoka Igaku Zasshi 92, 266–271 (2001).

48. Roise, D., Horvath, S. J., Tomich, J. M., Richards, J. H. & Schatz, G. A chemically

synthesized pre-sequence of an imported mitochondrial protein can form an amphiphilic

helix and perturb natural and artificial phospholipid bilayers. EMBO J. 5, 1327–34 (1986).

49. Popov-Čeleketić, D., Mapa, K., Neupert, W. & Mokranjac, D. Active remodelling of the

TIM23 complex during translocation of preproteins into mitochondria. EMBO J. 27,

1469–1480 (2008).

50. Ting, S. Y., Schilke, B. A., Hayashi, M. & Craig, E. A. Architecture of the TIM23 inner

mitochondrial translocon and interactions with the matrix import motor. J. Biol. Chem.

289, 28689–28696 (2014).

51. Kang, P. J. et al. Requirement for hsp70 in the mitochondrial matrix for translocation and

folding of precursor proteins. Nature 348, 137–143 (1990).

52. Vögtle, F. N. et al. Global Analysis of the Mitochondrial N-Proteome Identifies a

Processing Peptidase Critical for Protein Stability. Cell 139, 428–439 (2009).

64 53. Mossmann, D., Meisinger, C. & Vögtle, F. N. Processing of mitochondrial presequences.

Biochim. Biophys. Acta - Gene Regul. Mech. 1819, 1098–1106 (2012).

54. Davis, a J., Ryan, K. R. & Jensen, R. E. Tim23p contains separate and distinct signals for

targeting to mitochondria and insertion into the inner membrane. Mol. Biol. Cell 9, 2577–

2593 (1998).

55. Endres, M., Neupert, W. & Brunner, M. Transport of the ADP / ATP carrier of

mitochondria from the TOM complex to the TIM22 · 54 complex. 18, 3214–3221 (1999).

56. Leuenberger, D., Bally, N. A., Schatz, G. & Koehler, C. M. Different import pathways

through the mitochondrial intermembrane space for inner membrane proteins. EMBO J.

18, 4816–4822 (1999).

57. Webb, C. T., Gorman, M. A., Lazarou, M., Ryan, M. T. & Gulbis, J. M. Crystal structure

of the mitochondrial chaperone TIM9???10 reveals a six-bladed ??-propeller. Mol. Cell

21, 123–133 (2006).

58. Gebert, N. et al. Assembly of the three small Tim proteins precedes docking to the

mitochondrial carrier translocase. EMBO Rep. 9, 548–554 (2008).

59. Lionaki, E. et al. The essential function of Tim12 in vivo is ensured by the assembly

interactions of its C-terminal domain. J. Biol. Chem. 283, 15747–15753 (2008).

60. Paschen, S. A. The role of the TIM8-13 complex in the import of Tim23 into

mitochondria. EMBO J. 19, 6392–6400 (2000).

61. Murphy, M. P., Leuenberger, D., Curran, S. P., Oppliger, W. & Koehler, C. M. The

Essential Function of the Small Tim Proteins in the TIM22 Import Pathway Does Not

Depend on Formation of the Soluble 70-Kilodalton Complex. Mol. Cell. Biol. 21, 6132–

6138 (2001).

65 62. Tranebjaerg, L. et al. A new X linked recessive deafness syndrome with blindness,

dystonia, fractures, and mental deficiency is linked to Xq22. J. Med. Genet. 32, 257–263

(1995).

63. Koehler, C. M., Leuenberger, D., Merchant, S., Renold, A. & Junne, T. Human deafness

dystonia syndrome is a mitochondrial disease. Cell Biol. 96, 2141–2146 (1999).

64. Magen, D. et al. Mitochondrial Hsp60 Chaperonopathy Causes an Autosomal-Recessive

Neurodegenerative Disorder Linked to Brain Hypomyelination and Leukodystrophy. Am.

J. Hum. Genet. 83, 30–42 (2008).

65. Christensen, J. H. et al. Inactivation of the hereditary spastic paraplegia-associated Hspd1

gene encoding the Hsp60 chaperone results in early embryonic lethality in mice. Cell

Stress Chaperones 15, 851–863 (2010).

66. Richter-Dennerlein, R. et al. DNAJC19, a mitochondrial cochaperone associated with

cardiomyopathy, forms a complex with prohibitins to regulate cardiolipin remodeling.

Cell Metab. 20, 158–171 (2014).

67. Al Teneiji, A., Siriwardena, K., George, K., Mital, S. & Mercimek-Mahmutoglu, S.

Progressive Cerebellar Atrophy and a Novel Homozygous Pathogenic DNAJC19 Variant

as a Cause of Dilated Cardiomyopathy Ataxia Syndrome. Pediatr. Neurol. 62, 58–61

(2016).

68. Ojala, T. et al. New mutation of mitochondrial DNAJC19 causing dilated and

noncompaction cardiomyopathy, anemia, ataxia, and male genital anomalies. Pediatr. Res.

72, 432–437 (2012).

69. Lazarou, M., Jin, S. M., Kane, L. A. & Youle, R. J. Role of PINK1 Binding to the TOM

Complex and Alternate Intracellular Membranes in Recruitment and Activation of the E3

66 Ligase Parkin. Dev. Cell 22, 320–333 (2012).

70. Manczak, M. et al. Mitochondria are a direct site of A beta accumulation in Alzheimer’s

disease neurons: implications for free radical generation and oxidative damage in disease

progression. Hum. Mol. Genet. 15, 1437–49 (2006).

71. Mossmann, D. et al. Amyloid-β Peptide Induces Mitochondrial Dysfunction by Inhibition

of Preprotein Maturation. Cell Metab. (2014). doi:10.1016/j.cmet.2014.07.024

72. Gottschalk, W. K. et al. HHS Public Access. 1, 1–25 (2015).

73. Devi, L. Accumulation of Amyloid Precursor Protein in the Mitochondrial Import

Channels of Human Alzheimer’s Disease Brain Is Associated with Mitochondrial

Dysfunction. J. Neurosci. 26, 9057–9068 (2006).

74. Pagani, L. & Eckert, A. Amyloid-Beta Interaction with Mitochondria. Int. J. Alzheimers.

Dis. 2011, 1–12 (2011).

75. Baker, K. P. & Schatz, G. Mitochondrial proteins essential for viability mediate protein

import into yeast mitochondria. Nature 349, 205–8 (1991).

76. Hasson, S. a et al. Substrate specificity of the TIM22 mitochondrial import pathway

revealed with small molecule inhibitor of protein translocation. Proc. Natl. Acad. Sci. U.

S. A. 107, 9578–9583 (2010).

77. Dabir, D. V et al. A small molecule inhibitor of redox-regulated protein translocation into

mitochondria. Dev. Cell 25, 81–92 (2013).

78. Miyata, N. et al. Adaptation of a genetic screen reveals an inhibitor for mitochondrial

protein import component Tim44. J. Biol. Chem. 292, 5429–5442 (2017).

79. Hoogenraad, N. J., Ward, L. A. & Ryan, M. T. Import and assembly of proteins into

mitochondria of mammalian cells. Biochim. Biophys. Acta 1592, 97–105 (2002).

67 80. Kuznetsov, A. V. & Margreiter, R. Heterogeneity of mitochondria and mitochondrial

function within cells as another level of mitochondrial complexity. Int. J. Mol. Sci. 10,

1911–1929 (2009).

81. Bolender, N., Sickmann, A., Wagner, R., Meisinger, C. & Pfanner, N. Multiple pathways

for sorting mitochondrial precursor proteins. EMBO Rep. 9, 42–49 (2008).

82. van der Laan, M., Schrempp, S. G. & Pfanner, N. Voltage-coupled conformational

dynamics of mitochondrial protein-import channel. Nat. Struct. Mol. Biol. 20, 915–917

(2013).

83. Schulz, C., Schendzielorz, A. & Rehling, P. Unlocking the presequence import pathway.

Trends Cell Biol. 25, 265–275 (2015).

84. Meinecke, M. et al. Inner Membrane. 653, 2003–2006 (2006).

85. Lytovchenko, O. et al. Signal recognition initiates reorganization of the presequence

translocase during protein import. EMBO J. 32, 886–898 (2013).

86. Schulz, C. & Rehling, P. Remodelling of the active presequence translocase drives motor-

dependent mitochondrial protein translocation. Nat. Commun. 5, 1–9 (2014).

87. Wrobel, L., Sokol, A. M., Chojnacka, M. & Chacinska, A. The presence of disulfide

bonds reveals an evolutionarily conserved mechanism involved in mitochondrial protein

translocase assembly. Sci. Rep. 6, 27484 (2016).

88. Martinez-Caballero, S., Grigoriev, S. M., Herrmann, J. M., Campo, M. L. & Kinnally, K.

W. Tim17p regulates the twin pore structure and voltage gating of the mitochondrial

protein import complex TIM23. J. Biol. Chem. 282, 3584–3593 (2007).

89. Ramesh, A. et al. A disulfide bond in the TIM23 complex is crucial for voltage gating and

mitochondrial protein import. J. Cell Biol. 214, 417–431 (2016).

68 90. Rainbolt, T. K., Atanassova, N., Genereux, J. C. & Wiseman, R. L. Stress-regulated

translational attenuation adapts mitochondrial protein import through Tim17A

degradation. Cell Metab. 18, 908–919 (2013).

91. Ieva, R. et al. Mgr2 functions as lateral gatekeeper for preprotein sorting in the

mitochondrial inner membrane. Mol. Cell 56, 641–652 (2014).

92. Horst, M. et al. 1842.Full. 16, 1842–1849 (1997).

93. Kang, P. J. et al. Requirement for hsp70 in the mitochondrial matrix for translocation and

folding of precursor proteins. Nature 348, 137–143 (1990).

94. Banerjee, R., Gladkova, C., Mapa, K., Witte, G. & Mokranjac, D. Protein translocation

channel of mitochondrial inner membrane and matrix-exposed import motor communicate

via two-domain coupling protein. Elife 4, 1–17 (2015).

95. Liu, Q. Regulated Cycling of Mitochondrial Hsp70 at the Protein Import Channel. Science

(80-. ). 300, 139–141 (2003).

96. Slutsky-Leiderman, O. et al. The interplay between components of the mitochondrial

protein translocation motor studied using purified components. J. Biol. Chem. 282,

33935–33942 (2007).

97. Hasson, S. a. et al. Substrate specificity of the TIM22 mitochondrial import pathway

revealed with small molecule inhibitor of protein translocation. Proc. Natl. Acad. Sci. 107,

9578–9583 (2010).

98. Hurt, E. C. & van Loon, A. P. G. M. How proteins find mitochondria and

intramitochondrial compartments. Trends Biochem. Sci. 11, 204–207 (1986).

99. Hartl, F. U., Ostermann, J., Guiard, B. & Neupert, W. Successive translocation into and

out of the mitochondrial matrix: Targeting of proteins to the intermembrane space by a

69 bipartite signal peptide. Cell 51, 1027–1037 (1987).

100. Glick, B. S. et al. Cytochromes c1 and b2 are sorted to the intermembrane space of yeast

mitochondria by a stop-transfer mechanism. Cell 69, 809–822 (1992).

101. Koll, H. et al. Antifolding activity of hsp60 couples protein import into the mitochondrial

matrix with export to the intermembrane space. Cell 68, 1163–1175 (1992).

102. Gartner, F., Bomer, U., Guiard, B. & Pfanner, N. The sorting signal of cytochrome b2

promotes early divergence from the general mitochondrial import pathway and restricts

the unfoldase activity of matrix Hsp70. Embo J. 14, 6043–6057 (1995).

103. Gruhler, a, Ono, H., Guiard, B., Neupert, W. & Stuart, R. a. A novel intermediate on the

import pathway of cytochrome b2 into mitochondria: evidence for conservative sorting.

EMBO J. 14, 1349–1359 (1995).

104. Pratje, E. & Guiard, B. One nuclear. 5, 1313–1317 (1986).

105. Schneider, A., Behrens, M. & Scherer, P. Inner membrane protease I, an enzyme

mediating intramitochondrial protein sorting in yeast. EMBO … 10, 247–254 (1991).

106. Geissler, A. et al. Membrane potential-driven protein import into mitochondria. The

sorting sequence of cytochrome b(2) modulates the deltapsi-dependence of translocation

of the matrix-targeting sequence. Mol. Biol. Cell 11, 3977–91 (2000).

107. Claypool, S. M., Boontheung, P., McCaffery, J. M., Loo, J. A. & Koehler, C. M. The

Cardiolipin Transacylase, Tafazzin, Associates with Two Distinct Respiratory

Components Providing Insight into Barth Syndrome. Mol. Biol. Cell 19, 5143–5155

(2008).

108. Koehler, C. M. New Developments in Mitochondrial Assembly. Annu. Rev. Cell Dev.

Biol. 20, 309–335 (2004).

70 109. Alder, N. N., Jensen, R. E. & Johnson, A. E. Fluorescence Mapping of Mitochondrial

TIM23 Complex Reveals a Water-Facing, Substrate-Interacting Helix Surface. Cell 134,

439–450 (2008).

110. van der Laan, M. et al. Motor-free mitochondrial presequence translocase drives

membrane integration of preproteins. Nat. Cell Biol. 9, 1152–1159 (2007).

111. Neupert, W. & Herrmann, J. M. Translocation of Proteins into Mitochondria. Annu. Rev.

Biochem. 76, 723–749 (2007).

112. D’Silva, P. R., Schilke, B., Hayashi, M. & Craig, E. A. Interaction of the J-Protein

Heterodimer Pam18/Pam16 of the Mitochondrial Import Motor with the Translocon of the

Inner Membrane. Mol. Biol. Cell 19, 424–432 (2008).

113. Demishtein-Zohary, K. & Azem, A. The TIM23 mitochondrial protein import complex:

function and dysfunction. Cell Tissue Res. 367, 33–41 (2017).

114. Roesch, K., Curran, S. P., Tranebjaerg, L. & Koehler, C. M. Human deafness dystonia

syndrome is caused by a defect in assembly of the DDP1/TIMM8a-TIMM13 complex.

Hum. Mol. Genet. 11, 477–486 (2002).

115. Rothbauer, U. et al. Role of the Deafness Dystonia Peptide 1 (DDP1) in Import of Human

Tim23 into the Inner Membrane of Mitochondria. J. Biol. Chem. 276, 37327–37334

(2001).

116. Iacovino, M. et al. The conserved translocase Tim17 prevents mitochondrial DNA loss.

Hum. Mol. Genet. 18, 65–74 (2008).

117. Salhab, M., Patani, N., Jiang, W. & Mokbel, K. High TIMM17A expression is associated

with adverse pathological and clinical outcomes in human breast cancer. Breast Cancer

19, 153–160 (2012).

71 118. Xu, X. et al. Quantitative proteomics study of breast cancer cell lines isolated from a

single patient: Discovery of TIMM17A as a marker for breast cancer. Proteomics 10,

1374–1390 (2010).

119. Yang, X. et al. The impact of timm17a on aggressiveness of human breast cancer cells.

Anticancer Res. 36, 1237–1242 (2016).

120. Gao, S.-P. et al. Loss of TIM50 suppresses proliferation and induces apoptosis in breast

cancer. Tumor Biol. 37, 1279–1287 (2016).

121. Dabir, D. V. et al. A Small Molecule Inhibitor of Redox-Regulated Protein Translocation

into Mitochondria. Dev. Cell 25, 81–92 (2013).

122. Voos, W., Gambill, B. D., Guiard, B., Pfanner, N. & Craig, E. A. Presequence and mature

part of preproteins strongly influence the dependence of mitochondrial protein import on

heat shock protein 70 in the matrix. J. Cell Biol. 123, 119–126 (1993).

123. Beasley, E. M., Müller, S. & Schatz, G. The signal that sorts yeast cytochrome b2 to the

mitochondrial intermembrane space contains three distinct functional regions. EMBO J.

12, 2303–2311 (1993).

124. Pai, M. Y. et al. in 287–298 (2015). doi:10.1007/978-1-4939-2269-7_22

125. Waldmann, H. & Janning, P. Chemical Biology. 46, 0–340 (2009).

126. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene

deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

127. van der Laan, M. et al. A Role for Tim21 in Membrane-Potential-Dependent Preprotein

Sorting in Mitochondria. Curr. Biol. 16, 2271–2276 (2006).

128. de Rijk, M. C. et al. Prevalence of Parkinson’s disease in the elderly: the Rotterdam

Study. Neurology 45, 2143–6 (1995).

72 129. Matsuda, N. et al. PINK1 stabilized by mitochondrial depolarization recruits Parkin to

damaged mitochondria and activates latent Parkin for mitophagy. J. Cell Biol. 189, 211–

221 (2010).

130. Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate

Parkin. PLoS Biol. 8, (2010).

131. Vives-Bauza, C. et al. PINK1-dependent recruitment of Parkin to mitochondria in

mitophagy. Proc. Natl. Acad. Sci. U. S. A. 107, 378–383 (2010).

132. Greene, A. W. et al. Mitochondrial processing peptidase regulates PINK1 processing,

import and Parkin recruitment. EMBO Rep. 13, 378–85 (2012).

133. Jin, S. M. et al. Mitochondrial membrane potential regulates PINK1 import and

proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).

134. Meissner, C., Lorenz, H., Weihofen, A., Selkoe, D. J. & Lemberg, M. K. The

mitochondrial intramembrane protease PARL cleaves human Pink1 to regulate Pink1

trafficking. J. Neurochem. 117, 856–867 (2011).

135. Yamano, K. & Youle, R. J. PINK1 is degraded through the N-end rule pathway.

Autophagy 9, 1758–1769 (2013).

136. Okatsu, K. et al. A dimeric pink1-containing complex on depolarized mitochondria

stimulates parkin recruitment. J. Biol. Chem. 288, 36372–36384 (2013).

137. Tanida, I., Ueno, T. & Kominami, E. LC3 conjugation system in mammalian autophagy.

Int. J. Biochem. Cell Biol. 36, 2503–2518 (2004).

138. Tanida, I. Autophagosome Formation and Molecular Mechanism of Autophagy. 14,

(2011).

139. Hammerling, B. C. & Gustafsson, Å. B. Mitochondrial quality control in the myocardium:

73 Cooperation between protein degradation and mitophagy. J. Mol. Cell. Cardiol. 75, 122–

130 (2014).

140. Nath, S. et al. Lipidation of the LC3/GABARAP family of autophagy proteins relies on a

membrane-curvature-sensing domain in Atg3. Nat. Cell Biol. 16, 415–24 (2014).

141. Attaix, D. & Taillandier, D. The missing link: Mul1 signals mitophagy and muscle

wasting. Cell Metab. 16, 551–552 (2012).

142. Cilenti, L. et al. Inactivation of Omi/HtrA2 protease leads to the deregulation of

mitochondrial Mulan E3 ubiquitin ligase and increased mitophagy. Biochim. Biophys.

Acta 1843, 1295–1307 (2014).

143. Lokireddy, S. et al. The ubiquitin ligase Mul1 induces mitophagy in skeletal muscle in

response to muscle-wasting stimuli. Cell Metab. 16, 613–624 (2012).

144. Levesque, M. P., Krauss, J., Koehler, C., Boden, C. & Harris, M. P. New tools for the

identification of developmentally regulated enhancer regions in embryonic and adult

zebrafish. Zebrafish 10, 21–29 (2013).

145. Schiller, D., Cheng, Y. C., Liu, Q., Walter, W. & Craig, E. A. Residues of Tim44 involved

in both association with the translocon of the inner mitochondrial membrane and

regulation of mitochondrial Hsp70 tethering. Mol. Cell. Biol. 28, 4424–33 (2008).

146. Demishtein-Zohary, K. et al. Role of Tim17 in coupling the import motor to the

translocation channel of the mitochondrial presequence translocase. Elife 6, 1–11 (2017).

147. Glick, B. S. & Pon, L. A. Isolation of highly purified mitochondria from Saccharomyces

cerevisiae. Methods Enzymol. 260, 213–23 (1995).

74 Chapter 3: MB-20 reveals the role of Tim17 in stabilizing the TIM23/PAM complex

Abstract

Growing evidence reveals how mitochondrial dysfunction contributes to degenerative

diseases. Modulation of the mitochondrial protein import pathways can have regulatory effects

on mitochondrial function. Conventional methods using yeast genetics and RNAi approaches are

powerful, but limited. RNAi requires several days to take effect and during this time, cells can

alter metabolism to adapt. Therefore, we are committed to identify small molecule modulators

for mitochondrial protein translocation with the goal of dissecting the link of a defective

mitochondrial import system to diseases. This study utilizes a high-throughput small molecule

screening approach to identify modulators of the TIM23 import pathway, which transports

proteins across the inner membrane and into the matrix. This strategy will be useful in generating

a toolbox of small molecule modulators for mitochondrial translocation in order to understand

how defects in mitochondrial assembly contribute to disease and will advance future mechanistic

studies. Here, we identify a small molecule, designated MitoBloCK-20 (MB-20), which binds specifically to Tim17 in the TIM23 complex. Using a pulldown assay, as well as BN-PAGE, we have shown that MB-20 dissociates the PAM motor from the TIM23 channel through the disruption of Tim17-Tim44 interactions. Using zebrafish as a model organism, MB-20 also showed a critical role for mitochondria in development of the zebrafish neural system.

75 Introduction

Mitochondria play a complex multi-factorial role in cell metabolism and mitochondrial

dysfunction contributes to neural and muscular generative diseases. There are over 1500 proteins

within the mitochondria, but only 13 are encoded by the mitochondrial genome28,29. Most

mitochondrial proteins are encoded in the nucleus and imported into the mitochondria.

Mitochondria have developed a sophisticated translocation system for the import of nuclear-

encoded proteins30,108. Most mitochondrial matrix proteins contain a characteristic N-terminal

presequence, which forms a positively charged amphiphathic α-helix upon folding47. Once the

proteins are synthesized in the cytosol, the presequence directs the proteins to the mitochondria

where they are first transported across the outer mitochondrial membrane through the TOM

complex and then across the inner membrane through the TIM23 complex. Next, the presence of

the sorting signal or the lack thereof determines the destination of the preproteins10. The

preproteins can be inserted into the inner membrane or transported into the matrix using the

PAM motor. The presequence is cleaved by the mitochondrial processing peptidase (MPP)40,52.

Most carrier proteins are located in the inner membrane and are synthesized without a cleavable

presequence. The carrier proteins and some import components utilize the TIM22 import

pathway54–56. The small Tim proteins function as chaperones, escorting the substrates to the

insertion complex, which are then inserted into the inner membrane of the mitochondria61.

The TIM23 core complex consists of subunits Tim50, Tim23 and Tim17, where Tim23 and Tim17 form the translocation channel109 and Tim50 functions as a receptor84. Tim21

assembles with the TIM23 core complex to facilitate the transfer of substrates into the inner

membrane41,110. Translocation to the matrix requires the ATP-driven protein-associated motor

(PAM)111. Components of the PAM complex include Tim44, nucleotide exchange factor Mge1,

76 mitochondrial Hsp70 (mtHsp70), and J-proteins Pam16 and Pam18. Tim44 associates the PAM

complex to the TIM23 complex and helps transfer the preproteins from the TIM23 complex to

mtHsp7049,50. The stimulatory subunit Pam 18 and the regulatory subunit Pam16 regulate

ATPase activity of mtHsp70112. Finally, Mge1 is required for the completion and recycling of

mtHsp70 cycle.

Defects in the TIM23 protein import machinery are found to be associated with a variety

of diseases113. The human deafness dystonia syndrome (MTS/DFN-1) was shown to be caused

by truncation or deletion of DDP1/Tim862,114,115. Tim8, together with Tim13, is involved in the

import of Tim23 into the mitochondria. DNAJC19 (Pam18) mutant causes dilated

cardiomyopathy with ataxia syndrome66–68. A recent study showed that TIMM17a functions as a

suppressor of mitochondrial DNA instability and might be a potential target for disorders

associated with mitochondrial DNA loss116. Moreover, high expression of TIMM17a117–119 and

TIMM50120 have been observed in breast cancer cells, and suppression of the expression inhibits

the proliferation ability of the cancer cells.

Although there are numerous mitochondrial diseases affecting a lot of people, due to

inadequate tools and approaches, it has been difficult to make further progress in studying mitochondrial protein machineries and import processes. Thus, the development of small molecule modulators for the mitochondrial import system would aid the study of how protein translocation contributes to diseases. To this end, we have successfully identified inhibitors for

Tim10, Erv1 and Tim4476,78,121. From the same genetic screen that discovered MB-10, an

inhibitor of Tim44, another hit compound, designated MB-20, was characterized in detail. MB-

20 likely targets Tim17 of the TIM23 complex and inhibits the import of substrates that require

the TIM23 complex. MB-20 also reveals the role of Tim17 in stabilizing the PAM complex,

77 possibly through the Tim17-Tim44 interaction. Moreover, MB-20 treatment in mammalian cells

has shown LC3 lipidation and LC3 tubules formation, which suggests induction of mitophagy.

However, we did not see Pink1 accumulation or Parkin recruitment with MB-20 treatment,

suggesting that MB-20 might be inducing Pink1/Parkin independent mitophagy. Finally, MB-20

treatment in zebrafish supports an important role for Tim17 in neural development. MB-20 has

proven to be a useful tool to investigate the mechanistic role of Tim17 in protein translocation, as

well as its relationship to diseases.

Results

The discovery of MitoBloCK-20 (MB-20)

From the same genetic screen that resulted in the discovery of MitoBloCK-10 (MB-10), a

Tim44 inhibitor, we discovered another hit compound, designated MitoBloCK-20 (MB-20) for

further characterization. The detail and the rationale of this screen has been previously

described78. The chemical name for MB-20 is N-(3-(benzo[d]thiazol-2-yl)-6-methyl-4,5,6,7-

tetrahydrothieno[2,3-c]pyridin-2-yl)-2-(phenylsulfonyl)acetamide hydrochloride (Figure 1A).

We used another compound, designated MB-20.1 with similarity to MB-20 for structural-activity relationship (SAR) studies. MB-20.1 showed negative result in the screen with highly similar structure to MB-20, therefore it is an ideal molecule for testing specificity. Previous screens have shown nonspecific damages of mitochondria from the potential hits, therefore, secondary assays were performed to investigate the mitochondrial membrane integrity and oxidative phosphorylation properties in the presence of MB-20. To assess the integrity of the mitochondria, small molecules (100μM) were incubated with isolated mitochondria, and the

78 supernatant (S) and pellet (P) fractions were collected separately. The samples were then

analyzed by Coomassie staining (Figure S3-1B) and immunoblotting (Figure S3-1C). As a

positive control, 0.2% Trition X-100 (TX) was used to permeabilize mitochondrial membranes.

DMSO (vehicle) was used as a negative control. Treatment with MB-20 and MB-20.1 did not result in release of proteins from yeast mitochondria, in contrast to the Triton X-100 treatment.

Respiration of isolated mitochondria in the presence of the small molecule was measured using the Clarke-type oxygen electrode107 (Figure S3-1E). Respiration was initiated by addition of

Nicotinamide adenine dinucleotide (NADH), and the rate of oxygen consumption was monitored

using the oxygen electrode. The addition of DMSO or MB-20 showed similar effect, a 38%

decrease of the respiration rate, but the addition of carbonyl cyanide m-chlorophenyl hydrazine

(CCCP), a known uncoupler of mitochondria, increased the rate of oxygen consumption as

expected. Mitochondrial membrane potential is critical for maintaining the physiological

function of the respiratory chain for ATP production. To assess the membrane potential of the

mitochondria in the presence of the compounds, the cationic dye 3,3’-

Dipropylthiadicarbocyanine Iodide or DiSC3(5) was used. DiSC3(5) accumulates on

hyperpolarized membranes and is translocated into the lipid bilayer. When mitochondria are

coupled, DiSC3(5) accumulates in the membranes of the mitochondria and the fluorescence is

quenched. However, in the presence of depolarized mitochondria, DiSC3(5) is released to the

supernatant and high fluorescence is observed. Small molecules were incubated with isolated

mitochondria and DiSC3(5), and the fluorescence was then measured (Figure S3-1A). DMSO

(vehicle) was used as a negative control and CCCP was used as a positive control. Both MB20

and MB20.1 showed similar fluorescence measurements compared to DMSO, in contrast to the

high fluorescence reading observed with CCCP. We also tested the effect of MB-20 on the

79 mitochondrial membrane potential in vivo using HeLa cells. Results showed that the membrane

potential was not perturbed in the presence of MB-20, because staining with membrane potential dependent MitoTracker Red was robust (Figure S3-2). CCCP-treated cells were used as a negative control for MitoTracker staining, confirming that the membrane potential was dissipated.

MB-20 targets the Tim23 complex

The minimum inhibitory concentration required to inhibit the growth of 50% of the yeast

(MIC50) for MB-20 was determined for the WT and tim23-2 strains. These strains lacked the

multidrug resistance pumps Pdr5 and Snq2 to increase the intracellular concentration of the small molecules. Based on a previous screen for TIM22 modulators76, we expected that the translocon that MB-20 targets would exhibit increased sensitivity or lower MIC50 to MB-20. The MIC50 of

MB-20 is 5.6 μM for the tim23-2 strain, in contrast to the value 10.6 μM obtained for the WT

(Figure 3-1B, left panel). This suggests that the MB-20 likely targets the TIM23 translocon. The

assay was also performed with MB20.1, and the results showed that the compound does not

affect either strain (Figure 3-1B, right panel).

To further verify that MB-20 specifically targets the TIM23 translocon, in vitro import assays with selected radiolabeled substrates were performed. Isolated mitochondria were incubated with different concentration of MB-20 before the addition of radiolabeled substrates

Su9-DHFR (TIM23 substrate) or AAC (TIM22 substrate). The results indicated that 100 μM of

MB-20 inhibited Su9-DHFR import by 86%, while it did not block the import of AAC (Figure 3-

2A, top panel). Next, using cytochrome (cyt) b2 variants, we investigated the effect of MB-20 on

the sorting of precursors to the intermembrane space and the matrix. Cyt b2(167)-DHFR is

80 laterally sorted to the intermembrane space by the TIM23SORT complex100,110,122. The precursor of

cyt b2(167)-DHFR contains a stop transfer sequence, which prevents the mature portion of the

protein from entering the matrix122. The protein is arrested in the inner membrane, forming an intermediate (i) after cleavage by MPP. It is subsequently cleaved by IMP, forming the mature form (m) and released to the intermembrane space. Cyt b2(167)A63P-DHFR is targeted to the

mitochondrial matrix due to the point mutation A63P in the stop transfer sequence123. For the

import of cyt b2(167)-DHFR, we observed an inhibition of 86% for the intermediate form and

82% for the mature form in the presence of 100 μM MB-20 (Figure 3-2A, bottom panel).

Inhibition of cyt b2(167)A63P-DHFR import was also observed, but at a lower rate of 31% with

the same concentration of MB-20. Taken together, MB-20 showed inhibition of PAM motor-

independent import using the TIM23 pathway, as well as PAM motor-dependent import into the

matrix, suggesting that MB-20 targets specifically the TIM23 complex (Tim50, Tim21, Tim17

and Tim23).

MB-20 binds specifically to Tim17 of the TIM23 complex and disrupts the Tim17-Tim44 interaction The TIM23 complex includes Tim50, Tim21, Tim17 and Tim23. In order to identify

which subunit MB-20 specifically targets, a Drug Affinity Responsive Target Stability (DARTS)

assay was performed. DARTS is an approach to identify potential protein targets for small

molecules124,125. It relies on the protection against proteolysis of the target protein by binding to

the small molecule. The interaction of the target protein and the small molecule could make the

potential cleavage sites less accessible. This method can avoid immobilization or modification of

the small molecules, which could potentially affect its interaction with the target protein.

81 We used this assay to determine which subunits of TIM23 is the potential target of MB-

20. To optimize conditions in which the degradation of the protein could be captured in a time

course by immunoblotting, various proteases at varying concentrations were tested and

thermolysin gave the desired results. Isolated yeast mitochondria were lysed with buffer

containing 0.2% Triton X-100. The lysates were then incubated in the presence of 1% DMSO,

100μM of MB-20, followed by protease treatment and immunoblot analysis. In the presence of

DMSO, Tim23, Tim17 and Tim50 all showed increased degradation with increasing

concentration of the protease (Figure 3-3A). However, when treated with MB-20, only Tim17

showed resistance to protease degradation, suggesting a probable interaction of MB-20 with

Tim17. The same experiment was performed in mitochondria purified from HeLa cells.

TOMM40 was used as a control, and only TIMM17 showed insensitivity towards protease treatment in the presence of MB-20 (Figure 3-3C). Another version of the assay was performed with isolated yeast mitochondria to further verify the protein target of MB-20. Instead of increasing protease concentration with constant MB-20 concentration, we used a constant protease concentration with increasing MB-20 concentration. We expected that the protein target of MB-20 should be more resistant to protease degradation when we increase the concentration of MB-20 due to increased protein interaction with the small molecule. Indeed, the results showed an increased resistance for Tim17 to protease treatment with increasing concentrations of

MB-20, but not for Tim23 and Tim50 (Figure 3-3B). Thus, these data support that MB-20 likely targets Tim17 of the TIM23 complex.

82 MB-20 reveals the role of Tim17 in stabilizing the PAM complex formation To determine whether MB-20 treatment altered interactions between Tim17 and other

subunits of the TIM23 complex, we generated a yeast strain with a histidine tag integrated at the

3’end of TIM17, synthesizing Tim17-His protein for pulldown experiments with Ni2+-agarose beads126. Mitochondria were incubated with 1% DMSO or indicated concentration of MB-20 followed by solubilization in 1% digitonin. When Tim17-His was isolated, the interaction of

Tim17 and Tim23 was not disrupted by the addition of MB-20, up to 100 µM (Figure 3-4A).

However, at 25 µM MB-20, the interaction between Tim17 and Tim44 was significantly weakened. Hsp70 was used as a negative control. We used the same method to generate another yeast strain with a histidine tag integrated at the 3’ end of TIM23 and the same experiment was repeated using this strain. When Tim23-His was isolated, the interaction of Tim23 and Tim17 was not disrupted by the addition of MB-20 (Figure 3-4B), as seen with the Tim17-His pulldown experiment. As expected, the interaction between Tim23 and Tim44 was disrupted in the presence of MB-20. Results also showed disruption of interaction of Tim23 with other components of the PAM motor, Pam16 and Pam18 (Figure 3-4B), suggesting the possibility of

MB-20 did not disrupt the interaction within the TIM23 complex, but between the TIM23 complex and PAM motor. The subunits of the PAM motor (Tim44, Pam 18 and Pam16) are also shown to be precipitated and became insoluble upon dissociation induced by the presence of

MB-20 (Figure 3-4C). Isolated yeast mitochondria were incubated with DMSO or indicated concentration of MB-20, followed by solubilization with 1% digitonin. Both supernatant (S) and pellet (P) fractions were collected after centrifugation, and the samples were then separated by

83 SDS-PAGE. Taken together, MB-20 disrupts the Tim17-Tim44 interaction and causes the PAM

motor to dissociate and to become insoluble.

Next, we investigated the effect of MB-20 treatment on the assembly of the complexes

using Blue Native (BN)-PAGE. It has been shown that there exist two different forms of the

presequence translocase for distinct transport routes of pre-proteins: a TIM23CORE complex,

consisting of Tim17, Tim23 and Tim50, that associates with the PAM motor for matrix

translocation of pre-proteins; and a TIM23SORT complex that contains Tim21 with no association

to the PAM motor, which mediates the lateral release of sorted pre-proteins into the inner

membrane41,127. The results showed that the assembly of Tim17 to both the TIM23CORE and

TIM23SORT complex was significantly decreased (Figure 3-4D), which supports the in vitro

import assay discussed earlier that MB-20 inhibits protein imports for both Cyt b2(167)-DHFR

CORE SORT (TIM23 ) and Cyt b2(167)A63P-DHFR (TIM23 ) precursors. Based on the BN-PAGE, the TIM23 complex formation was disrupted in the presence of MB-20, although the individual

Tim23-Tim17 interaction was not affected (Figure 3-4A). The results also showed that the assembly of the PAM motor (Tim44, Pam16 and Pam18) was strongly affected in the presence of MB-20 (Figure 3-4E), supporting the observation from the pulldown assay.

MB-20 treatment does not induce or affect the Pink1/Parkin pathway Parkinson’s disease (PD) is the second most common neurodegenerative disease128.

Evidence has linked the disease to defective mitophagy, a mechanism that selectively removes

damaged mitochondria in the cells. The main players include PTEN-induced putative kinase

protein 1 (PINK1) and the E3 ubiquitin ligase Parkin. PINK1 accumulates on depolarized

84 mitochondria specifically, marking them for elimination, and Parkin is subsequently translocated

to mitochondria129–131 followed by recruitment of the autophagy machinery. In healthy

mitochondria, PINK1 is imported through the TOM complex of the outer mitochondrial

membrane and into the TIM 23 complex of the inner mitochondrial membrane, where it is

cleaved first by the mitochondrial processing peptidase132 (MPP), then by the presenilin- associated rhomboid-like protein133,134 (PARL). Cleaved PINK1 is then released to the cytosol

and degraded by the ubiquitin proteasome system135, thus the PINK1 level on healthy

mitochondria is very low or undetectable. On the other hand, in damaged mitochondria where

import is disrupted, PINK1 processing is disrupted because import into the inner membrane

where PARL and MPP reside is blocked, resulting in uncleaved PINK1 accumulation on the

outer membrane of the mitochondria bound to the TOM complex69,136. Since rapid and constitutive degradation of PINK1 in healthy mitochondria relies on the TIM23 import pathway, we set out to investigate whether blocking the pathway leads to accumulation of PINK1 on mitochondria, subsequently inducing mitophagy.

First, we tested whether MB-20 inhibits PINK1 protein import using an in vitro mitochondria import assay. In the presence of MB-20, import of PINK1 was inhibited as expected (Figure 3-5A). Su9-DHFR was used as a positive control. Using HeLa cells, we then investigated the accumulation of PINK1 and subsequent Parkin recruitment to the mitochondria in the presence of MB-20. Surprisingly, results showed no PINK1 accumulation on the mitochondria (Figure 3-5B) or Parkin recruitment (Figure 3-5C). CCCP was used as a positive control. Taken together, MB-20 treatment does not induce the PINK1/Parkin pathway. We then tested the effect of combining MB-20 and CCCP treatment. HeLa cells were pre-incubated with

MB-20, followed by CCCP treatment. Results show that MB-20 treatment did not affect Parkin

85 recruitment to the mitochondria. Same experiment was done for immunoblots analysis, and MB-

20 does not affect PINK1 accumulation on mitochondria (Figure S3-6B). It is very intriguing that PINK1 import was inhibited in the presence of MB-20, yet PINK1 accumulation is not affected. This raised the question on the precise mechanism of PINK1 import and how PINK1 detects damaged mitochondria.

MB-20 treatment induces LC3 lipidation and LC3 tubules formation While investigating the Pink1/Parkin pathway using MB-20, we also looked at an

autophagy marker LC3. The LC3 proteins are essential in the all autophagic process including

mitophagy and are involved in the formation of autophagosomes137–139. It was shown that LC3-I is conjugated to phosphatidylethanolamine (PE) to form lipidated LC3-II during mitophagy and remains associated with mitochondria until after lysosomal fusion occurs and subsequent elimination of the mitochondria140. Therefore, the presence or increased level of LC3-II is often

used as a mitophagy marker and is indicative of mitophagy activation.

Although Pink1 accumulation and Parkin recruitment to the mitochondria was not

observed, we saw increased level of LC3-II upon MB-20 treatment (Figure 3-6A), but not with

MB20.1 (Figure 3-6B). We also visualized LC3 in HeLa cells by tranfecting fluorescently labeled LC3 into the cells and perform live imaging with a fluorescence microscope. Under condition with minimum autophagy, GFP-RFP-LC3-I is mostly diffused in the cytosol. When autophagy is induced, GFP-RFP-LC3-I is modified by lipidation and form GFP-RFP-LC3-II puncta in the cells. As autophagosomes fuse with lysosomes, the outer membrane GFP-RFP-

LC3-II is quenched for the GFP signal but not the RFP signal due to the lysosomal acidic

86 environment that protonates the fluorophore of the GFP not the RFP signal. Therefore, there

should be yellow and red only puncta under normal autophagy condition. In conditions inhibiting

lysosomal fusion to the autophagasome, red only puncta will not be observed. Upon MB-20

treatment, we can see LC3 tubules formation in the cells, which is not observed with DMSO or

MB20.1 (Figure 3-6C). We repeated the experiment with MEF cells and MEF cells stably

expressing Flag-Parkin. LC3 tubules formation was also observed in these cell lines (Figure S3-

5). As shown above, Pink1 accumulation and Parkin recruitment to mitochondria were not

observed, yet an increased level of LC3-II was detected, suggesting MB-20 might be inducing a

Pink1/Parkin independent mitophagy.

Recent studies have identified another mitochondrial E3 ubiquitin ligase, Mulan,

involved in mitophagy141–143. Therefore, we next investigated whether MB-20 is inducing Mulan

specific mitophagy. HeLa cells with Mulan knocked out were tranfected with LC3-RFP and

MLS-DsRed (mitochondria marker) for 24 hours. Cells were then treated with DMSO, CCCP or

MB-20 for 3 hours, and live images were taken using a fluorescence microscope. LC3 tubules

formation was observed upon MB-20 treatment with or without Mulan in the cells (Figure S3-

3A). Experiment was repeated without the transfection for immunoblot analysis, and increased

level of LC3-II was observed with all cell lines (Figure S3-3B). These results suggest that MB-

20 does not induce Mulan specific mitophagy.

Next, we want to investigate the location of the LC3 tubules observed. We co-transfected

LC3-GFP with MLS-DsRed (mitochondria marker) or LC3-RFP with Sec61b-GFP (ER marker), followed by MB-20 treatment to visualize whether the LC3 tubules formed co-localize with mitochondria or ER. Results show that the LC3 tubules formed under MB-20 treatment does not co-localize with either mitochondria or ER (Figure S3-4). This is a very intriguing result,

87 because MB-20 causes LC3 lipidation and tubules formation, which is indicative of autophagy

activation. MB-20 targets mitochondria specifically, therefore it is likely that it is inducing

mitophagy, rather than general autophagy. However, the LC3 tubules do not seem to localize to

mitochondria, suggesting defect in mitochondrial import system might be causing some

unknown cargo-specific autophagy.

MB-20 treatment showed defect of neural development in zebrafish Defects in mitochondrial protein import have been associated with numerous neural

degenerative diseases. Therefore, we applied MB-20 to zebrafish embryos to determine how

impaired TIM23 pathway altered development, particularly focusing on the motor neuron. In the

zebrafish line we used, Gal4-driven GFP expression marks primary motor neuron, and

mitochondria are marked by a TOL2-mediated insertion of a MLS-DsRed construct that is

expressed by the Gal4-UAS promotor144. Embryos were incubated with either 1% DMSO, MB-

20 or MB20.1 at 4 hpf and allowed to develop until 72 hpf. Aberrant neuronal cells accumulation

at the tail tip was observed in embryos incubated with 8 µM of MB-20 (Figure 3-5A).

Interestingly, accumulated neuronal cells at the tail tip showed loss of mitochondria (Figure 3-

5C). In addition, embryos incubated with MB-20.1 did not show similar developmental defects to MB-20 (Figure 3-5B), suggesting that the phenotype observed in zebrafish is specific to

TIM23 pathway defect induced by MB-20 treatment.

88 Discussion

Using the genetic screen described previously78, we discovered a small molecule,

designated MB-20 that targets Tim17 of the TIM23 translocon. Combining a genetic approach

using the tim23-2 mutant and a battery of import assays using substrates of different import

pathways, we narrowed the target to four subunits of the TIM23 complex. Subsequent studies

using the DARTS assay showed that MB-20 likely targets Tim17. Unfortunately, the structure of

Tim17 is unknown, and therefore it is difficult to verify the target and identify the binding sites.

Further effort using His-tagged Tim17 and BN-PAGE showed that MB-20 disrupts the Tim17-

Tim44 interaction and such disruption destabilizes the PAM motor formation. It has been shown

that the translocation channel and the import motor of the TIM23 complex communicate through

Tim44. The N-terminal domain of Tim44 binds to the components of the import motor, whereas

its C-terminal domain binds to the translocation channel, possibly through Tim1794,145. Tim17

contains four predicted transmembrane (TM) helices, spanning the inner membrane of the

mitochondria. Desmishtein-Zohary and colleagues showed that mutations in TM1 and TM2 of

Tim17 impairs the interaction of Tim17 and Tim23, and the second half of Tim17 (in particular, a highly conserved arginine residue at position 105) plays an essential role in recruitment of

Tim4450,146. Taken together, we propose that the possible binding site of MB-20 lies between the

second half of the Tim17 protein and the C-terminal domain of Tim44, where Tim44 and Tim17

interact. Tim17 is an essential subunit of the translocase, yet its function remains unclear. Hence,

MB-20 will be a useful tool in mechanistic studies, thereby providing insights into Tim17

function.

The mitochondrial protein translocation system is highly conserved throughout higher

eukaryotes; therefore the small molecules developed can also be used in different model systems,

89 including zebrafish and mammalian cells. There are significant numbers of diseases such as

Parkinson’s disease and Alzhemier’s disease that are associated with the import system, yet the

detailed mechanisms are still unclear. Here, we deliberately introduced damage to mitochondria

in cultured cells using MB-20. Surprisingly, the results show LC3 lipidation without induction of the PINK1/Parkin dependent mitophagy, indicating that the mechanism is more complicated than what is currently known. Besides single cell models, it is also very interesting to see the effect on a whole organism. Zebrafish is a great model for developmental study, and MB-20 treated embryos showed aberrant neuronal phenotype that can be related to neurodegenerative diseases in human. Further investigation is underway to explain these observations, but we are certain that

MB-20 can be served as an effective tool to continue mechanistic studies of the TIM23 translocon.

Some of the import components are essential, therefore studies on these components have been very challenging using conventional genetic methods like gene deletion. Moreover, these components are assembled in complexes, expression of one component often alter the expression of the other components in the same complex, making them difficult to study by RNAi. Using small molecules, we can investigate the effect of the loss of a particular protein under different cellular stresses, for varying lengths of time. We can also study the recovery of such effect because most treatments with small molecules are reversible. Hence, small molecules can be an extremely useful tool for future mechanistic studies on the mitochondrial protein translocation system.

90 Materials and Methods

Monitoring the rate of oxygen consumption using Clarke-type oxygen electrode. Oxygen

consumption of isolated mitochondria were performed as described previously107. Briefly, 100

μg of mitochondria isolated from yeast were incubated in 1 ml of respiration buffer with gentle

stirring. Respiration was initiated with addition of 2 mM NADH. Once a steady-state level was

reached, small molecule or DMSO (vehicle control) is added. As a control, mitochondria were

uncoupled by addition of 20µM Carbonyl cyanide 4-(trifluoromethoxy) phenlhydrazone (CCCP)

at the end of the experiment. Slope indicates the rates of oxygen consumption.

Membrane potential measurements using DiSC3(5). Membrane potential measurements of

purified mitochondria were performed with fluorescent 3,3’-Dipropylthiadicarbocyanine Iodide

dye DiSC3(5). 1% DMSO, CCCP or small molecules were added to mitochondria in import

buffer and incubated for 10 minutes, 0.2 μM of dye was then added. 5 minutes later, fluorescence

was measured at excitation and emission length of 620 nm and 670 nm respectively.

Mitochondria integrity assay. 25 μg of yeast isolated mitochondria were incubated in import buffer (0.6 M sorbitol, 2 mM KH2PO4, 60 mM KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 2.5

mM EDTA,5 mM L-methionine, pH 7.1) the presence of small molecules at 25ºC for 30

minutes. Mitochondria were then pelleted by centrifugation at 8,000 x g for 10 minutes at 4ºC.

Supernatant containing released proteins from mitochondria were TCA precipitated on ice for 30

minutes. Precipitated proteins were recovered by centrifugation at maximum speed for 15

minutes at 4ºC. Both mitochondrial pellet and released proteins were resuspended in 5x-Laemmli

91 sample buffer (0.25 M Tris-Cl pH 6.8, 10% SDS, 30% glycerol, 0.02% bromophenol blue) with

5% β-mercaptoethanol (βME) and analyzed on SDS-PAGE. Resulting gel were stained with

coomassie blue for 30 minutes and destained in destaining solution (20% methanol, 10% acetic

acid) overnight in order to visualize the proteins.

Import of radiolabeled proteins into isolated yeast mitochondria. Mitochondria were purified

from yeast grown in ethanol-glycerol media as described in previous studies126,147. Mitochondria

concentration was measured by bicinchoninic acid (BCA) assay, and flash-freezed in 25 mg/ml

aliquots using liquid nitrogen. Mitochondria were stored at -80°C and thawed in 25°C water bath

before experiment. 35S-labeled precursors were synthesized in vitro using TNT Quick Coupled

Transcription/Translation kits (Promega) from purified plasmid. 15 µg mitochondria were used per import reaction. The mitochondria were then incubated with small molecules or DMSO vehicle control for 15 minutes at 25 ºC in import buffer (0.6 M sorbitol, 2 mM KH2PO4, 60 mM

KCl, 50 mM HEPES-KOH, 5 mM MgCl2, 2.5 mM EDTA,5 mM L-methionine, pH 7.1) supplemented with 2 mM NADH. Import reactions were initiated by precursor addition. Import was stopped by adding 50 µg/mL trypsin in cold buffer, and the samples were transferred to ice for 15 minutes. 250 µg/mL soybean trypsin inhibitor was then added. Mitochondria were isolated by centrifugation at 8,000 x g at 4ºC for 10 minutes. Final mitochondria pellet were dissolved in

5x Laemlli sample buffer. Samples were resolved on SDS-PAGE. Gels were dried prior to exposure to film.

Mitochondria isolation from mammalian cells for protein import. Cultured cells were grown

to confluency. One day prior to mitochondria isolation, cells were fed with fresh media. Cells

92 were then harvested and homogenized in 20 mM HEPES pH 7.6, 220 mM mannitol, 70 mM

sucrose, 2 mg/ml BSA, and 0.5 mM PMSF. Lysates were dounced with Teflon dounce.

Homogenates were centrifuged at 770 x g at 4ºC for 5 minutes. Post-nuclear supernatants were

centrifuged at 10,000 x g for 10 minutes to obtain mitochondria pellets. Pellets were further

washed with homogenization buffer without BSA. Protein concentrations were measured using

BCA assay (Thermo Scientific).

Import of radiolabeled proteins into isolated mammalian mitochondria. Mitochondria were

isolated as described above. 35S-labeled precursors were synthesized using the TNT Quick

Coupled Transcription/Translation kits (Promega). 20 µg mitochondria were added to import

buffer (20 mM HEPES pH 7.6, 220 mM mannitol, 70 mM sucrose) supplemented with 1 mM

ATP, 0.5 mM magnesium acetate, 5 mM NADH, and 20 mM sodium succinate. Small molecules

or DMSO vehicle control were added and samples were incubated for 15 minutes at 25ºC. 10 µL

precursor was added to initiate import. Import was stopped by adding cold import buffer with 25

µg/mL trypsin after 15 minutes. Soybean trypsin inhibitor (50 µg/mL) was added following 15 minutes trypsin treatment. Mitochondria were pelleted by centrifugation at 12,000 x g at 4ºC for

5 minutes. Final mitochondria pellet were dissolved in 5x Laemnli sample buffer. Samples were resolved on SDS-PAGE. Gels were dried prior to exposure to film for autoradiography.

Cell culture. HeLa and HeLa cells stably expressing EGFP-Parkin were cultured in Dulbecco’s modified eagle medium with pyruvate (DMEM) (Life Technologies) supplemented with 10%

FBS (v/v) and 1% penicillin/streptomycin (Life Technologies) in humidified atmosphere with

93 5% CO2 at 37°C. For small molecules treatment, cells were seeded at 70% confluency and

treated the next day.

Cell viability measurement. Cells were seeded at 50% confluency, incubated for 24 hrs, then

treated with the DMSO or the indicated MB-20 concentrations. After 24 hrs, cell viability was

measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicology assay (Sigma), according to the manufacturer’s protocol.

Small molecule treatment and mitochondria isolation. HeLa cells were seeded at 50% confluency, incubated for 24 hrs, then transfected with indicated plasmid. After 16 hrs, cells

were treated with the indicated small molecule for 3 hrs, and cells were then harvested. For

mitochondria isolation, cells were homogenized in 20 mM HEPES pH 7.6, 220 mM mannitol, 70

mM sucrose, 2 mg/ml BSA, 10 mM NEM and 0.5 mM PMSF and 1 μM MG132. Cells were

passed through a 25-gauge needle using a 1 mL syringe to lyse the cells. Homogenates were centrifuged at 770 x g at 4ºC for 5 min. To isolate mitochondria, post-nuclear supernatants were centrifuged at 10,000 x g at 4ºC for 10 min. Pellets were washed with homogenization buffer lacking BSA. Protein concentrations were measured using BCA assay (Thermo Scientific).

Samples were resolved on SDS-PAGE and transferred to polyvinyl difluoride (PVDF) membrane (EMD Millipore) for immunoblot analysis.

Immunofluorescence. Cells were seeded on a 6-wells plate containing glass coverslips and treated the next day with indicated small molecules or DMSO vehicle control. Cells were fixed in 3.7% formaldehyde for 15 minutes followed by 3 times PBS wash. For immunostaining, cells

94 were permeabilized in 0.2% Triton X-100 in PBS for 10 minutes after fixation. Subsequently,

cells were incubated with appropriate antibodies diluted in PBS-BSA (2% BSA in PBS).

Following PBS washes, cells were incubated with secondary antibodies conjugated Alexa Fluor

dyes 350, 488, or 568 (Life Technologies) diluted in PBS-BSA. Images were obtained using

Yokogawa spinning disc with Zeiss 3i system or Axiovert 200M Carl Zeiss inverted microscope.

For MitoTracker Red staining, cells were incubated in 25 nM MitoTracker Red CMXROS (Life

Technologies) for 30 minutes prior to fixation.

Yeast MIC50 assay. An early-log growth of the indicated yeast strains in YPEG media was

76 diluted to an initial OD600 of 0.01 in fresh YPEG media . This diluted stock was dispensed in 50

µl aliquots into individual wells of a 96-well glass bottom plate. MB-20/MB-20.1/DMSO was serially diluted with YPEG by a factor of 2 to the indicated concentration. The final concentration of DMSO was 1%. Plates were then incubated at 25°C in a humidified chamber for

24 hours. Before OD600 measurement of each well was taken, the plates were shaken in a

Beckman orbital shaker to resuspend settled cells. The OD600 in YPEG with 1% DMSO was set

as 100% survival.

Drug Affinity Responsive Target Stability (DARTS) assay. 40 µg of isolated yeast

mitochondria were solubilized with buffer containing 20 mM HEPES-KOH pH 7.4, 50 mM KCl

and 0.2% Triton X-100 for 30 minutes on ice, followed by centrifugation at maximum speed for

15 minutes. Lysate was treated with 1% DMSO or indicated concentration of MB-20 at 25°C for

15 minutes. Various concentration of thermolysin (Sigma) was added to the samples and after 10

minutes incubation, the reaction was immediately quenched by the addition of TCA to a final

95 concentration of 15%. Proteins were recovered by centrifugation at maximum speed for 15

minutes at 4°C, and the protein pellets were resuspend in Laemmli sample buffer containing 15%

β-mercaptoethanol. The samples were separated by SDS-PAGE and analyzed by immunoblotting.

Generation of Tim17-His and Tim23-His yeast strains. The TIM17-His10 strain was generated using PCR-based integration at the 3’ end of TIM17 and integration was selected using the HISMX6 selection cassette126. The TIM23-His10 strain was generated the same way. The

strains were verified using PCR with isolated genomic DNA, as well as immunoblotting with His

antibody (Abcam).

Pulldown assay using Tim17-His and Tim23-His yeast strains. Isolated yeast mitochondria

(400 µg) were treated with 1% DMSO or indicated concentration of MB-20 for 30 minutes and

subsequently lysed in pulldown buffer (1% digitonin from AG scientific, 20 mM HEPES-KOH pH 7.4, 80 mM KCl, 105 glycerol, 20 mM imidazole and 1 mM PMSF) for 30 minutes on ice.

Lysate was centrifuged at maximum speed for 30 minutes to remove insoluble material. 50 µg lysate was removed from the supernatant to use as a reference for the total sample (T). The remaining 350 µg was diluted to 0.4 µg/µl in pulldown buffer. Samples were incubated with 30

µl of HisPur Ni2+ beads (Thermo Scientific) overnight at 4°C with rotation. After binding, 50 µg

of lysate was removed as a reference for the material that did not bind (denoted flow-through,

FT). The beads were then washed 3 times with buffer. The beads were then boiled with Laemmli sample buffer supplemented with 15% β-mercaptoethanol and 300 mM imidazole, and the

96 proteins bound (B) to the beads were released. The samples collected throughout the experiment

(T, FT, B) were separated by SDS-PAGE and analyzed by immunoblotting.

Blue Native (BN)-PAGE analysis. 100 – 200 µg of isolated yeast mitochondria were lysed in

BN lysis buffer (20 mM HEPES-KOH pH7.4, 50 mM NaCl, 2.5 mM MgCl2, 10% glycerol, 0.1 mM EDTA, 0.5 mM PMSF, 1% digitonin) at a concentration of 2.5 mg/ml for 30 minutes on ice.

After centrifugation at maximum speed for 10 minutes, the mitochondrial extract was separated on 6-16% BN-PAGE. Proteins were transferred to a PVDF membrane and detected by immunoblotting using indicated antibodies.

Zebrafish manipulations. Zebrafish lines derived from the characterized TL background were

maintained in a 14-hr light/10-hr dark cycle and mated for 1 hour to obtain synchronized

embryonic development. Embryos were grown for 3hpf in E3 buffer (5mM NaCl, 0.17 mM KCl,

0.33 mM CaCl2, 0.33 mM MgSO4) and then incubated with E3 buffer supplemented with 1%

DMSO or MB-20/MB-20.1 in 1% DMSO for 3 days at 28.5°C. Following treatment, embryos

were imaged using a Leica MZ16F fluorescent stereoscope at 5X magnification. Images were

resized to 300dpi without resampling using Photoshop software (Adobe).

Antibodies. The following antibodies were used in this study: PINK 494 (Novus Biologicals),

Tom20 (sc-11415), Tim 17 (sc-13293), Parkin (sc-32282) (Santa Cruz Biotechnology, Inc.), LC3

(Cell Signaling), Mortalin (NeuroMab).

97 Miscellaneous. Mitochondrial proteins were analyzed by SDS-PAGE using a 12 or 15% polyacrylamide gel and a Tricine-based running buffer. Proteins were detected by immunoblotting using PVDF membranes and visualized with HRP labeled goat secondary antibody against rabbit or mouse IgG. Chemiluminescent and autoradiographic imaging was performed on film.

98 Figures

Figure 3-1. Structure and MIC50 of MB-20 and MB20.1. (A) Chemical structures of MB-20

and MB-20.1. (B) MIC50 measurements of MB-20 and MB-20.1 in WT and tim23-2 strains.

Results showed increased sensitivity of MB-20 in tim23-2 strain. No MIC50 value can be

obtained with MB-20.1 because it does not affect the growth of yeast cells. (C) MTT viability 99 assay was used to measure the MIC50 of MB-20 and MB20.1 in HeLa cells. Results showed similar value, 9.1 μM, to the MIC50 in yeast, which is 10.6 μM. No MIC50 value can be obtained with MB-20.1 because it has very little effect on the growth of HeLa cells.

100

Figure 3-2. MB-20 targets the TIM23 complex. (A) In vitro mitochondrial import of different

precursors with MB-20 titration. Results showed MB-20 blocks import of Su9-DHFR, Cyt

b2(167)-DHFR, Cyt b2(167)A63P-DHFR but not AAC. Results suggested that MB-20 targets the

TIM23 complex. (B) In vitro mitochondrial import of Su9-DHFR and AAC with MB-20 and

MB-20.1 titration. MB20.1 showed decreased inhibition in contrast to MB-20. (C) As in (A),

same experiment was performed with mitochondria isolated from HeLa cells. Again, results

showed that MB-20 blocks import of Su9-DHFR, but not AAC. 101

Figure 3-3. MB-20 targets Tim17 specifically. (A) Yeast mitochondrial lysate was incubated with MB-20, and treated with increasing concentration of thermolysin. Tim17 is stabilized but not Tim23 in the presence of MB-20. (B) As in (A), MB-20 was titrated with 2ng/ul of thermolysin. Again, results showed that Tim17 is protected from protease degradation, but not

Tim23. (C) As in (A), the experiment was repeated with purified mitochondria from HeLa cells.

TOMM40 was used as the negative control. Again, results showed that TIMM17 is protected from protease degradation.

102

Figure 3-4. MB-20 disrupts the Tim44-Tim17 interaction and destabilizes the PAM motor.

(A) Pulldown experiment was conducted in the presence and absence of MB-20 with Tim17-His to test the interaction of the TIM23 and PAM complex. Immunoblots show that Tim23 and

Tim17 interacted but Tim44 interaction was disrupted in the presence of MB-20. (B) Another

103 pulldown experiment was conducted in the presence and absence of MB-20 with mitochondria isolated from the Tim23-His strain to test the interaction of the TIM23 and PAM complex.

Results show that Tim23 and Tim17 interact but Tim44, Pam18 and Pam16 interactions were disrupted in the presence of MB-20. (C) Isolated mitochondria were incubated with increasing concentration of MB-20 and solubilized with 1% digitonin. Soluble proteins (S) were separated from the insoluble proteins (P) by centrifugation. Treatment with the Triton X-100 was included as a positive control. 50 μM of MB-20 resulted in PAM complex precipitation. (D) Isolated mitochondria were incubated with increasing concentration of MB-20 and solubilized with 1% digitonin. BN-PAGE analysis showed that MB-20 treatment caused perturbation in TIM23CORE

complex and loss of PAM complex.

104

105

Figure 3-5. MB-20 treatment does not induce or affect mitophagy. (A) Pink1 was imported

into isolated yeast mitochondria in the presence of different concentrations of MB-20. (B) HeLa

cells were treated with DMSO, CCCP, MG132 or varying concentration of MB-20 for 3 hours.

Cells were then collected, and mitochondria were isolated from the cells. Immunoblots showed

MB-20 does not cause Pink1 accumulation. CCCP was used as a positive control. (C) HeLa cells

stably expressing eGFP-Parkin were treated with MB-20 for 3 hours and fixed for 15 minutes

with 4% formaldehyde in PBS. Mitochondria were visualized by immunostaining using

TOMM20 antibody, followed by anti-rabbit IgG antibody conjugated with Alexa Fluor 568.

Results show that Parkin is not recruited to the mitochondria. (E) HeLa cells stably expressing

eGFP-Parkin were incubated either with 5 µM MB-20 or DMSO for 30 minutes prior to 10 µM

106 CCCP treatment. The cells were treated the same way as in (D). Results showed that MB-20 does not affect Parkin recruitment to the cells.

107

Figure 3-6. MB-20 treatment induces LC3 lipidation and LC3 tubules formation. (A) HeLa

cells were treated with DMSO, CCCP, MG132 or varying concentration of MB-20 for 3 hours.

Cells were then collected, and mitochondria were isolated from the cells. Immunoblots show

MB-20 causes LC3 lipidation. (B) Experiment in (A) was repeated with MB20.1, and results show that MB20.1 does not cause LC3 lipidation as expected. (C) HeLa cells were transfected with either GFP-RFP-LC3 or eGFP-mcherry-MLS for 24 hours. Cells were then treated with 8 108 µM MB20 or MB20.1 for 3 hours, and live images were taken. Results show that MB-20 causes

LC3 tubules formation in cells, but not MB20.1. Mitochondria were more fragmented with MB-

20 treatment, but not MB-20.1.

109

Figure 3-7. MB-20 treatment in zebrafish showed aberrant neuronal phenotype. (A)

Zebrafish expressing GFP in primary motor neurons was used to investigate the neuronal

development. Embryos (4 hpf) were treated with MB-20 or 1% DMSO. Motor neurons were visualized by fluorescence microscope at 72 hpf. Neuronal cells accumulated at the tail tip with

MB-20 treatment. (B) As in (A), embryos were treated with MB-20.1 instead, and no neuronal

110 accumulation was observed. (C) As in (A), mitochondria were visualized by DsRed fused with mitochondrial targeting signal. Accumulated neuronal cells at tail tip showed loss of mitochondria.

111 Supplementary Figures

Figure S3-1. MB-20 does not impair general mitochondrial functions. (A) Mitochondrial membrane potential (Δψ) was evaluated using the DiSC3(5) dye. The dye is taken up and quenched in coupled mitochondria. Purified mitochondria were incubated with 50 or 100 μM of

MB-20 or MB-20.1 and DiSC3(5) for 5 minutes. Fluorescence was measured at excitation and

112 emission wavelengths of 620nm and 670nm respectively. CCCP addition caused release of the dye, but both MB-20 and MB-20.1 did not impair Δψ. (B) (Left panel) 50 μM or 100 μM of MB-

20 was added to purified mitochondria for 30 min at 25°C. Released proteins (S) were separated from mitochondria (P) by centrifugation. Coomassie blue staining was used to visualized proteins. As a control, treatment with the vehicle (1% DMSO) was included. The mitochondrial membranes remained intact in the presence of MB-20 and MB20.1. (C) As in (B), same experiment was performed, and immunoblot analysis was used to determine fractionation of key mitochondrial proteins. Again, results show that the mitochondrial membranes remained intact in the presence of MB-20 and MB20.1. (D) Respiration measurements were performed with an oxygen electrode using mitochondria from WT yeast in the presence of the small molecule. The slope indicates the rate of oxygen consumption. Respiration was initiated with NADH addition.

DMSO was used as a vehicle control and addition of DMSO did not significantly affect the respiration of mitochondria. As a control, CCCP was added to uncouple the mitochondria. (E) As in (D), 100 μM MB-20 was added instead of DMSO, and MB-20 addition did not alter steady state respiration hat was induced with NADH.

113

Figure S3-2. MB-20 does not affect mitochondrial membrane potential. HeLa cells were

treated with DMSO, CCCP or varying concentration of MB-20 for 3 hours. An antibody against

TOMM20 marked mitochondria and the membrane potential was verified by MitoTracker staining. MB-20 treatment does not uncouple mitochondria.

114

115

Figure S3-3. Mulan KO cells exhibit the same phenotype as WT. (A) Mulan KO cells from 2

different colonies were transfected with LC3-RFP and MLS-DsRed for 24 hours. Cells were then treated with MB-20 and live images were taken. Results show mitochondrial fragmentation as

well as LC3 tubules formation, which is the same as WT. (B) Experiment in (A) was repeated for immunoblot analysis. Cells treated with MB-20 showed LC3 lipidation, and same results were obtained with Mulan KO cells.

116

Figure S3-4. LC3 does not localize to the mitochondria or to the ER. (A) HeLa cells were co-

transfected with Sec61b-GFP (ER marker) and LC3-RFP for 24 hours. Cells were then treated

with DMSO or 8µM of MB-20, and live images were taken. Results show that LC3 does not co- localized with the ER. (B) HeLa cells were co-transfected with MLS-DsRed (mitochondria marker) and LC3-RFP for 24 hours. Cells were then treated with DMSO or 8µM of MB-20, and live images were taken. Results show that LC3 does not co-localized with the mitochondria.

117

Figure S3-5. LC3 tubules formation is also observed in MEF cells and MEF cells stably expressing Parkin-flag. Both cell lines (MEF WT and MEF stably expressing Parkin) were transfected with GFP-RFP-LC3 for 24 hours. Cells were then treated with DMSO or 8µM MB-

20, and live images were taken. LC3 tubules formation was observed.

118

Figure S3-6. MB-20 does not affect mitophagy. (A) HeLa cells stably expressing Parkin were

transfected with RFP-LC3 for 24 hours. Cells were then treated with DMSO, MB-20 alone, or in

11 9 combination with CCCP after 30 mins preincubation. Live images were taken and results show

that MB-20 does not affect Parkin recruitment to mitochondria with CCCP. (B) Same experiment

was repeated for immunoblot analysis, and results show that MB-20 does not affect PINK1 accumulation on mitochondria.

120 References

1. McBride, H. M., Neuspiel, M. & Wasiak, S. Mitochondria: More Than Just a Powerhouse.

Curr. Biol. 16, 551–560 (2006).

2. Kroemer, G. & Reed, J. C. Mitochondrial control of cell death. Nat Med 6, 513–519

(2000).

3. Newmeyer, D. D. & Ferguson-Miller, S. Mitochondria: Releasing power for life and

unleashing the machineries of death. Cell 112, 481–490 (2003).

4. Contreras, L., Drago, I., Zampese, E. & Pozzan, T. Mitochondria: The calcium

connection. Biochim. Biophys. Acta - Bioenerg. 1797, 607–618 (2010).

5. Rizzuto, R., De Stefani, D., Raffaello, A. & Mammucari, C. Mitochondria as sensors and

regulators of calcium signalling. Nat. Rev. Mol. Cell Biol. 13, 566–578 (2012).

6. Ott, M., Gogvadze, V., Orrenius, S. & Zhivotovsky, B. Mitochondria, oxidative stress and

cell death. Apoptosis 12, 913–922 (2007).

7. Lenaz, G. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta -

Bioenerg. 1366, 53–67 (1998).

8. Murley, A. & Nunnari, J. The Emerging Network of Mitochondria-Organelle Contacts.

Mol. Cell 61, 648–653 (2016).

9. Karbowski, M., Norris, K. L., Cleland, M. M., Jeong, S.-Y. & Youle, R. J. Role of Bax

and Bak in mitochondrial morphogenesis. Nature 443, 658–662 (2006).

10. Kuznetsov, A. V., Janakiraman, M., Margreiter, R. & Troppmair, J. Regulating cell

survival by controlling cellular energy production: Novel functions for ancient signaling

pathways? FEBS Lett. 577, 1–4 (2004).

121 11. Kuznetsov, A. V et al. Survival signaling by C-RAF: mitochondrial reactive oxygen

species and Ca2+ are critical targets. Mol. Cell. Biol. 28, 2304–2313 (2008).

12. Riedl, S. J. & Salvesen, G. S. The apoptosome: signalling platform of cell death. Nat. Rev.

Mol. Cell Biol. 8, 405–413 (2007).

13. Youle, R. J. CELL BIOLOGY: Cellular Demolition and the Rules of Engagement.

Science (80-. ). 315, 776–777 (2007).

14. Kotiadis, V. N., Duchen, M. R. & Osellame, L. D. Mitochondrial quality control and

communications with the nucleus are important in maintaining mitochondrial function and

cell health. Biochim. Biophys. Acta - Gen. Subj. 1840, 1254–1265 (2014).

15. van Vliet, A. R., Verfaillie, T. & Agostinis, P. New functions of mitochondria associated

membranes in cellular signaling. Biochim. Biophys. Acta - Mol. Cell Res. 1843, 2253–

2262 (2014).

16. Meisinger, C. et al. Contents, Ed. Board + Forthc. articles. Trends Biochem. Sci. 30, i

(2005).

17. Csordás, G. et al. Structural and functional features and significance of the physical

linkage between ER and mitochondria. J. Cell Biol. 174, 915–921 (2006).

18. AhYoung, A. P. et al. Conserved SMP domains of the ERMES complex bind

phospholipids and mediate tether assembly. Proc. Natl. Acad. Sci. 112, E3179–E3188

(2015).

19. Murley, A. et al. ER-associated mitochondrial division links the distribution of

mitochondria and mitochondrial DNA in yeast. Elife 2013, 1–16 (2013).

20. Sun, N., Youle, R. J. & Finkel, T. The Mitochondrial Basis of Aging. Mol. Cell 61, 654–

666 (2016).

122 21. Zhao, Q. et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21,

4411–4419 (2002).

22. Martinus, R. D. et al. Selective Induction of Mitochondrial Chaperones in Response to

Loss of the Mitochondrial Genome. Eur. J. Biochem. 240, 98–103 (1996).

23. Eiyama, A. & Okamoto, K. PINK1/Parkin-mediated mitophagy in mammalian cells. Curr.

Opin. Cell Biol. 33, 95–101 (2015).

24. Pickrell, A. M. & Youle, R. J. The roles of PINK1, Parkin, and mitochondrial fidelity in

parkinson’s disease. Neuron 85, 257–273 (2015).

25. Jin, S. M. & Youle, R. J. The accumulation of misfolded proteins in the mitochondrial

matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized

mitochondria. Autophagy 9, 1750–1757 (2013).

26. Pellegrino, M. W. & Haynes, C. M. Mitophagy and the mitochondrial unfolded protein

response in neurodegeneration and bacterial infection. BMC Biol. 13, 22 (2015).

27. Nunnari, J. & Suomalainen, A. Mitochondria: In sickness and in health. Cell 148, 1145–

1159 (2012).

28. Dyall, S. D. Ancient Invasions: From Endosymbionts to Organelles. Science (80-. ). 304,

253–257 (2004).

29. Gray, M. W., Gray, M. W., Burger, G. & Lang, B. F. Mitochondrial Evolution. 1476, 1–

16 (2008).

30. Chacinska, A., Koehler, C. M., Milenkovic, D., Lithgow, T. & Pfanner, N. Importing

Mitochondrial Proteins: Machineries and Mechanisms. Cell 138, 628–644 (2009).

31. Koehler, C. M. New Developments in Mitochondrial Assembly. Annu. Rev. Cell Dev.

Biol. 20, 309–335 (2004).

123