DOCSLIB.ORG

An in Vitro Vesicle Formation Assay Reveals Cargo Clients and Factors That Mediate Vesicular Trafficking

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
An in Vitro Vesicle Formation Assay Reveals Cargo Clients and Factors That Mediate Vesicular Trafficking An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking Yan Huanga,1, Haidi Yinb,c,1, Baiying Lid,1, Qian Wub,c, Yang Liua, Kristina Poljake, Julija Maldutytee, Xiao Tanga, Mo Wanga, Zhixiao Wua, Elizabeth A. Millere, Liwen Jiangd, Zhong-Ping Yaob,c,2, and Yusong Guoa,f,g,2 aDivision of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China; bState Key Laboratory of Chemical Biology and Drug Discovery, Research Institute for Future Food, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region, China; cState Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Instituteof Hong Kong Polytechnic University, Shenzhen 518057, China; dSchool of Life Sciences, Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; eCell Biology Division, Medical Research Council Laboratory of Molecular Biology, CB2 0QH, Cambridge, United Kingdom; fShenzhen Research Institute, The Hong Kong University of Science and Technology, 518057 Shenzhen, China; and gHong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China Edited by Peter J. Novick, University of California San Diego, La Jolla, CA, and approved July 13, 2021 (received for review January 21, 2021) The fidelity of protein transport in the secretory pathway relies on II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the accurate sorting of proteins to their correct destinations. To the lumen of the ER cannot be directly recognized by COPII coat deepen our understanding of the underlying molecular mechanisms, and such proteins are thought to be linked to the cargo sorting it is important to develop a robust approach to systematically machinery on the cytosolic side by transmembrane cargo receptors. reveal cargo proteins that depend on specific sorting machinery to One cargo receptor in mammalian cells, ERGIC53, is a mannose be enriched into transport vesicles. Here, we used an in vitro assay lectin and functions in capturing specific N-linked glycoproteins in that reconstitutes packaging of human cargo proteins into vesicles the lumen of the ER (3). ERGIC53 regulates ER export of blood to quantify cargo capture. Quantitative mass spectrometry (MS) coagulation factors V and VIII, a cathepsin-Z–related protein, and analyses of the isolated vesicles revealed cytosolic proteins that alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds are associated with vesicle membranes in a GTP-dependent man- amino-terminal tripeptide motifs of soluble cargo proteins and CELL BIOLOGY ner. We found that two of them, FAM84B (also known as LRAT regulates ER export of soluble cargo proteins, including the yolk domain containing 2 or LRATD2) and PRRC1, contain proline-rich protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and domains and regulate anterograde trafficking. Further analyses apolipoprotein B in mammalian cells (9–11). revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit Although significant progress has been made in understanding sites, interacts with the inner COPII coat, and its absence increases the general steps of cargo sorting, the spectrum of cargo clients membrane association of COPII. In addition, we uncovered cargo of a specific Arf family member, cargo adaptor, or cargo receptor proteins that depend on GTP hydrolysis to be captured into vesicles. remains largely underinvestigated. To deepen our understand- Comparing control cells with cells depleted of the cargo receptors, ing of protein sorting in the secretory pathway, it is important to SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation as- Significance say in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular traf- ficking and to uncover cargo clients of specific cellular factors. Protein sorting in the secretory pathway is a fundamentally im- portant cellular process, but the clients of a specific cargo sorting machinery remains largely underinvestigated. Here, utilizing a cargo sorting | secretory pathway | intracellular protein transport | vesicle formation assay to profile proteins associated with vesi- COPII | cargo receptor cles, we identified cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact he eukaryotic secretory pathway plays important roles in de- with GTP-bound Sar1A. We found that two of them, FAM84B and Tlivering a variety of newly synthesized proteins to their specific PRRC1, regulate anterograde trafficking. Moreover, we revealed resident compartments. The fidelity of protein transport in the specific clients of two export adaptors, SURF4 and ERGIC53. These secretory pathway depends on accurate sorting of specific cargo analyses demonstrate that our approach is powerful to identify proteins into transport vesicles. Defects in cargo sorting cause protein factors that regulate vesicular trafficking and to uncover clients mistargeting and induce defects in establishing cell polarity, im- of specific cargo receptors, providing a robust method to reveal munity, as well as other physiological processes (1). insights into the secretory pathway. A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic Author contributions: Y.H. and Y.G. designed research; Y.H., H.Y., B.L., Q.W., Y.L., K.P., proteins include small GTPases of the Arf family and cargo adaptors J.M., X.T., M.W., Z.W., and Y.G. performed research; E.A.M., L.J., Z.-P.Y., and Y.G. contrib- uted new reagents/analytic tools; Y.H., H.Y., E.A.M., L.J., Z.-P.Y., and Y.G. analyzed data; (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic and Y.H., H.Y., E.A.M., Z.-P.Y., and Y.G. wrote the paper. state and a GTP-bound state. Upon GTP binding, Arf proteins un- The authors declare no competing interest. dergo conformational changes in which the N-terminal amphipathic This article is a PNAS Direct Submission. helix is exposed to bind membranes and the switch domains change This open access article is distributed under Creative Commons Attribution License 4.0 their conformation to recruit various cytosolic cargo adaptors. Once (CC BY). recruited onto the membranes, these cargo adaptors recognize sort- 1Y.H., H.Y., and B.L. contributed equally to this work. ing motifs on the cargo proteins. This recognition step is important 2To whom correspondence may be addressed. Email: [email protected] or zhongping. for efficiently capturing cargo proteins into vesicles. [email protected]. The Arf family protein, Sar1, regulates packaging of cargo pro- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ teins into vesicles at the endoplasmic reticulum (ER). GTP-bound doi:10.1073/pnas.2101287118/-/DCSupplemental. Sar1 mediates membrane recruitment of the coat protein complex Published August 25, 2021. PNAS 2021 Vol. 118 No. 35 e2101287118 https://doi.org/10.1073/pnas.2101287118 | 1of12 Downloaded by guest on September 24, 2021 develop a robust approach to systematically reveal cargo proteins (13). This is possibly due to an unappreciated requirement for that depend on a specific factor to be efficiently packaged into other cytosolic factors in addition to the COP coats. Affinity vesicles. Revealing this will provide significant insight into the chromatography has been utilized to reveal cytosolic proteins that functions and the specificity of cargo sorting. Since distinct cyto- specifically interact with GTP-bound Arf or Rab proteins (14–16). solic proteins are recruited to membranes by different GTP-bound In this approach, the membranes are disrupted, which might Arf family proteins, systematic approaches are needed to char- preclude identification of membrane-associated effectors. Thus, it acterize budding events associated with a specific GTP-bound Arf is important to develop additional approaches to reveal novel family protein. cytosolic proteins that associate with GTP-bound Arf proteins on A cellular imaging approach, pairing analysis of cargo recep- membranes. tors (PAIRS), has been utilized to identify the spectrum of cargo Here, we used an in vitro assay to reconstitute packaging of cargo proteins that depend on a specific cargo receptor for ER export proteins into transport vesicles utilizing rat liver cytosol (RLC) as a in yeast. This analysis focused on around 150 cargo molecules source of cytosolic proteins. Analysis of vesicle fractions by quan- labeled with fluorescent tags (12). An in vitro assay that recon- titative mass spectrometry (MS) revealed cytosolic proteins that are stitutes packaging of cargo proteins into vesicles has been used to associated with vesicles dependent on GTP or GTP-bound Sar1A, reveal protein profiles of vesicles budded with purified COPII or and that regulate protein trafficking. One of the identified proteins, COPI proteins (13). However, this analysis did not identify any PRRC1, regulates membrane association of the COPII coat
Load more

Recommended publications
  • Cyclin D1/Cyclin-Dependent Kinase 4 Interacts with Filamin a and Affects the Migration and Invasion Potential of Breast Cancer Cells
    Published OnlineFirst February 28, 2010; DOI: 10.1158/0008-5472.CAN-08-1108 Tumor and Stem Cell Biology Cancer Research Cyclin D1/Cyclin-Dependent Kinase 4 Interacts with Filamin A and Affects the Migration and Invasion Potential of Breast Cancer Cells Zhijiu Zhong, Wen-Shuz Yeow, Chunhua Zou, Richard Wassell, Chenguang Wang, Richard G. Pestell, Judy N. Quong, and Andrew A. Quong Abstract Cyclin D1 belongs to a family of proteins that regulate progression through the G1-S phase of the cell cycle by binding to cyclin-dependent kinase (cdk)-4 to phosphorylate the retinoblastoma protein and release E2F transcription factors for progression through cell cycle. Several cancers, including breast, colon, and prostate, overexpress the cyclin D1 gene. However, the correlation of cyclin D1 overexpression with E2F target gene regulation or of cdk-dependent cyclin D1 activity with tumor development has not been identified. This suggests that the role of cyclin D1 in oncogenesis may be independent of its function as a cell cycle regulator. One such function is the role of cyclin D1 in cell adhesion and motility. Filamin A (FLNa), a member of the actin-binding filamin protein family, regulates signaling events involved in cell motility and invasion. FLNa has also been associated with a variety of cancers including lung cancer, prostate cancer, melanoma, human bladder cancer, and neuroblastoma. We hypothesized that elevated cyclin D1 facilitates motility in the invasive MDA-MB-231 breast cancer cell line. We show that MDA-MB-231 motility is affected by disturbing cyclin D1 levels or cyclin D1-cdk4/6 kinase activity.
    [Show full text]
  • Molecular Subtypes of Diffuse Large B-Cell Lymphoma Arise by Distinct Genetic Pathways
    Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways Georg Lenza, George W. Wrightb, N. C. Tolga Emrea, Holger Kohlhammera, Sandeep S. Davea, R. Eric Davisa, Shannon Cartya, Lloyd T. Lama, A. L. Shaffera, Wenming Xiaoc, John Powellc, Andreas Rosenwaldd, German Ottd,e, Hans Konrad Muller-Hermelinkd, Randy D. Gascoynef, Joseph M. Connorsf, Elias Campog, Elaine S. Jaffeh, Jan Delabiei, Erlend B. Smelandj,k, Lisa M. Rimszal, Richard I. Fisherm,n, Dennis D. Weisenburgero, Wing C. Chano, and Louis M. Staudta,q aMetabolism Branch, bBiometric Research Branch, cCenter for Information Technology, and hLaboratory of Pathology, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892; dDepartment of Pathology, University of Wu¨rzburg, 97080 Wu¨rzburg, Germany; eDepartment of Clinical Pathology, Robert-Bosch-Krankenhaus, 70376 Stuttgart, Germany; fBritish Columbia Cancer Agency, Vancouver, British Columbia, Canada V5Z 4E6; gHospital Clinic, University of Barcelona, 08036 Barcelona, Spain; iPathology Clinic and jInstitute for Cancer Research, Rikshospitalet Hospital, Oslo, Norway; kCentre for Cancer Biomedicine, Faculty Division the Norwegian Radium Hospital, N-0310, Oslo, Norway; lDepartment of Pathology, University of Arizona, Tucson, AZ 85724; mSouthwest Oncology Group, 24 Frank Lloyd Wright Drive, Ann Arbor, MI 48106 ; nJames P. Wilmot Cancer Center, University of Rochester, Rochester, NY 14642; and oDepartments of Pathology and Microbiology, University of Nebraska, Omaha, NE 68198 Edited by Ira
    [Show full text]
  • Of GTP-Binding Proteins: ARF, ARL, and SAR Proteins
    Published Online: 27 February, 2006 | Supp Info: http://doi.org/10.1083/jcb.200512057 JCB: COMMENT Downloaded from jcb.rupress.org on May 9, 2019 <doi>10.1083/jcb.200512057</doi><aid>200512057</aid>Nomenclature for the human Arf family of GTP-binding proteins: ARF, ARL, and SAR proteins Richard A. Kahn,1 Jacqueline Cherfi ls,2 Marek Elias,3 Ruth C. Lovering,4 Sean Munro,5 and Annette Schurmann6 1Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322 2Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifi que, 91198 Gif-sur-Yvette, France 3Department of Plant Physiology, Faculty of Science, Charles University, 128 44 Prague 2, Czech Republic 4Human Genome Organisation Gene Nomenclature Committee, Galton Laboratory, Department of Biology, University College London, London NW1 2HE, United Kingdom 5Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom 6Department of Pharmacology, German Institute of Human Nutrition Potsdam-Rehbrücke, D-14558 Nuthetal, Germany The Ras superfamily is comprised of at least four large they have been found to be ubiquitous regulators of membrane families of regulatory guanosine triphosphate–binding traffi c and phospholipid metabolism in eukaryotic cells (for re- views and discussion of Arf actions see Nie et al., 2003; Burd proteins, including the Arfs. The Arf family includes three et al., 2004; Kahn, 2004). Arfs are soluble proteins that translocate different groups of proteins: the Arfs, Arf-like (Arls), and onto membranes in concert with their activation, or GTP bind- SARs. Several Arf family members have been very highly ing. The biological actions of Arfs are thought to occur on mem- conserved throughout eukaryotic evolution and have branes and to result from their specifi c interactions with a large orthologues in evolutionally diverse species.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Location Analysis of Estrogen Receptor Target Promoters Reveals That
    Location analysis of estrogen receptor ␣ target promoters reveals that FOXA1 defines a domain of the estrogen response Jose´ e Laganie` re*†, Genevie` ve Deblois*, Ce´ line Lefebvre*, Alain R. Bataille‡, Franc¸ois Robert‡, and Vincent Gigue` re*†§ *Molecular Oncology Group, Departments of Medicine and Oncology, McGill University Health Centre, Montreal, QC, Canada H3A 1A1; †Department of Biochemistry, McGill University, Montreal, QC, Canada H3G 1Y6; and ‡Laboratory of Chromatin and Genomic Expression, Institut de Recherches Cliniques de Montre´al, Montreal, QC, Canada H2W 1R7 Communicated by Ronald M. Evans, The Salk Institute for Biological Studies, La Jolla, CA, July 1, 2005 (received for review June 3, 2005) Nuclear receptors can activate diverse biological pathways within general absence of large scale functional data linking these putative a target cell in response to their cognate ligands, but how this binding sites with gene expression in specific cell types. compartmentalization is achieved at the level of gene regulation is Recently, chromatin immunoprecipitation (ChIP) has been used poorly understood. We used a genome-wide analysis of promoter in combination with promoter or genomic DNA microarrays to occupancy by the estrogen receptor ␣ (ER␣) in MCF-7 cells to identify loci recognized by transcription factors in a genome-wide investigate the molecular mechanisms underlying the action of manner in mammalian cells (20–24). This technology, termed 17␤-estradiol (E2) in controlling the growth of breast cancer cells. ChIP-on-chip or location analysis, can therefore be used to deter- We identified 153 promoters bound by ER␣ in the presence of E2. mine the global gene expression program that characterize the Motif-finding algorithms demonstrated that the estrogen re- action of a nuclear receptor in response to its natural ligand.
    [Show full text]
  • Familial Multiple Coagulation Factor Deficiencies
    Journal of Clinical Medicine Article Familial Multiple Coagulation Factor Deficiencies (FMCFDs) in a Large Cohort of Patients—A Single-Center Experience in Genetic Diagnosis Barbara Preisler 1,†, Behnaz Pezeshkpoor 1,† , Atanas Banchev 2 , Ronald Fischer 3, Barbara Zieger 4, Ute Scholz 5, Heiko Rühl 1, Bettina Kemkes-Matthes 6, Ursula Schmitt 7, Antje Redlich 8 , Sule Unal 9 , Hans-Jürgen Laws 10, Martin Olivieri 11 , Johannes Oldenburg 1 and Anna Pavlova 1,* 1 Institute of Experimental Hematology and Transfusion Medicine, University Clinic Bonn, 53127 Bonn, Germany; [email protected] (B.P.); [email protected] (B.P.); [email protected] (H.R.); [email protected] (J.O.) 2 Department of Paediatric Haematology and Oncology, University Hospital “Tzaritza Giovanna—ISUL”, 1527 Sofia, Bulgaria; [email protected] 3 Hemophilia Care Center, SRH Kurpfalzkrankenhaus Heidelberg, 69123 Heidelberg, Germany; ronald.fi[email protected] 4 Department of Pediatrics and Adolescent Medicine, University Medical Center–University of Freiburg, 79106 Freiburg, Germany; [email protected] 5 Center of Hemostasis, MVZ Labor Leipzig, 04289 Leipzig, Germany; [email protected] 6 Hemostasis Center, Justus Liebig University Giessen, 35392 Giessen, Germany; [email protected] 7 Center of Hemostasis Berlin, 10789 Berlin-Schöneberg, Germany; [email protected] 8 Pediatric Oncology Department, Otto von Guericke University Children’s Hospital Magdeburg, 39120 Magdeburg, Germany; [email protected] 9 Division of Pediatric Hematology Ankara, Hacettepe University, 06100 Ankara, Turkey; Citation: Preisler, B.; Pezeshkpoor, [email protected] B.; Banchev, A.; Fischer, R.; Zieger, B.; 10 Department of Pediatric Oncology, Hematology and Clinical Immunology, University of Duesseldorf, Scholz, U.; Rühl, H.; Kemkes-Matthes, 40225 Duesseldorf, Germany; [email protected] B.; Schmitt, U.; Redlich, A.; et al.
    [Show full text]
  • Supporting Information
    Supporting Information Edgar et al. 10.1073/pnas.1601895113 SI Methods (Actimetrics), and recordings were analyzed using LumiCycle Mice. Sample size was determined using the resource equation: Data Analysis software (Actimetrics). E (degrees of freedom in ANOVA) = (total number of exper- – Cell Cycle Analysis of Confluent Cell Monolayers. NIH 3T3, primary imental animals) (number of experimental groups), with −/− sample size adhering to the condition 10 < E < 20. For com- WT, and Bmal1 fibroblasts were sequentially transduced − − parison of MuHV-4 and HSV-1 infection in WT vs. Bmal1 / with lentiviral fluorescent ubiquitin-based cell cycle indicators mice at ZT7 (Fig. 2), the investigator did not know the genotype (FUCCI) mCherry::Cdt1 and amCyan::Geminin reporters (32). of the animals when conducting infections, bioluminescence Dual reporter-positive cells were selected by FACS (Influx Cell imaging, and quantification. For bioluminescence imaging, Sorter; BD Biosciences) and seeded onto 35-mm dishes for mice were injected intraperitoneally with endotoxin-free lucif- subsequent analysis. To confirm that expression of mCherry:: Cdt1 and amCyan::Geminin correspond to G1 (2n DNA con- erin (Promega E6552) using 2 mg total per mouse. Following < ≤ anesthesia with isofluorane, they were scanned with an IVIS tent) and S/G2 (2 n 4 DNA content) cell cycle phases, Lumina (Caliper Life Sciences), 15 min after luciferin admin- respectively, cells were stained with DNA dye DRAQ5 (abcam) and analyzed by flow cytometry (LSR-Fortessa; BD Biosci- istration. Signal intensity was quantified using Living Image ences). To examine dynamics of replicative activity under ex- software (Caliper Life Sciences), obtaining maximum radiance perimental confluent conditions, synchronized FUCCI reporter for designated regions of interest (photons per second per − − − monolayers were observed by time-lapse live cell imaging over square centimeter per Steradian: photons·s 1·cm 2·sr 1), relative 3 d (Nikon Eclipse Ti-E inverted epifluorescent microscope).
    [Show full text]
  • Conserved and Novel Properties of Clathrin-Mediated Endocytosis in Dictyostelium Discoideum" (2012)
    Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2012 Conserved and Novel Properties of Clathrin- Mediated Endocytosis in Dictyostelium Discoideum Laura Macro Follow this and additional works at: http://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons Recommended Citation Macro, Laura, "Conserved and Novel Properties of Clathrin-Mediated Endocytosis in Dictyostelium Discoideum" (2012). Student Theses and Dissertations. Paper 163. This Thesis is brought to you for free and open access by Digital Commons @ RU. It has been accepted for inclusion in Student Theses and Dissertations by an authorized administrator of Digital Commons @ RU. For more information, please contact [email protected]. CONSERVED AND NOVEL PROPERTIES OF CLATHRIN- MEDIATED ENDOCYTOSIS IN DICTYOSTELIUM DISCOIDEUM A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Laura Macro June 2012 © Copyright by Laura Macro 2012 CONSERVED AND NOVEL PROPERTIES OF CLATHRIN- MEDIATED ENDOCYTOSIS IN DICTYOSTELIUM DISCOIDEUM Laura Macro, Ph.D. The Rockefeller University 2012 The protein clathrin mediates one of the major pathways of endocytosis from the extracellular milieu and plasma membrane. Clathrin functions with a network of interacting accessory proteins, one of which is the adaptor complex AP-2, to co-ordinate vesicle formation. Disruption of genes involved in clathrin-mediated endocytosis causes embryonic lethality in multicellular animals suggesting that clathrin-mediated endocytosis is a fundamental cellular process. However, loss of clathrin-mediated endocytosis genes in single cell eukaryotes, such as S.cerevisiae (yeast), does not cause lethality, suggesting that clathrin may convey specific advantages for multicellularity.
    [Show full text]
  • LMAN1 Mouse Monoclonal Antibody [Clone ID: OTI1E3] – TA502138
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA502138 LMAN1 Mouse Monoclonal Antibody [Clone ID: OTI1E3] Product data: Product Type: Primary Antibodies Clone Name: OTI1E3 Applications: FC, IF, IHC, WB Recommended Dilution: WB 1:500~2000, IHC 1:150, IF 1:100, FLOW 1:100 Reactivity: Human, Mouse, Rat, Dog Host: Mouse Isotype: IgG1 Clonality: Monoclonal Immunogen: Full length human recombinant protein of human LMAN1 (NP_005561) produced in HEK293T cell. Formulation: PBS (PH 7.3) containing 1% BSA, 50% glycerol and 0.02% sodium azide. Concentration: 0.24 mg/ml Purification: Purified from mouse ascites fluids or tissue culture supernatant by affinity chromatography (protein A/G) Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 54.2 kDa Gene Name: lectin, mannose binding 1 Database Link: NP_005561 Entrez Gene 70361 MouseEntrez Gene 116666 RatEntrez Gene 476186 DogEntrez Gene 3998 Human P49257 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 6 LMAN1 Mouse Monoclonal Antibody [Clone ID: OTI1E3] – TA502138 Background: The protein encoded by this gene is a type I integral membrane protein localized in the intermediate region between the endoplasmic reticulum and the Golgi, presumably recycling between the two compartments. The protein is a mannose-specific lectin and is a member of a novel family of plant lectin homologs in the secretory pathway of animal cells.
    [Show full text]
  • Supplementary Figures 1-14 and Supplementary References
    SUPPORTING INFORMATION Spatial Cross-Talk Between Oxidative Stress and DNA Replication in Human Fibroblasts Marko Radulovic,1,2 Noor O Baqader,1 Kai Stoeber,3† and Jasminka Godovac-Zimmermann1* 1Division of Medicine, University College London, Center for Nephrology, Royal Free Campus, Rowland Hill Street, London, NW3 2PF, UK. 2Insitute of Oncology and Radiology, Pasterova 14, 11000 Belgrade, Serbia 3Research Department of Pathology and UCL Cancer Institute, Rockefeller Building, University College London, University Street, London WC1E 6JJ, UK †Present Address: Shionogi Europe, 33 Kingsway, Holborn, London WC2B 6UF, UK TABLE OF CONTENTS 1. Supplementary Figures 1-14 and Supplementary References. Figure S-1. Network and joint spatial razor plot for 18 enzymes of glycolysis and the pentose phosphate shunt. Figure S-2. Correlation of SILAC ratios between OXS and OAC for proteins assigned to the SAME class. Figure S-3. Overlap matrix (r = 1) for groups of CORUM complexes containing 19 proteins of the 49-set. Figure S-4. Joint spatial razor plots for the Nop56p complex and FIB-associated complex involved in ribosome biogenesis. Figure S-5. Analysis of the response of emerin nuclear envelope complexes to OXS and OAC. Figure S-6. Joint spatial razor plots for the CCT protein folding complex, ATP synthase and V-Type ATPase. Figure S-7. Joint spatial razor plots showing changes in subcellular abundance and compartmental distribution for proteins annotated by GO to nucleocytoplasmic transport (GO:0006913). Figure S-8. Joint spatial razor plots showing changes in subcellular abundance and compartmental distribution for proteins annotated to endocytosis (GO:0006897). Figure S-9. Joint spatial razor plots for 401-set proteins annotated by GO to small GTPase mediated signal transduction (GO:0007264) and/or GTPase activity (GO:0003924).
    [Show full text]
  • CD29 Identifies IFN-Γ–Producing Human CD8+ T Cells With
    + CD29 identifies IFN-γ–producing human CD8 T cells with an increased cytotoxic potential Benoît P. Nicoleta,b, Aurélie Guislaina,b, Floris P. J. van Alphenc, Raquel Gomez-Eerlandd, Ton N. M. Schumacherd, Maartje van den Biggelaarc,e, and Monika C. Wolkersa,b,1 aDepartment of Hematopoiesis, Sanquin Research, 1066 CX Amsterdam, The Netherlands; bLandsteiner Laboratory, Oncode Institute, Amsterdam University Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands; cDepartment of Research Facilities, Sanquin Research, 1066 CX Amsterdam, The Netherlands; dDivision of Molecular Oncology and Immunology, Oncode Institute, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands; and eDepartment of Molecular and Cellular Haemostasis, Sanquin Research, 1066 CX Amsterdam, The Netherlands Edited by Anjana Rao, La Jolla Institute for Allergy and Immunology, La Jolla, CA, and approved February 12, 2020 (received for review August 12, 2019) Cytotoxic CD8+ T cells can effectively kill target cells by producing therefore developed a protocol that allowed for efficient iso- cytokines, chemokines, and granzymes. Expression of these effector lation of RNA and protein from fluorescence-activated cell molecules is however highly divergent, and tools that identify and sorting (FACS)-sorted fixed T cells after intracellular cytokine + preselect CD8 T cells with a cytotoxic expression profile are lacking. staining. With this top-down approach, we performed an un- + Human CD8 T cells can be divided into IFN-γ– and IL-2–producing biased RNA-sequencing (RNA-seq) and mass spectrometry cells. Unbiased transcriptomics and proteomics analysis on cytokine- γ– – + + (MS) analyses on IFN- and IL-2 producing primary human producing fixed CD8 T cells revealed that IL-2 cells produce helper + + + CD8 Tcells.
    [Show full text]
  • Association of Gene Ontology Categories with Decay Rate for Hepg2 Experiments These Tables Show Details for All Gene Ontology Categories
    Supplementary Table 1: Association of Gene Ontology Categories with Decay Rate for HepG2 Experiments These tables show details for all Gene Ontology categories. Inferences for manual classification scheme shown at the bottom. Those categories used in Figure 1A are highlighted in bold. Standard Deviations are shown in parentheses. P-values less than 1E-20 are indicated with a "0". Rate r (hour^-1) Half-life < 2hr. Decay % GO Number Category Name Probe Sets Group Non-Group Distribution p-value In-Group Non-Group Representation p-value GO:0006350 transcription 1523 0.221 (0.009) 0.127 (0.002) FASTER 0 13.1 (0.4) 4.5 (0.1) OVER 0 GO:0006351 transcription, DNA-dependent 1498 0.220 (0.009) 0.127 (0.002) FASTER 0 13.0 (0.4) 4.5 (0.1) OVER 0 GO:0006355 regulation of transcription, DNA-dependent 1163 0.230 (0.011) 0.128 (0.002) FASTER 5.00E-21 14.2 (0.5) 4.6 (0.1) OVER 0 GO:0006366 transcription from Pol II promoter 845 0.225 (0.012) 0.130 (0.002) FASTER 1.88E-14 13.0 (0.5) 4.8 (0.1) OVER 0 GO:0006139 nucleobase, nucleoside, nucleotide and nucleic acid metabolism3004 0.173 (0.006) 0.127 (0.002) FASTER 1.28E-12 8.4 (0.2) 4.5 (0.1) OVER 0 GO:0006357 regulation of transcription from Pol II promoter 487 0.231 (0.016) 0.132 (0.002) FASTER 6.05E-10 13.5 (0.6) 4.9 (0.1) OVER 0 GO:0008283 cell proliferation 625 0.189 (0.014) 0.132 (0.002) FASTER 1.95E-05 10.1 (0.6) 5.0 (0.1) OVER 1.50E-20 GO:0006513 monoubiquitination 36 0.305 (0.049) 0.134 (0.002) FASTER 2.69E-04 25.4 (4.4) 5.1 (0.1) OVER 2.04E-06 GO:0007050 cell cycle arrest 57 0.311 (0.054) 0.133 (0.002)
    [Show full text]