Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts

Chad D. Williamson1, Daniel S. Wong2, Petros Bozidis3, Aiping Zhang4, Anamaris M. Colberg‐Poley5

1 Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, 2 Cellular and Molecular Physiology Program, Sackler School for Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts, 3 Laboratory of Microbiology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece, 4 Center for Genetic Medicine Research, Children's Research Institute, Washington, D.C., 5 Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C.
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.27
DOI:  10.1002/0471143030.cb0327s68
Online Posting Date:  September, 2015
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Abstract

Increasingly mechanistic virology studies require dependable and sensitive methods for isolating purified organelles containing functional cellular sub‐domains. The mitochondrial network is, in part, closely apposed to the endoplasmic reticulum (ER). The mitochondria‐associated membrane (MAM) fraction provides direct physical contact between the ER and mitochondria. Characterization of the dual localization and trafficking of human cytomegalovirus (HCMV) UL37 proteins required establishing protocols in which the ER and mitochondria could be reliably separated. Because of its documented role in lipid and ceramide transfer from the ER to mitochondria, a method to purify MAM from infected cells was also developed. Two robust procedures were developed to efficiently isolate mitochondria, ER, and MAM fractions while providing substantial protein yields from HCMV‐infected primary fibroblasts and from transfected HeLa cells. Furthermore, this unit includes protocols for isolation of detergent resistant membranes from subcellular fractions as well as techniques that allow visualization of the mitochondrial network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. © 2015 by John Wiley & Sons, Inc.

Keywords: subcellular fractionation; human fibroblasts; ER; mitochondria; MAM; HCMV; protein localization; sucrose gradient; Percoll gradient; differential centrifugation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Differential Sucrose Gradient Isolation of ER and Mitochondria
  • Basic Protocol 2: Separation of Mitochondria and Mitochondria‐Associated Membrane Fraction
  • Basic Protocol 3: Flotation of Detergent‐Resistant MAM Membranes from Crude ER or Mitochondrial Fractions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Differential Sucrose Gradient Isolation of ER and Mitochondria

  Materials
  • Human foreskin fibroblasts (HFFs; Viromed SF cells)
  • HeLa cells (ATCC CCL‐2)
  • HCMV (desired strain) or DNA for transfection
  • 2% and 10% (v/v) FBS
  • Lipofectamine 2000 (Invitrogen; unit 20.6; Hawley‐Nelson and Ciccarone, )
  • Opti‐MEM I (Invitrogen)
  • 1.0, 1.3, 1.5, 1.7, and 2.0 M sucrose solutions (see reciperecipes), sterile
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.25% trypsin/EDTA (Life Technologies, cat. no. 25200114)
  • 1× mannitol/Tris/EDTA (1× MTE) buffer (see recipe)
  • 100 mM PMSF stock (see recipe)
  • Ultrapure water
  • 70% ethanol
  • 175‐cm2 flasks (∼0.8–2 × 107 cells/flask)
  • 37°C CO 2 incubator
  • Pre‐sterilized (autoclaved) Beckman polyallomer centrifuge tubes: 14 × 89–mm (cat. no. 331372) or 11 × 60–mm (cat. no. 328874)
  • 5‐ml serological pipets
  • Aspirator
  • Sterile 15‐ml conical tubes
  • Beckman GS‐6R centrifuge with GH‐3.8 swinging‐bucket rotor
  • Analog sonicator with 1/8‐in. microtip (Branson Ultrasonics model 250)
  • 250‐ml glass beakers
  • 1.5‐ml microcentrifuge tubes
  • 14‐ml polypropylene, round‐bottom, snap‐cap tubes (17 × 100–mm; Falcon cat. no. 2059)
  • Beckman J2‐MI centrifuge with JA20.1 rotor
  • Beckman XL‐90 ultracentrifuge with SW60 Ti and SW41 Ti rotors
  • 1‐ml syringes and 20‐G needles
  • Parafilm
  • Beckman GS‐15R centrifuge with F2402 rotor

Basic Protocol 2: Separation of Mitochondria and Mitochondria‐Associated Membrane Fraction

  Materials
  • Untransfected or transfected HeLa cells (3 × 107 cells, ten 100 × 20–mm tissue culture dishes, 100% confluent); uninfected or HCMV‐infected HFF cells (5 × 107 cells, four 850‐cm2 roller bottles, 90% confluent); or twenty 100 × 20‐mm tissue culture dishes, 70% to 90% confluent), and appropriate complete medium for the cell line
  • Phosphate‐buffered saline (PBS), pH 7.4 ( appendix 2A)
  • 0.25% trypsin/EDTA (e.g., Life Technologies)
  • Sucrose homogenization medium (SHM; see recipe), ice cold
  • MSF (optional but recommended, see recipe)
  • 70% ethanol
  • Mannitol buffer A (see recipe), ice cold
  • 30% (v/v) Percoll solution (see recipe), ice cold
  • Mannitol buffer B (see recipe), ice cold
  • Cell lifter, sterile
  • 50‐ and 15‐ml conical tubes (e.g., Corning Falcon)
  • Bright‐field microscope
  • Beckman GS‐15R tabletop centrifuge with swinging‐bucket rotor (e.g., S4180) and fixed‐angle rotor (e.g., F2402)
  • 12 × 75‐mm polypropylene tubes (e.g., Corning Falcon)
  • 2‐ml Potter‐Elvehjem plastic‐coasted tissue grinder with PTFE pestle (e.g., Wheaton safe‐grind type, cat. no. 358003)
  • Overhead stirrer for tissue grinder (Wheaton, cat no. 903475)
  • Phase‐contrast microscope
  • 16 × 76–mm Ultraclear ultracentrifuge tubes (Beckman, cat. no. 344085)
  • Sorvall Legend RT centrifuge or equivalent
  • Millipore Amicon Ultra‐15 Centrifugal Filter Units with Ultracel‐3 membrane (3‐Da cutoff; 24‐pk: Millipore cat. no. 900324)
  • Beckman XL‐90 ultracentrifuge with Ti 70 fixed‐angle rotor
  • 14 × 89–mm Ultraclear ultracentrifuge tubes (Beckman, cat. no. 344059)
  • 50‐ml beaker
  • 1‐ml syringes and 20‐G needles

Basic Protocol 3: Flotation of Detergent‐Resistant MAM Membranes from Crude ER or Mitochondrial Fractions

  Materials
  • MBST buffer, ice cold (see recipe)
  • 5%, 30%, and 90% sucrose in MBS buffer, ice cold (see reciperecipes)
  • Analog sonicator with 1/8‐in. microtip (optional; Branson Ultrasonics model 250)
  • Polyallomer ultracentrifuge tubes, 11 × 60–mm (Beckman, cat. no. 328874), sterilized by autoclaving and then pre‐chilled
  • Beckman XL‐90 ultracentrifuge with SW60 Ti rotor
  • Additional reagents and equipment for isolation of ER/MAM pellet ( protocol 1, step 24) or a MAM/mitochondria pellet ( protocol 2, step 20)
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Figures

Videos

Literature Cited

Literature Cited
  Ardail, D., Popa, I., Bodennec, J., Louisot, P., Schmitt, D., and Portoukalian, J. 2003. The mitochondria‐associated endoplasmic‐reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid‐specific glycosyltransferases. Biochem. J. 371:1013‐1019.
  Bartolome, A., Guillen, C., and Benito, M. 2012. Autophagy plays a protective role in endoplasmic reticulum stress‐mediated pancreatic beta cell death. Autophagy 8:1757‐1768.
  Bereiter‐Hahn, J. and Voth, M. 1994. Dynamics of mitochondria in living cells: Shape changes, dislocations, fusion, and fission of mitochondria. Microsc. Res. Tech. 27:198‐219.
  Bhuvanendran, S., Salka, K., Rainey, K., Sreetama, S.C., Williams, E., Leeker, M., Prasad, V., Boyd, J., Patterson, G.H., Jaiswal, J.K., and Colberg‐Poley, A.M. 2014. Superresolution imaging of human cytomegalovirus vMIA localization in sub‐mitochondrial compartments. Viruses 6:1612‐1636.
  Bionda, C., Portoukalian, J., Schmitt, D., Rodriguez‐Lafrasse, C., and Ardail, D. 2004. Subcellular compartmentalization of ceramide metabolism: MAM (mitochondria‐associated membrane) and/or mitochondria? Biochem. J. 382:527‐533.
  Bononi, A., Missiroli, S., Poletti, F., Suski, J.M., Agnoletto, C., Bonora, M., De Marchi, E., Giorgi, C., Marchi, S., Patergnani, S., Rimessi, A., Wieckowski, M.R., and Pinton, P. 2012. Mitochondria‐associated membranes (MAMs) as hotspot Ca(2+) signaling units. Adv. Exp. Med. Biol. 740:411‐437.
  Bozidis, P., Williamson, C.D., and Colberg‐Poley, A.M. 2008. Mitochondrial and secretory human cytomegalovirus UL37 proteins traffic into mitochondrion‐associated membranes of human cells. J. Virol. 82:2715‐2726.
  Bozidis, P., Williamson, C.D., Wong, D.S., and Colberg‐Poley, A.M. 2010. Trafficking of UL37 proteins into mitochondrion‐associated membranes during permissive human cytomegalovirus infection. J. Virol. 84:7898‐7903.
  Browman, D.T., Resek, M.E., Zajchowski, L.D., and Robbins, S.M. 2006. Erlin‐1 and erlin‐2 are novel members of the prohibitin family of proteins that define lipid‐raft‐like domains of the ER. J. Cell Sci. 119:3149‐3160.
  Calaminus, J.M., Bruggen, J., and Sorg, C. 1979. Isolation, characterization and cultivation of human trophoblastic cells. Immunobiology 156:287.
  Carman, C.V., Lisanti, M.P., and Benovic, J.L. 1999. Regulation of G protein‐coupled receptor kinases by caveolin. J. Biol. Chem. 274:8858‐8864.
  Colberg‐Poley, A.M. and Williamson, C.D. 2013. Intracellular sorting and trafficking of cytomegalovirus proteins during permissive infection. In Cytomegaloviruses: From Molecular Pathogenesis to Intervention, 2nd ed., Vol. I (M.J. Reddehase, ed.) pp. 196‐229. Caister Academic Press/Horizon, Norwich, U.K.
  Colberg‐Poley, A.M., Patel, M.B., Erezo, D.P., and Slater, J.E. 2000. Human cytomegalovirus UL37 immediate‐early regulatory proteins traffic through the secretory apparatus and to mitochondria. J. Gen. Virol. 81:1779‐1789.
  Colberg‐Poley, A.M., Patterson, G.H., Salka, K., Bhuvanendran, S., Yang, D., and Jaiswal, J.K. 2015. Superresolution imaging of viral protein trafficking. Med. Microbiol. Immunol. 204:449‐460.
  Collins, T.J., Berridge, M.J., Lipp, P., and Bootman, M.D. 2002. Mitochondria are morphologically and functionally heterogeneous within cells. EMBO J. 21:1616‐1627.
  de Brito, O.M. and Scorrano, L. 2008. Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456:605‐610.
  Friedman, J.R., Lackner, L.L., West, M., DiBenedetto, J.R., Nunnari, J., and Voeltz, G.K. 2011. ER tubules mark sites of mitochondrial division. Science 334:358‐362.
  Fujimoto, M., Hayashi, T., and Su, T.P. 2012. The role of cholesterol in the association of endoplasmic reticulum membranes with mitochondria. Biochem. Biophys. Res. Commun. 417:635‐639.
  Gasnier, F., Rousson, R., Lerme, F., Vaganay, E., Louisot, P., and Gateau‐Roesch, O. 1992. Mitochondrial dolichyl‐phosphate mannose synthase. Purification and immunogold localization by electron microscopy. Eur. J. Biochem. 206:853‐858.
  Gelebart, P., Opas, M., and Michalak, M. 2005. Calreticulin, a Ca2+‐binding chaperone of the endoplasmic reticulum. Int. J. Biochem. Cell Biol. 37:260‐266.
  Gilady, S.Y., Bui, M., Lynes, E.M., Benson, M.D., Watts, R., Vance, J.E., and Simmen, T. 2010. Ero1alpha requires oxidizing and normoxic conditions to localize to the mitochondria‐associated membrane (MAM). Cell Stress Chaperones 15:619‐629.
  Goldmacher, V.S., Bartle, L.M., Skaletskaya, A., Dionne, C.A., Kedersha, N.L., Vater, C.A., Han, J.W., Lutz, R.J., Watanabe, S., Cahir McFarland, E.D., Kieff, E.D., Mocarski, E.S., and Chittenden, T. 1999. A cytomegalovirus‐encoded mitochondria‐localized inhibitor of apoptosis structurally unrelated to Bcl‐2. Proc. Natl. Acad. Sci. U.S.A. 96:12536‐12541.
  Goswami, R., Majumdar, T., Dhar, J., Chattopadhyay, S., Bandyopadhyay, S.K., Verbovetskaya, V., Sen, G.C., and Barik, S. 2013. Viral degradasome hijacks mitochondria to suppress innate immunity. Cell Res. 23:1025‐1042.
  Guardia‐Laguarta, C., Area‐Gomez, E., Rub, C., Liu, Y., Magrane, J., Becker, D., Voos, W., Schon, E.A., and Przedborski, S. 2014. alpha‐Synuclein is localized to mitochondria‐associated ER membranes. J. Neurosci. 34:249‐259.
  Hawley‐Nelson, P. and Ciccarone, V. 2003. Transfection of cultured eukaryotic cells using cationic lipid reagents. Curr. Protoc. Cell Biol. 20:20.6.1‐20.6.17.
  Hayajneh, W.A., Colberg‐Poley, A.M., Skaletskaya, A., Bartle, L.M., Lesperance, M.M., Contopoulos‐Ioannidis, D.G., Kedersha, N.L., and Goldmacher, V.S. 2001. The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains. Virology 279:233‐240.
  Hayashi, T. and Fujimoto, M. 2010. Detergent‐resistant microdomains determine the localization of sigma‐1 receptors to the endoplasmic reticulum‐mitochondria junction. Mol. Pharmacol. 77:517‐528.
  Hayashi, T. and Su, T.P. 2003. Sigma‐1 receptors (sigma(1) binding sites) form raft‐like microdomains and target lipid droplets on the endoplasmic reticulum: Roles in endoplasmic reticulum lipid compartmentalization and export. J. Pharmacol. Exp. Ther. 306:718‐725.
  Hayashi, T. and Su, T.P. 2007. Sigma‐1 receptor chaperones at the ER‐mitochondrion interface regulate Ca(2+) signaling and cell survival. Cell 131:596‐610.
  Horner, S.M., Liu, H.M., Park, H.S., Briley, J., and Gale, M., Jr. 2011. Mitochondrial‐associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proc. Natl. Acad. Sci. U.S.A. 108:14590‐14595.
  Hovius R., Lambrechts, H., Nicolay, K., and de Kruijff, B. 1990. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim. Biophys. Acta. 1021:217‐226.
  Johnson, S., Michalak, M., Opas, M., and Eggleton, P. 2001. The ins and outs of calreticulin: From the ER lumen to the extracellular space. Trends Cell Biol. 11:122‐129.
  Kenan, D.J. and Wahl, M.L. 2005. Ectopic localization of mitochondrial ATP synthase: A target for anti‐angiogenesis intervention? J. Bioenerg. Biomembr. 37:461‐465.
  Kim, W.S., Kagedal, K., and Halliday, G.M. 2014. Alpha‐synuclein biology in Lewy body diseases. Alzheimers Res. Ther. 6:73.
  Lynes, E.M., Bui, M., Yap, M.C., Benson, M.D., Schneider, B., Ellgaard, L., Berthiaume, L.G., and Simmen, T. 2012. Palmitoylated TMX and calnexin target to the mitochondria‐associated membrane. EMBO J. 31:457‐470.
  Maeda, Y., Tomita, S., Watanabe, R., Ohishi, K., and Kinoshita, T. 1998. DPM2 regulates biosynthesis of dolichol phosphate‐mannose in mammalian cells: Correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate. EMBO J. 17:4920‐4929.
  Maeda, Y., Tanaka, S., Hino, J., Kangawa, K., and Kinoshita, T. 2000. Human dolichol‐phosphate‐mannose synthase consists of three subunits, DPM1, DPM2 and DPM3. EMBO J. 19:2475‐2482.
  Manning‐Krieg, U.C., Scherer, P.E., and Schatz, G. 1991. Sequential action of mitochondrial chaperones in protein import into the matrix. EMBO J. 10:3273‐3280.
  Marchi, S., Patergnani, S., and Pinton, P. 2014. The endoplasmic reticulum‐mitochondria connection: One touch, multiple functions. Biochim. Biophys. Acta 1837:461‐469.
  Matsuo, Y., Akiyama, N., Nakamura, H., Yodoi, J., Noda, M., and Kizaka‐Kondoh, S. 2001. Identification of a novel thioredoxin‐related transmembrane protein. J. Biol. Chem. 276:10032‐10038.
  Mavinakere, M.S. and Colberg‐Poley, A.M. 2004a. Dual targeting of the human cytomegalovirus UL37 exon 1 protein during permissive infection. J. Gen. Virol. 85:323‐329.
  Mavinakere, M.S. and Colberg‐Poley, A.M. 2004b. Internal cleavage of the human cytomegalovirus UL37 immediate‐early glycoprotein and divergent trafficking of its proteolytic fragments. J. Gen. Virol. 85:1989‐1994.
  Mavinakere, M.S., Williamson, C.D., Goldmacher, V.S., and Colberg‐Poley, A.M. 2006. Processing of human cytomegalovirus UL37 mutant glycoproteins in the endoplasmic reticulum lumen prior to mitochondrial importation. J. Virol. 80:6771‐6783.
  McCormick, A.L., Smith, V.L., Chow, D., and Mocarski, E.S. 2003. Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion‐localized inhibitor of apoptosis. J. Virol. 77:631‐641.
  Morita, S.Y., Shirakawa, S., Kobayashi, Y., Nakamura, K., Teraoka, R., Kitagawa, S., and Terada, T. 2012. Enzymatic measurement of phosphatidylserine in cultured cells. J. Lipid Res. 53:325‐330.
  Myhill, N., Lynes, E.M., Nanji, J.A., Blagoveshchenskaya, A.D., Fei, H., Carmine Simmen, K., Cooper, T.J., Thomas, G., and Simmen, T. 2008. The subcellular distribution of calnexin is mediated by PACS‐2. Mol. Biol. Cell 19:2777‐2788.
  Pearce, M.M., Wang, Y., Kelley, G.G., and Wojcikiewicz, R.J. 2007. SPFH2 mediates the endoplasmic reticulum‐associated degradation of inositol 1,4,5‐trisphosphate receptors and other substrates in mammalian cells. J. Biol. Chem. 282:20104‐20115.
  Pertoft, H., Laurent, T.C., Laas, T., and Kagedal, L. 1978. Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll). Anal. Biochem. 88:271‐282.
  Piccini, M., Vitelli, F., Bruttini, M., Pober, B.R., Jonsson, J.J., Villanova, M., Zollo, M., Borsani, G., Ballabio, A., and Renieri, A. 1998. FACL4, a new gene encoding long‐chain acyl‐CoA synthetase 4, is deleted in a family with Alport syndrome, elliptocytosis, and mental retardation. Genomics 47:350‐358.
  Poston, C.N., Duong, E., Cao, Y., and Bazemore‐Walker, C.R. 2011. Proteomic analysis of lipid raft‐enriched membranes isolated from internal organelles. Biochem. Biophys. Res. Commun. 415:355‐360.
  Rizzuto, R., Duchen, M.R., and Pozzan, T. 2004. Flirting in little space: The ER/mitochondria Ca2 +liaison. Sci. STKE 2004:re1.
  Rizzuto, R., Pinton, P., Carrington, W., Fay, F.S., Fogarty, K.E., Lifshitz, L.M., Tuft, R.A., and Pozzan, T. 1998. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280:1763‐1766.
  Ruhle, T. and Leister, D. 2015. Assembly of FF‐ATP synthases. Biochim. Biophys. Acta. pii:S0005‐2728(15)00030‐4. [Epub ahead of print].
  Rusiñol, A.E., Cui, Z., Chen, M.H., and Vance, J.E. 1994. A unique mitochondria‐associated membrane fraction from rat liver has a high capacity for lipid synthesis and contains pre‐Golgi secretory proteins including nascent lipoproteins. J. Biol. Chem. 269:27494‐27502.
  Rutter, J., Winge, D.R., and Schiffman, J.D. 2010. Succinate dehydrogenase: Assembly, regulation and role in human disease. Mitochondrion 10:393‐401.
  Sadacharan, S.K., Singh, B., Bowes, T., and Gupta, R.S. 2005. Localization of mitochondrial DNA encoded cytochrome c oxidase subunits I and II in rat pancreatic zymogen granules and pituitary growth hormone granules. Histochem. Cell Biol. 124:409‐421.
  Scheffler, I.E. 2001. A century of mitochondrial research: Achievements and perspectives. Mitochondrion 1:3‐31.
  Schon, E.A. and Area‐Gomez, E. 2013. Mitochondria‐associated ER membranes in Alzheimer disease. Mol. Cell. Neurosci. 55:26‐36.
  Simmen, T., Aslan, J.E., Blagoveshchenskaya, A.D., Thomas, L., Wan, L., Xiang, Y., Feliciangeli, S.F., Hung, C.H., Crump, C.M., and Thomas, G. 2005. PACS‐2 controls endoplasmic reticulum‐mitochondria communication and Bid‐mediated apoptosis. EMBO J. 24:717‐729.
  Song, K.S., Scherer, P.E., Tang, Z., Okamoto, T., Li, S., Chafel, M., Chu, C., Kohtz, D.S., and Lisanti, M.P. 1996. Expression of caveolin‐3 in skeletal, cardiac, and smooth muscle cells. Caveolin‐3 is a component of the sarcolemma and co‐fractionates with dystrophin and dystrophin‐associated glycoproteins. J. Biol. Chem. 271:15160‐15165.
  Stone, S.J. and Vance, J.E. 2000. Phosphatidylserine synthase‐1 and ‐2 are localized to mitochondria‐associated membranes. J. Biol. Chem. 275:34534‐34540.
  Suski, J.M., Karkucinska‐Wieckowska, A., Lebiedzinska, M., Giorgi, C., Szczepanowska, J., Szabadkai, G., Duszynski, J., Pronicki, M., Pinton, P., and Wieckowski, M.R. 2011. p66Shc aging protein in control of fibroblasts cell fate. Int. J. Mol. Sci. 12:5373‐5389.
  Szabadkai, G., Bianchi, K., Varnai, P., De Stefani, D., Wieckowski, M.R., Cavagna, D., Nagy, A.I., Balla, T., and Rizzuto, R. 2006. Chaperone‐mediated coupling of endoplasmic reticulum and mitochondrial Ca2+ channels. J. Cell Biol. 175:901‐911.
  Vance, J.E. 1990. Phospholipid synthesis in a membrane fraction associated with mitochondria. J. Biol. Chem. 265:7248‐7256.
  Vance, J.E. 2014. MAM (mitochondria‐associated membranes) in mammalian cells: Lipids and beyond. Biochim. Biophys. Acta 1841:595‐609.
  Wieckowski, M.R., Giorgi, C., Lebiedzinska, M., Duszynski, J., and Pinton, P. 2009. Isolation of mitochondria‐associated membranes and mitochondria from animal tissues and cells. Nat. Protoc. 4:1582‐1590.
  Williamson, C.D. and Colberg‐Poley, A.M. 2010. Intracellular sorting signals for sequential trafficking of human cytomegalovirus UL37 proteins to the endoplasmic reticulum and mitochondria. J. Virol. 84:6400‐6409.
  Williamson, C.D., Zhang, A., and Colberg‐Poley, A.M. 2011. The human cytomegalovirus protein UL37 exon 1 associates with internal lipid rafts. J. Virol. 85:2100‐2111.
  Zhang, A., Hildreth, R.L., and Colberg‐Poley, A.M. 2013. Human cytomegalovirus inhibits apoptosis by proteasome‐mediated degradation of Bax at endoplasmic reticulum‐mitochondrion contacts. J. Virol. 87:5657‐5668.
  Zhang, A., Williamson, C.D., Wong, D.S., Bullough, M.D., Brown, K.J., Hathout, Y., and Colberg‐Poley, A.M. 2011. Quantitative proteomic analyses of human cytomegalovirus‐induced restructuring of endoplasmic reticulum‐mitochondrial contacts at late times of infection. Mol. Cell Proteomics 10:M111.009936.
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