Unit

UNIT 3.27 Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts

  1. Petros Bozidis1,
  2. Chad D. Williamson1,2,
  3. Anamaris M. Colberg-Poley1,2,3

Published Online: 1 DEC 2007

DOI: 10.1002/0471143030.cb0327s37

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Bozidis, P., Williamson, C. D. and Colberg-Poley, A. M. 2007. Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts. Current Protocols in Cell Biology. 37:3.27:3.27.1–3.27.23.

Author Information

  1. 1

    Center for Cancer and Immunology Research, Children's Research Institute, Washington, DC

  2. 2

    Department of Biochemistry and Molecular Biology, George Washington University School of Medicine and Health Sciences, Washington, DC

  3. 3

    Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, DC

Publication History

  1. Published Online: 1 DEC 2007
  2. Published Print: DEC 2007

This is not the most recent version of the article. View current version (1 SEP 2015)

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 the substantial protein yields from HCMV-infected primary fibroblasts and from transfected HeLa cells. Moreover, this unit includes a protocol that allows visualization of the mitochondria network disruption that occurs in permissively infected cells by their optimal resolution in Percoll gradients. Curr. Protoc. Cell Biol. 37:3.27.1-3.27.23. © 2007 by John Wiley & Sons, Inc.

Keywords:

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