Open AccessIsolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria‐Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus‐Infected Primary Fibroblasts
Author(s) -
Williamson Chad D.,
Wong Daniel S.,
Bozidis Petros,
Zhang Aiping,
ColbergPoley Anamaris M.
Publication year - 2015
Publication title -
current protocols in cell biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.149
H-Index - 38
eISSN - 1934-2616
pISSN - 1934-2500
DOI - 10.1002/0471143030.cb0327s68
Subject(s) - endoplasmic reticulum , mitochondrion , microbiology and biotechnology , transfection , biology , organelle , mfn2 , human cytomegalovirus , percoll , biochemistry , mitochondrial dna , in vitro , mitochondrial fusion , gene
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.