Isolation of Endoplasmic Reticulum, Mitochondria, and Mitochondria-Associated Membrane and Detergent Resistant Membrane Fractions from Transfected Cells and from Human Cytomegalovirus-Infected Primary Fibroblasts.
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.
Center for Genetic Medicine Research, Children's Research Institute, Washington, D.C.|Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Chad D
CD
Williamson
Cellular and Molecular Physiology Program, Sackler School for Graduate Biomedical Sciences, Tufts University, Boston, Massachusetts.
Daniel S
DS
Wong
Laboratory of Microbiology, Department of Medicine, School of Health Sciences, University of Ioannina, Ioannina, Greece.
Petros
P
Bozidis
Center for Genetic Medicine Research, Children's Research Institute, Washington, D.C.
Aiping
A
Zhang
Center for Genetic Medicine Research, Children's Research Institute, Washington, D.C.|Department of Integrative Systems Biology, George Washington University School of Medicine and Health Sciences, Washington, D.C.|Department of Biochemistry and Molecular Medicine, George Washington University School of Medicine and Health Sciences, Washington, D.C.|Department of Pediatrics, George Washington University School of Medicine and Health Sciences, Washington, D.C.
Anamaris M
AM
Colberg-Poley
D003587:Cytomegalovirus / Q000472:pathogenicity; D003902:Detergents / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry*; D051336:Mitochondrial Membranes / Q000737:chemistry*
D003902:Detergents; D017874:Immediate-Early Proteins; C420323:UL37 protein, Human herpesvirus 5
ER; HCMV; MAM; Percoll gradient; differential centrifugation; human fibroblasts; mitochondria; protein localization; subcellular fractionation; sucrose gradient