Quantitative Proteomic Analyses of Human Cytomegalovirus-Induced Restructuring of Endoplasmic Reticulum-Mitochondrial Contacts at Late Times of Infection

Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon...

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Published in:Molecular & cellular proteomics 2011-10, Vol.10 (10), p.M111.009936-M111.009936, Article M111.009936
Main Authors: Zhang, Aiping, Williamson, Chad D., Wong, Daniel S., Bullough, Matthew D., Brown, Kristy J., Hathout, Yetrib, Colberg-Poley, Anamaris M.
Format: Article
Language:English
Subjects:
Chromatography, Liquid
Cytomegalovirus
Cytomegalovirus Infections - metabolism
Endoplasmic Reticulum - metabolism
Fibroblasts
Herpesvirus
Human cytomegalovirus
Humans
Isotope Labeling
Mitochondria - metabolism
Mitochondrial Membranes - metabolism
Proteome - analysis
Proteomics - methods
Tandem Mass Spectrometry
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Summary:Endoplasmic reticulum-mitochondrial contacts, known as mitochondria-associated membranes, regulate important cellular functions including calcium signaling, bioenergetics, and apoptosis. Human cytomegalovirus is a medically important herpesvirus whose growth increases energy demand and depends upon continued cell survival. To gain insight into how human cytomegalovirus infection affects endoplasmic reticulum-mitochondrial contacts, we undertook quantitative proteomics of mitochondria-associated membranes using differential stable isotope labeling by amino acids in cell culture strategy and liquid chromatography-tandem MS analysis. This is the first reported quantitative proteomic analyses of a suborganelle during permissive human cytomegalovirus infection. Human fibroblasts were uninfected or human cytomegalovirus-infected for 72 h. Heavy mitochondria-associated membranes were isolated from paired unlabeled, uninfected cells and stable isotope labeling by amino acids in cell culture-labeled, infected cells and analyzed by liquid chromatography-tandem MS analysis. The results were verified by a reverse labeling experiment. Human cytomegalovirus infection dramatically altered endoplasmic reticulum-mitochondrial contacts by late times. Notable is the increased abundance of several fundamental networks in the mitochondria-associated membrane fraction of human cytomegalovirus-infected fibroblasts. Chaperones, including HSP60 and BiP, which is required for human cytomegalovirus assembly, were prominently increased at endoplasmic reticulum-mitochondrial contacts after infection. Minimal translational and translocation machineries were also associated with endoplasmic reticulum-mitochondrial contacts and increased after human cytomegalovirus infection as were glucose regulated protein 75 and the voltage dependent anion channel, which can form an endoplasmic reticulum-mitochondrial calcium signaling complex. Surprisingly, mitochondrial metabolic enzymes and cytosolic glycolytic enzymes were confidently detected in the mitochondria-associated membrane fraction and increased therein after infection. Finally, proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa1, were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. Together, these results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitoch
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.M111.009936