Abstract
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-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production.
Highlights
From the ‡Center for Cancer and Immunology Research and §Research Center for Genetic Medicine, Children’s Research Institute, Children’s National Medical Center, 111 Michigan Avenue, NW, Washington, DC 20010, ¶Departments of Integrative Systems Biology, ʈBiochemistry and Molecular Biology, George Washington University School of Medicine and Health Sciences, Washington DC 20037
Proapoptotic regulatory proteins, including Bax, cytochrome c, and Opa[1], were augmented in endoplasmic reticulum-mitochondrial contacts after infection, suggesting attenuation of proapoptotic signaling by their increased presence therein. These results suggest that human cytomegalovirus infection restructures the proteome of endoplasmic reticulum-mitochondrial contacts to bolster protein translation at these junctions, calcium signaling to mitochondria, cell survival, and bioenergetics and, thereby, allow for enhanced progeny production
Quantitative Proteomic Analyses of the mitochondria-associated membranes (MAM) Proteome in Uninfected and human cytomegalovirus (HCMV)-Infected human foreskin fibroblasts (HFFs)—The enriched MAM fraction can be reproducibly isolated by banding in Percoll gradients (Fig. 1B) (47, 51–53)
Summary
Cells and Viruses—HFFs were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal calf serum (FCS), 100 U/ml of penicillin, 100 g/ml of streptomycin (Invitrogen) as described (45). The unlabeled HCMV-infected cells were mixed 1:1 with SILAC-labeled uninfected cells. The Percoll method used for this analysis is one of the most rigorous procedures to obtain banded MAM and mitochondria It relies on multiple fractionation steps, each used to separate organelles based upon their connectivity, size, shape, and density. The MAM and mitochondria were further separated by homogenization and resolved by banding in Percoll density gradients. Protein Separation and Mass Spectrometry Analysis—One hundred micrograms of the heavy MAM fraction obtained from the mixture of unlabeled and SILAC-labels cells were further resolved by SDS-PAGE (Fig. 1C). The mixed cells were fractionated to obtain heavy MAM as previously described (28, 47). Heavy MAM proteins (100 g), obtained from mixed unlabeled and SILAC-labeled cells, were resolved by SDS-PAGE.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
