Abstract
The human cytomegalovirus (HCMV) protein US2 hijacks the endoplasmic reticulum (ER)-associated degradation machinery to dispose of MHC class I heavy chain (HC) at the ER. This process requires retrotranslocation of newly synthesized HC molecules from the ER membrane into the cytosol, but the mechanism underlying the dislocation reaction has been elusive. Here we establish an in vitro permeabilized cell assay that recapitulates the retrotranslocation of MHC HC in US2-expressing cells. Using this assay, we demonstrate that the dislocation process requires ATP and ubiquitin, as expected. The retrotranslocation also involves the p97 ATPase. However, the mechanism by which p97 dislocates MHC class I HC in US2 cells is distinct from that in US11 cells: the dislocation reaction in US2 cells is independent of the p97 cofactor Ufd1-Npl4. Our results suggest that different retrotranslocation mechanisms can employ distinct p97 ATPase complexes to dislocate substrates.
Highlights
The endoplasmic reticulum (ER)2 is a major site of protein synthesis for secretory and membrane proteins
The p97 ATPase complex, which comprises of the AAA (ATPase associated with various cellular activities) ATPase p97 and the dimeric adaptor complex Ufd1-Npl4, use a dual recognition mode to recognize both polypeptide chain and the attached polyubiquitin conjugates, leading to the extraction of the polypeptide from the ER membrane [7, 8]. p97 subsequently delivers the sub
The salt-treated first fractionated into pellet and membrane pellet was resuspended in cow liver cytosol contain- supernatant fractions before immunoing 10 M bovine ubiquitin and ATP regenerating system (ARS)
Summary
The endoplasmic reticulum (ER) is a major site of protein synthesis for secretory and membrane proteins. To ensure that only properly folded polypeptides and correctly assembled protein complexes enter the secretory pathway to reach their final destinations, eukaryotic cells have evolved a conserved quality control mechanism This quality control program selectively retains polypeptides that fail to reach native conformation and export them from the ER into the cytosol for degradation by the 26 S proteasome. The elimination of the antigen presenting MHC HC molecules from the ER membrane apparently allows viruses to escape detection by the host immune system Both US11 and US2 directly bind MHC class I HC to initiate the retrotranslocation process, and both pathways involve ubiquitin, the 26 S proteasome, and a translocating chain-associated membrane protein-1 [15,16,17,18,19]. It is unclear whether or not the p97 ATPase participates in this process, and if so, how it
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