E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system.
In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.
Flierman
Dennis
D
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Coleman
Catherine S
CS
Pickart
Cecile M
CM
Rapoport
Tom A
TA
Chau
Vincent
V
D000818:Animals; D001681:Biological Assay; D002417:Cattle; D045744:Cell Line, Tumor; D003587:Cytomegalovirus; D019281:Dimerization; D005805:Genes, MHC Class I; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D007143:Immunoglobulin Heavy Chains; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C074546:US11 protein, herpesvirus; D025801:Ubiquitin; D014764:Viral Proteins; C100879:UBE2K protein, human; D044763:Ubiquitin-Conjugating Enzymes