Ubiquitinylation of the Cytosolic Domain of a Type I Membrane Protein Is Not Required to Initiate Its Dislocation from the Endoplasmic Reticulum

Abstract

Human cytomegalovirus US2 and US11 target newly synthesized class I major histocompatibility complex (MHC) heavy chains for rapid degradation by the proteasome through a process termed dislocation. The presence of US2 induces the formation of class I MHC heavy chain conjugates of increased molecular weight that are recognized by a conformation-specific monoclonal antibody, W6/32, suggesting that these class I MHC molecules retain their proper tertiary structure. These conjugates are properly folded glycosylated heavy chains modified by attachment of an estimated one, two, and three ubiquitin molecules. The folded ubiquitinated class I MHC heavy chains are not observed in control cells or in cells transfected with US11, suggesting that US2 targets class I MHC heavy chains for dislocation in a manner distinct from that used by US11. This is further supported by the fact that US2 and US11 show different requirements in terms of the conformation of the heavy chain molecule. Although ubiquitin conjugation may occur on the cytosolic tail of the class I MHC molecule, replacement of lysines in the cytosolic tail of heavy chains with arginine does not prevent their degradation by US2. In an in vitro system that recapitulates US2-mediated dislocation, heavy chains that lack these lysines still occur in an ubiquitin-modified form, but in the soluble (cytoplasmic) fraction. Such ubiquitin conjugation can only occur on the class I MHC lumenal domain and is likely to take place once class I MHC heavy chains have been discharged from the endoplasmic reticulum. We conclude that ubiquitinylation of class I MHC heavy chain is not required during the initial step of the US2-mediated dislocation reaction.

The p97 ATPase Dislocates MHC Class I Heavy Chain in US2-expressing Cells via a Ufd1-Npl4-independent Mechanism

“…3c, lane 8). Together, these observations suggest that dislocation of MHC class I HC in permeabilized US2 cells also involves ubiquitination in agreement with previous studies in intact cells, which showed that polyubiquitinated MHC class I HC is present in US2 cells treated with a proteasome inhibitor (17), and that removal of potential ubiquitin conjugation sites in MHC class I HC abolished its dislocation (18).…”

“…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). Nonetheless, these two processes each posses its own unique features, indicating that these two viral proteins may use different retrotranslocation routes to dislocate MHC class I HC from the ER membrane.…”

MHC class I molecules are preferentially ubiquitinated on endoplasmic reticulum luminal residues during HRD1 ubiquitin E3 ligase-mediated dislocation

“…Mutation of cytosolic tail lysines results in a marked loss of US2-induced ubiquitination of membrane-associated MHC I HCs, implying that TRC8 preferentially targets lysine residues in the MHC I tail (26). The mutant HC is, however, still dislocated, suggesting US2 may use alternative mechanisms of dislocation in the absence of accessible cytosolic lysines (26).…”

“…US2 interacts with the luminal domain of conformational, β2m-associated MHC I HC, but requires the MHC I transmembrane domain, and possibly tail residues, to mediate dislocation (22,25). Mutation of cytosolic tail lysines results in a marked loss of US2-induced ubiquitination of membrane-associated MHC I HCs, implying that TRC8 preferentially targets lysine residues in the MHC I tail (26). The mutant HC is, however, still dislocated, suggesting US2 may use alternative mechanisms of dislocation in the absence of accessible cytosolic lysines (26).…”

Characterization of a Novel Ubiquitin-Conjugating Enzyme That Regulates β1,4-Galactosyltransferase-1 in Embryonic Stem Cells

“…Using this system, cells can remove unwanted, degenerated, or excessively expressed proteins, thus maintaining cellular protein homeostasis. The ubiquitination pathway is known to regulate critical control points in multiple cellular processes, including receptor internalization, endosomal degradation, nuclear transport, and the turnover of several key cell cycle-regulating proteins [3][4][5]. The pathway involves a complex enzymatic cascade responsible for protein ubiquitination and degradation.…”