MHC class I molecules display peptides from endogenous and viral proteins for immunosurveillance by cytotoxic T lymphocytes (CTL). The importance of the class I pathway is emphasised by the remarkable strategies employed by different viruses to downregulate surface class I and avoid CTL recognition. The K3 gene product from Kaposi's sarcoma-associated herpesvirus (KSHV) is a viral ubiquitin E3 ligase which ubiquitinates and degrades cell surface MHC class I molecules. We now show that modification of K3-associated class I by lysine-63-linked polyubiquitin chains is necessary for their efficient endocytosis and endolysosomal degradation and present three lines of evidence that monoubiquitination of class I molecules provides an inefficient internalisation signal. This lysine-63-linked polyubiquitination requires both UbcH5b/c and Ubc13-conjugating enzymes for initiating mono-and subsequent polyubiquitination of class I, and the clathrin-dependent internalisation is mediated by the epsin endocytic adaptor. Our results explain how lysine-63-linked polyubiquitination leads to degradation by an endolysosomal pathway and demonstrate a novel mechanism for endocytosis and endolysosomal degradation of class I, which may be applicable to other receptors.
Models for protein sorting at multivesicular bodies in the endocytic pathway of mammalian cells have relied largely on data obtained from yeast. These data suggest the essential role of four ESCRT complexes in multivesicular body protein sorting. However, the putative mammalian ESCRTII complex (hVps25p, hVps22p, and hVps36p) has no proven functional role in endosomal transport. We have characterized the human ESCRTII complex and investigated its function in endosomal trafficking. The human ESCRTII proteins interact with one another, with hVps20p (a component of ESCRTIII), and with their yeast homologues. Our interaction data from yeast two-hybrid studies along with experiments with purified proteins suggest an essential role for the N-terminal domain of hVps22p in the formation of a heterotetrameric ESCRTII complex. Although human ESCRTII is found in the cytoplasm and in the nucleus, it can be recruited to endosomes upon overexpression of dominant-negative hVps4Bp. Interestingly, we find that small interference RNA depletion of mammalian ESCRTII does not affect degradation of epidermal growth factor, a known cargo of the multivesicular body protein sorting pathway. We also show that depletion of the deubiquitinating enzymes AMSH (associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)) and UBPY (ubiquitin isopeptidase Y) have opposite effects on epidermal growth factor degradation, with UBPY depletion causing dramatic swelling of endosomes. Down-regulation of another cargo, the major histocompatibility complex class I in cells expressing the Kaposi sarcoma-associated herpesvirus protein K3, is unaffected in ESCRTII-depleted cells. Our data suggest that mammalian ESCRTII may be redundant, cargo-specific, or not required for protein sorting at the multivesicular body.
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