Lysine-63-linked ubiquitination is required for endolysosomal degradation of class I molecules

Abstract

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.

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…Instead, it was claimed based on overexpression of a supposedly GTP-locked dominant-negative Arf6 mutant that MHCI follows an Arf6-specific clathrin-independent endocytosis pathway (6,40,52). Recent data from several other laboratories have called this conclusion into question because MHCI endocytosis is unaffected by knockdown of Arf6 (41) but depends on the clathrin/ AP-2 binding endocytic protein epsin 1 (42,43), consistent with our data reported here. Because Arf6 regulates rapid recycling, it is possible that the previously reported effects of overexpression of dominant-negative mutant Arf6 on MHCI uptake (6, 40, 52) may have resulted from general perturbations of the endosomal system rather than reflecting clathrin-independent MHCI internalization.…”

“…These data demonstrate that MHCI endocytosis strictly depends on clathrin/AP-2 and epsin (43), in agreement with earlier data from a number of laboratories (41)(42)(43) and with its inhibition by Pitstop compounds but not by clathrin-inactive analogs. The specific effect of Pitstop compounds on CME is further underscored by the observation that plasma membrane dynamics of GPI-eGFP in FRAP experiments are nearly unaffected by application of Pitstop-2 or Pitstop-2-100 (Fig.…”

“…The effect of the Pitstop compounds on MHCI endocytosis could reflect off target side effects. Alternatively, MHCI internalization may indeed depend on clathrin and/or AP-2, in agreement with several recent studies reporting a crucial role for clathrin/AP-2 and associated epsins in MHCI endocytosis (41)(42)(43). To distinguish between these possibilities, we treated HeLa cells with well characterized clathrin heavy chain (CHC)-or AP-2 -specific siRNAs resulting in depletion of clathrin or AP-2 to less than 10% of the levels expressed in control cells (Fig.…”

Autoregulation of MARCH1 Expression by Dimerization and Autoubiquitination

“…To investigate the type of ubiquitination taking place in these conditions, we coexpressed MARCH1 and a GFP-tagged lysineless ubiquitin (GFP-ubiK-0) that prevents further elongation of a chain once linked to the substrate (36,37). As a control, we used an inactive ubiquitin molecule (GFP-ubiG76) that cannot be conjugated to any target (36).…”

Polyubiquitination of Prolactin Receptor Stimulates Its Internalization, Postinternalization Sorting, and Degradation via the Lysosomal Pathway

“…Each individual ubiquitin moiety harbors seven lysine residues, allowing for the formation of ubiquitin chains linked through its internal lysine residues such as K48 and K63, etc. While the overall role of ubiquitination in mediating the internalization of many yeast receptors (6, 21) and mammalian receptors (such as the nerve growth factor receptor TrkA [16], major histocompatibility complex class II proteins [14], growth hormone receptor [42], model fusion proteins [3], and many others) has been reported previously, the requirements of ubiquitination specificity differ substantially between the various receptors.We have recently demonstrated that linkages of both K48 and K63 types were required for efficient internalization of the alpha/beta interferon receptor chain IFNAR1 (24). We pro-* Corresponding author.…”