Stabilization of an E3 ligase-E2-ubiquitin complex increases cell surface MHC class I expression.
The Kaposi's sarcoma-associated herpesvirus-encoded ubiquitin E3 ligase K3 ubiquitinates cell-surface MHC class I molecules (MHC I), causing the internalization and degradation of MHC I via the endolysosomal pathway. K3 recruits the cellular E2 ubiquitin-conjugating enzyme Ubc13 to generate lysine-63-linked polyubiquitin chains on MHC I, leading to the clathrin-mediated endocytosis and lysosomal degradation of MHC I. In this study, we identify a ubiquitin isoleucine-44-alanine mutant (I44A) that inhibits K3-mediated downregulation of MHC I by preventing MHC I polyubiqitination. This E3-specific inhibition by I44A prevents dissociation of the MHC I-K3-Ubc13-ubiquitin complex, allows the in vivo visualization of a transient substrate-E3-E2-ubiquitin complex interaction, and highlights a potential substrate hierarchy between the different MHC I alleles downregulated by K3. The I44A mutant also increases cell-surface MHC I expression in control cells in the absence of K3, predicting the presence of an endogenous E3 ubiquitin ligase required for cell-surface MHC I regulation.
Duncan
Lidia M
LM
Department of Medicine, Cambridge Institute for Medical Research, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom.
Nathan
James A
JA
Lehner
Paul J
PJ
D000071443:12E7 Antigen; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000956:Antigens, Viral / Q000276:immunology; D016667:Bacterial Capsules / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism*; D002469:Cell Separation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D015398:Signal Transduction; D014162:Transfection; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054875:Ubiquitination
D000071443:12E7 Antigen; D015703:Antigens, CD; D000956:Antigens, Viral; C061828:CD99 protein, human; D015815:Cell Adhesion Molecules; D015395:Histocompatibility Antigens Class I; D025801:Ubiquitin; C098418:capsular polysaccharide K3; D044767:Ubiquitin-Protein Ligases