Efficient Internalization of MHC I Requires Lysine‐11 and Lysine‐63 Mixed Linkage Polyubiquitin Chains

Jessica M Boname,Mair Thomas,Junmin Peng,Paul J Lehner,Ping Xu,Helen R Stagg

doi:10.1111/j.1600-0854.2009.01011.x

Jessica M Boname, Mair Thomas + Show 4 more

Open Access

https://doi.org/10.1111/j.1600-0854.2009.01011.x

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Journal: Traffic	Publication Date: Jan 4, 2010
Citations: 117	License type: cc-by

Affiliation: University of Cambridge, Emory University

Abstract

The downregulation of cell surface receptors by endocytosis is a fundamental requirement for the termination of signalling responses and ubiquitination is a critical regulatory step in receptor regulation. The K5 gene product of Kaposi's sarcoma-associated herpesvirus is an E3 ligase that ubiquitinates and downregulates several cell surface immunoreceptors, including major histocompatibility complex (MHC) class I molecules. Here, we show that K5 targets the membrane proximal lysine of MHC I for conjugation with mixed linkage polyubiquitin chains. Quantitative mass spectrometry revealed an increase in lysine-11, as well as lysine-63, linked polyubiquitin chains on MHC I in K5-expressing cells. Using a combination of mutant ubiquitins and MHC I molecules expressing a single cytosolic lysine residue, we confirm a functional role for lysines-11 and -63 in K5-mediated MHC I endocytosis. We show that lysine-11 linkages are important for receptor endocytosis, and that complex mixed linkage polyubiquitin chains are generated in vivo.

Highlights

In addition to its role in proteolysis, ubiquitin provides a signal for endocytosis and trafficking of internalized receptors [1,2]
While the monoubiquitinated major histocompatibility complex (MHC) I species appears predominant in HeLa-K5 cells, this is not seen with HeLa-K3 cells
This dominance of the monoubiquitinated class I species in HeLa-K5 cells was further exaggerated following IP of cell surface compared with total major histocompatibility complex class I (MHC I) (Figure 1B)

Summary

Introduction

In addition to its role in proteolysis, ubiquitin provides a signal for endocytosis and trafficking of internalized receptors [1,2]. For other ubiquitin chain linkages, only a limited number of examples of in vivo or in vitro outcomes are reported This does not reflect a low frequency of these chain types, as a recent quantitative proteomic analysis in yeast found Lys11-linked chains to be as abundant as Lys chains, with all non-Lys48-linked chains being well represented [5]. K3 and K5 of Kaposi’s sarcoma-associated herpesvirus and mK3 of the murine gammaherpesvirus-68 were identified by their ability to downregulate MHC I [1,14] Subsequent analysis showed they function as membranebound ubiquitin E3 ligases with an N-terminal RING-CH domain [15] required for targeting MHC I for polyubiquitination and degradation [16,17,18]. Both K3 and K5 downregulate MHC I, while K5 is more promiscuous and targets a wide range of immunoreceptors, including CD31, intercellular adhesion molecule type 1 (ICAM-1), B7.2, MHC class I-related chains A and B (MICA, MICB), and activationinduced C-type lectin (AICL) [19,20,21,22]

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