The Viral E3 Ubiquitin Ligase mK3 Uses the Derlin/p97 Endoplasmic Reticulum-associated Degradation Pathway to Mediate Down-regulation of Major Histocompatibility Complex Class I Proteins

Xiaoli Wang,Yihong Ye,Wayne Lencer,Ted H Hansen

doi:10.1074/jbc.m513920200

Abstract

Ubiquitin E3 ligases are important cellular components for endoplasmic reticulum (ER)-associated degradation due to their role in substrate-specific ubiquitination, which is required for retrotranslocation (dislocation) of most unwanted proteins from the ER to the cytosol for proteasome degradation. However, our understanding of the molecular mechanisms of how E3 ligases confer substrate-specific recognition, and their role in substrate retrotranslocation is limited especially in mammalian cells. mK3 is a type III ER membrane protein encoded by murine gamma herpesvirus 68. As conferred by its N-terminal RING-CH domain, mK3 has E3 ubiquitin ligase activity. In its role as an immune evasion protein, mK3 specifically targets nascent major histocompatibility complex class I heavy chains (HC) for rapid degradation. The mechanism by which mK3 extracts HC from the ER membrane into the cytosol for proteasome-mediated degradation is unknown. Evidence is presented here that HC down-regulation by mK3 is dependent on the p97 AAA-ATPase. By contrast, the kK5 protein of Kaposi's sarcoma-associated herpesvirus is p97-independent despite the fact that it is highly homologous to mK3. mK3 protein was also found in physical association with Derlin1, an ER protein recently implicated in the retrotranslocation of HC by immune evasion protein US11, but not US2, of human cytomegalovirus. The mechanistic implications of these findings are discussed.

Highlights

To exist in the presence of an active host immune system, herpesviruses in particular have elaborate mechanisms of immune evasion
Ubiquitination is required for retrotranslocation of most luminal ERAD substrates including integral endoplasmic reticulum (ER) membrane proteins
Hrd1p and Doa10p are responsible for ubiquitination of distinct subsets of ERAD substrates that may contain misfolded domains on the lumenal or cytosolic faces of the ER membrane respectively [41, 44]

Summary

EXPERIMENTAL PROCEDURES

Cell Lines—Murine B6/WT3 (WT3, H-2b) and L cell (H-2k) fibroblasts as well as their transductants (Ld, human B7.2, mK3, and kK5) have been described previously [8]. 293T cells [30] were used for production of ecotropic retrovirus (to transduce B6/WT3 cells) and ampotropic retrovirus (to transduce L cells). After blocking in phosphate-buffered saline containing 0.5% saponin and 3% bovine serum albumin, fixed cells were incubated with mAb to RGS-His (0.1 ␮g/ml, Qiagen) and Alexa 594 goat anti-mouse IgG1 (6.5 ␮g/ml, Molecular Probes, Inc., Eugene, OR) sequentially. Beads were washed four times with 0.05% Tween 20-Trisbuffered saline (pH 7.4) and proteins were eluted with an excess of HA. peptide (Covance Research Products) for anti-HA beads or by boiling in non-reducing sample buffer (Pierce) for anti-myc beads. Immunoblotting was performed following SDS-PAGE separation of precipitated proteins or cell lysates as described previously [2]. Metabolic Labeling and Pulse-Chase—After 30 min of preincubation in cysteine- and methionine-free medium (Dulbecco’s modified Eagle’s medium with 5% dialyzed fetal calf serum), cells were pulse labeled with Express [35S]Cys/Met labeling mix (PerkinElmer Life Sciences) at 150. Samples were subjected to SDSPAGE, and gels were treated with Amplify (Amersham Biosciences), dried, and exposed to BioMax-MR film (Eastman Kodak Co.)

RESULTS

Findings

DISCUSSION