E2-25K mediates US11-triggered retro-translocation of MHC class I heavy chains in a permeabilized cell system

Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2006; 103(31):11589-94. DOI: 10.1073/pnas.0605215103
Source: PubMed


In cells expressing human cytomegalovirus US11 protein, newly synthesized MHC class I heavy chains (HCs) are rapidly dislocated from the endoplasmic reticulum (ER) and degraded in the cytosol, a process that is similar to ER-associated degradation (ERAD), the pathway used for degradation of misfolded ER proteins. US11-triggered movement of HCs into the cytosol requires polyubiquitination, but it is unknown which ubiquitin-conjugating and ubiquitin-ligase enzymes are involved. To identify the ubiquitin-conjugating enzyme (E2) required for dislocation, we used a permeabilized cell system, in which endogenous cytosol can be replaced by cow liver cytosol. By fractionating the cytosol, we show that E2-25K can serve as the sole E2 required for dislocation of HCs in vitro. Purified recombinant E2-25K, together with components that convert this E2 to the active E2-ubiquitin thiolester form, can substitute for crude cytosol. E2-25K cannot be replaced by the conjugating enzymes HsUbc7/Ube2G2 or Ube2G1, even though HsUbc7/Ube2G2 and its yeast homolog Ubc7p are known to participate in ERAD. The activity of E2-25K, as measured by ubiquitin dimer formation, is strikingly enhanced when added to permeabilized cells, likely by membrane-bound ubiquitin protein ligases. To identify these ligases, we tested RING domains of various ligases for their activation of E2-25K in vitro. We found that RING domains of gp78/AMFR, a ligase previously implicated in ERAD, and MARCHVII/axotrophin, a ligase of unknown function, greatly enhanced the activity of E2-25K. We conclude that in permeabilized, US11-expressing cells polyubiquitination of the HC substrate can be catalyzed by E2-25K, perhaps in cooperation with the ligase MARCHVII/axotrophin.

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Available from: Dennis Flierman
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    • "Unlike the Derlin-independent mechanism proposed for US2, studies of the US11 protein facilitated identification of Derlin-1 and SEL1L as ERAD components (Figure 1; Lilley and Ploegh, 2004; Ye et al., 2004; Mueller et al., 2006). US11 does not require SPP for MHC-I degradation (Loureiro et al., 2006), but appears to interact with the E3 ligase MARCHVII/axotrophin (Flierman et al., 2006). The cytosolic domain of MHC-I is required for US11-mediated ERAD targeting (Story et al., 1999; Barel et al., 2003), and deletion of the C-terminal valine of MHC-I reduced interaction with Derlin-1 (Cho et al., 2013a). "
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    ABSTRACT: Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. This review focuses on ERAD and its components as well as how viruses use this process to promote their replication and to avoid the immune response.
    Full-text · Article · Jul 2014 · Frontiers in Microbiology
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    • "Studies showed that Derlin-1 is part of a multi-protein complex that mediates the dislocation, ubiquitination and extraction of ERAD substrates from the ER membrane [19,20]. Flierman reported that MHC-I molecules are rapidly dislocated and ubiquitinated once captured by Derlin-1 [21]. Therefore, the functional overlap of p62 and Derlin-1 in the degradation of ubiquitinated proteins may indicate their potential links. "
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    ABSTRACT: Background Recent findings indicated that Derlin-1 has an important function in tumour progression. In this study, we aimed to determine whether Derlin-1 has an oncogene function as a cross-talk molecule with autophagy. Methods Cancer cells were treated with tunicamycin (TM) for 8 and 24 h. The expression of Derlin-1 and autophagy-related genes was determined by western blot. Autophagy was analysed by fluorescence microscopy after staining the cancer cells with monodansylcadaverine. The interaction between Derlin-1 and other proteins was identified using co-immunoprecipitation assay. Results Our study demonstrated high Derlin-1 expression levels in most non-small lung cancer cell lines. Derlin-1 expression was enhanced under endoplasmic reticulum (ER) stress. Previous studies revealed that TM triggers the initiation of autophagy by activating Beclin 1, converting LC3I to LC3II and degrading p62. Knockdown of Derlin-1 did not affect Beclin 1 and LC3II expression but disrupted the degradation of p62 under ER stress, which resulted in the blockage of autophagy flux. Furthermore, Derlin-1 and p62 were observed to interact under ER stress. Conclusion This study is the first report about the interaction between Derlin-1 and p62. Derlin-1 may function in tumour progression partially by interacting with p62.
    Full-text · Article · Jun 2014 · Cancer Cell International
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    • "Derlin-1 forms part of multi-protein complex that mediates the dislocation, ubiquitination, and extraction of ERAD substrates from the ER membrane [13,14]. Thus, once captured by Derlin-1, MHC-I molecules are rapidly dislocated, ubiquitinated by an as-yet unidentified E3 ligase [24] and, finally, extracted from the ER membrane and released into the cytosol by the cytosolic AAA ATPase p97, which provides the energy required for the process [15,25]. We recently found that replacing the TMD of MHC-I molecules with that of US11 forced them to interact with Derlin-1, whereupon they were rapidly dislocated to the cytosol and subsequently degraded by the proteasome [16]. "
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    ABSTRACT: Derlin-1 plays a critical role in endoplasmic reticulum-associated protein degradation (ERAD) of a particular subset of proteins. Although it is generally accepted that Derlin-1 mediates the export of ERAD substrates from the ER to the cytosol, little is known about how Derlin-1 interacts with these substrates. Human cytomegalovirus (HCMV) US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules and evade immune surveillance. US11 requires the cytosolic tail of the MHC-I heavy chain to divert MHC-I molecules into the ERAD pathway for degradation; however, the underlying mechanisms remain unknown. Here, we show that the cytosolic tail of the MHC-I heavy chain, although not required for interaction with US11, is required for tight binding to Derlin-1 and thus for US11-induced dislocation of the MHC-I heavy chain to the cytosol for proteasomal degradation. Surprisingly, deletion of a single C-terminal amino acid from the cytosolic tail disrupted the interaction between MHC-I molecules and Derlin-1, rendering mutant MHC-I molecules resistant to US11-induced degradation. Consistently, deleting the C-terminal cytosolic region of Derlin-1 prevented it from binding to MHC-I molecules. Taken together, these results suggest that the cytosolic region of Derlin-1 is involved in ERAD substrate binding and that this interaction is critical for the Derlin-1-mediated dislocation of the MHC-I heavy chain to the cytosol during US11-induced MHC-I degradation.
    Full-text · Article · Aug 2013 · PLoS ONE
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