Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin–proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood. Here using a high-coverage genome-wide shRNA library, we identify the uncharacterized protein TMEM129 and the ubiquitin-conjugating E2 enzyme UBE2J2 to be essential for US11-mediated HLA class I downregulation. TMEM129 is an unconventional C4C4-type RING finger E3 ubiquitin ligase that resides within a complex containing various other ERAD components, including Derlin-1, Derlin-2, VIMP and p97, indicating that TMEM129 is an integral part of the ER-resident dislocation complex mediating US11-induced HLA class I degradation.

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Summary Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.

Misfolded, potentially toxic proteins in the lumen and membrane of the endoplasmic reticulum (ER) are eliminated by proteasomes in the cytosol through ER-associated degradation (ERAD). The ERAD process involves the recognition of substrates in the lumen and membrane of the ER, their translocation into the cytosol, ubiquitination, and delivery to the proteasome for degradation. These ERAD steps are performed by membrane-embedded ubiquitin-ligase complexes of different specificity that together cover a wide range of substrates. Besides misfolded proteins, ERAD further contributes to quality control by targeting unassembled and mislocalized proteins. ERAD also targets a restricted set of folded proteins to influence critical ER functions such as sterol biosynthesis, calcium homeostasis, or ER contacts with other organelles. This review describes the ubiquitin-ligase complexes and the principles guiding protein degradation by ERAD.