Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2’s impact on HCMV pathogenesis.

The US11 gene product of human cytomegalovirus promotes viral immune evasion by hijacking the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. US11 initiates dislocation of newly translocated MHC I from the ER to the cytosol for proteasome-mediated degradation. Despite the critical role for ubiquitin in this degradation pathway, the responsible E3 ligase is unknown. In a forward genetic screen for host ERAD components hijacked by US11 in near-haploid KBM7 cells, we identified TMEM129, an uncharacterized polytopic membrane protein. TMEM129 is essential and rate-limiting for US11-mediated MHC-I degradation and acts as a novel ER resident E3 ubiquitin ligase. TMEM129 contains an unusual cysteine-only RING with intrinsic E3 ligase activity and is recruited to US11 via Derlin-1. Together with its E2 conjugase Ube2J2, TMEM129 is responsible for the ubiquitination, dislocation, and subsequent degradation of US11-associated MHC-I. US11 engages two degradation pathways: a Derlin-1/TMEM129-dependent pathway required for MHC-I degradation and a SEL1L/HRD1-dependent pathway required for "free" US11 degradation. Our data show that TMEM129 is a novel ERAD E3 ligase and the central component of a novel mammalian ERAD complex. P roteins inserted into the endoplasmic reticulum (ER) must fold and acquire their native state before further trafficking through the secretory pathway (1, 2). To avoid the toxicity associated with misfolded gene products, all proteins must pass the ER quality-control checkpoint. Misfolded proteins are rejected and dislocated across the ER-membrane for cytosolic proteasome degradation in a process known as ER-associated degradation (ERAD). ERAD degrades misfolded and unassembled proteins and regulates turnover of ER-resident proteins (3).Ubiquitination of the protein substrate provides a critical step in protein dislocation (4). The RING family constitutes the largest family of E3 ligases, including those involved in ERAD (5). The RING domain creates a binding platform for the E2 conjugase and consists of two zinc atoms coordinated in a cross-brace motif via interspersed cysteine (C) and histidine (H) residues in a C3HC4, C3H2C3, or C4HC3 conformation (5). Most ERAD E3 ligases are integral membrane proteins; they form the core of the ERAD machinery, nucleating functionally distinct ERAD complexes. The mammalian system is more complex than yeast and has undergone an expansion of the ERAD E3 ligase family, with the Hrd1p homologs Hrd1 and Gp78, the Doa10p homolog MARCH6, RNF5, TRC8, and CHIP (3, 4).Many pathogens appropriate the ubiquitin-proteasome system, particularly to degrade components of the host immune system (6, 7). The human cytomegalovirus (HCMV) US2 and US11 gene products have been instrumental in studies of the mammalian ERAD system. These viral proteins hijack separate components of the ERAD system to degrade MHC-I, thus preventing cytotoxic T lymphocyte recognition of infected cells (8,9). US2 appropriates the TRC8 E3 ubiquitin ligase (10), whereas dislocation induced by US1...

Misfolded MHC class I heavy chains (MHC I HCs) are targeted for endoplasmic reticulum (ER)-associated degradation (ERAD) by the ubiquitin E3 ligase HRD1, and E2 ubiquitin conjugating enzyme UBE2J1, and represent one of the few known endogenous ERAD substrates. The mechanism by which misfolded proteins are dislocated across the ER membrane into the cytosol is unclear. Here, we investigate the requirements for MHC I ubiquitination and degradation and show that endogenous misfolded MHC I HCs are recognized in the ER lumen by EDEM1 in a glycan-dependent manner and targeted to the core SEL1L/HRD1/UBE2J1 complex. A soluble MHC I HC lacking its transmembrane domain and cytosolic tail uses the same ERAD components and is degraded as efficiently as wildtype MHC I. Unexpectedly, HRD1-dependent polyubiquitination is preferentially targeted to the ER luminal domain of full-length MHC I HCs, despite the presence of an exposed cytosolic C-terminal tail. MHC I luminal domain ubiquitination occurs before p97 ATPase-mediated extraction from the ER membrane and can be targeted to nonlysine, as well as lysine, residues. A subset of integral membrane proteins, therefore, requires an early dislocation event to expose part of their luminal domain to the cytosol, before HRD1-mediated polyubiquitination and dislocation.T he assembly and regulated expression of plasma membrane and secreted proteins is fundamentally reliant on effective endoplasmic reticulum quality control (ERQC). Endoplasmic reticulum (ER)-associated degradation (ERAD) is central to ERQC, selectively disposing of misfolded or surplus proteins to maintain ER homeostasis. ERAD involves recognition, ubiquitination, and retrotranslocation of substrates from the ER to the cytosol for proteasome-mediated degradation (1). Polyubiquitination of the substrate is critical, both for facilitating ER membrane extraction by the p97 ATPase-ubiquitin fusion degradation 1 (Ufd1)-nuclear protein localization 4 (Npl4) complex (2-4) and for delivery to the proteasome. However, the precise role of ubiquitin in the actual dislocation event is unclear.The cellular ERAD machinery consists of multiprotein complexes, typically comprising a membrane-embedded ubiquitin E3 ligase, which engages substrates directly or via ER luminal adaptors (1). Different substrates degraded by the same E3 may use distinct ERAD cofactors that facilitate substrate delivery to the ligase and E2-conjugating enzyme for ubiquitination.Attempts to delineate distinct degradation pathways according to the site of the defect in a misfolded protein have been made in yeast. Proteins with cytosolic defects (ERAD-C) require Doa10p, whereas proteins with transmembrane (ERAD-M) or luminal (ERAD-L) lesions use Hrd1p (5, 6). The expanded repertoire of ERAD E3s in mammalian cells makes developing analogous rules more challenging. 3-hydroxy-3-methylglutaryl-CoA reductase degradation protein 1 (HRD1) and its homolog gp78/autocrine motility factor receptor (AMFR) are the best-characterized mammalian ERAD ligases. The association of HR...

Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export.

HighlightsE3 ubiquitin ligases play a central role in viral and cellular degradation of MHC-I.HCMV US2 and US11 hijack the mammalian ERAD machinery to induce MHC-I degradation.We identified the TRC8 and TMEM129 E3 ligases as crucial for US2/11 function.The US2/11 degradation hubs are flexible and enable viral evasion of different immune functions.Cellular quality control of MHC-I is controlled by the HRD1/SEL1L E3 ligase complex.