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The transporter associated with antigen processing (TAP) is a key player in the MHC class I-restricted antigen presentation and an attractive target for immune evasion by viruses. Bovine herpesvirus 1 (BoHV-1) impairs TAP-dependent antigenic peptide transport through a two-pronged mechanism in which binding of the UL49.5 gene product to TAP both inhibits peptide transport and promotes its proteasomal degradation. How UL49.5 promotes TAP degradation is unknown. Here, we use high-content siRNA and genome-wide CRISPR-Cas9 screening to identify CLR2 KLHDC3 as the E3 ligase responsible for UL49.5-triggered TAP disposal in human cells. We propose that the C-terminus of UL49.5 mimics a C-end rule degron that recruits the E3 to TAP and engages the CRL2 E3 in ER-associated degradation., Competing Interests: Declaration of interests The authors declare no competing interests.

Over 80% of people with cystic fibrosis (CF) carry the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride ion channel at the apical plasma membrane (PM) of epithelial cells. F508del impairs CFTR folding causing it to be destroyed by endoplasmic reticulum associated degradation (ERAD). Small molecule correctors, which act as pharmacological chaperones to divert CFTR-F508del from ERAD, are the primary strategy for treating CF, yet corrector development continues with only a rudimentary understanding of how ERAD targets CFTR-F508del. We conducted genome-wide CRISPR/Cas9 knockout screens to systematically identify the molecular machinery that underlies CFTR-F508del ERAD. Although the ER-resident ubiquitin ligase, RNF5 was the top E3 hit, knocking out RNF5 only modestly reduced CFTR-F508del degradation. Sublibrary screens in an RNF5 knockout background identified RNF185 as a redundant ligase, demonstrating that CFTR-F508del ERAD is highly buffered. Gene-drug interaction experiments demonstrated that correctors tezacaftor (VX-661) and elexacaftor (VX-445) stabilize sequential, RNF5-resistant folding states. We propose that binding of correctors to nascent CFTR-F508del alters its folding landscape by stabilizing folding states that are not substrates for RNF5-mediated ubiquitylation.

Ribosomes that stall while translating cytosolic proteins are incapacitated by incomplete nascent chains, termed "arrest peptides" (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as the ribosome-associated quality control (RQC) pathway. By contrast, APs on ribosomes that stall while translocating secretory proteins into the endoplasmic reticulum (ER-APs) are shielded from cytosol by the ER membrane and the tightly sealed ribosome-translocon junction (RTJ). How this junction is breached to enable access of cytosolic UPS machinery and 26S proteasomes to translocon- and ribosome-obstructing ER-APs is not known. Here, we show that UPS and RQC-dependent degradation of ER-APs strictly requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to 60S ribosomal subunits at the RTJ. Therefore, UFMylation of translocon-bound 60S subunits modulates the RTJ to promote access of proteasomes and RQC machinery to ER-APs., Significance Statement: UFM1 is a ubiquitin-like protein that is selectively conjugated to the large (60S) subunit of ribosomes bound to the endoplasmic reticulum (ER), but the specific biological function of this modification is unclear. Here, we show that UFMylation facilitates proteasome-mediated degradation of arrest polypeptides (APs) which are generated following splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER. We propose that UFMylation weakens the tightly sealed ribosome-translocon junction, thereby allowing the cytosolic ubiquitin-proteasome and ribosome-associated quality control machineries to access ER-APs.