NEMF-mediated CAT tailing facilitates translocation-associated quality control.

Ribosome stalling during co-translational translocation at the ER causes translocon clogging and impairs ER protein biogenesis. Mammalian cells resolve translocon clogging via a poorly characterized translocation-associated quality control (TAQC) process. Here, we combine a genome-wide CRISPR screen with live-cell imaging to dissect the molecular linchpin of TAQC. We show that TAQC substrates translated from mRNAs bearing a ribosome-stalling poly(A) sequence are degraded by lysosomes and the proteasome. By contrast, the degradation of defective nascent chains encoded by nonstop (NS) mRNAs involves an unconventional ER-associated protein degradation (ERAD) mechanism depending on ER-to-Golgi trafficking, KDEL-mediated substrate retrieval at the Golgi, and a tRNA-binding factor NEMF that appends an aggregation-prone carboxyl tail to stalled NS nascent chains. We propose that NEMF-mediated CAT tailing targets a subset of TAQC substrates via Golgi retrieval for ERAD, safeguarding ER homeostasis.