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uL24/RPL26 UFMylation Is Essential for Ribosome Quality Control at the ER
Francesco Scavone1, Samantha C. Gumbin2, Paul A. Da Rosa1, Ivan Penchev3, Joao A Paulo4, Vinay V Eapen4, Alban Ourdureau4, Joshua J. Peter5, Yogesh Kulathu5, Wade J. Harper4, Thomas Becker3, Roland Beckmann3, and Ron R. Kopito1
1Department of Biology and 2Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA
3Department of Biochemistry, Gene Center Munich, Munich, Germany
4Department of Cell Biology, Harvard Medical School, Boston, MA, USA
5Medical Research Council Protein Phosphorylation & Ubiquitylation Unit (MRC-PPU), School of Life Sciences, University of Dundee, Dundee, UK.
Ribosomes that stall while translating cytosolic proteins are incapacitated by nascent chains, termed “arrest peptides” (APs) that are destroyed by the ubiquitin proteasome system (UPS) via a process known as ribosome-associated quality control (RQC). 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), yet their degradation is dependent on RQC machinery and 26S proteasome. How the RTJ is breached to enable access of cytosolic UPS to translocon- and ribosome-obstructing ER-APs is not known. Our recent findings indicate that RQC-dependent degradation of ER-APs requires conjugation of the ubiquitin-like (Ubl) protein UFM1 to one or two specific lysine residues of a single ribosomal protein, uL24/RPL26, on 60S ribosomal subunits docked at ER translocons. This exquisite substrate selectivity is conferred by the trimeric ER-bound UFM1 E3 ligase complex (E3UFM1: UFL1, CDK5RAP3, and DDRGK1), which is anchored to the cytosolic face of the ER membrane. Here, we show that ribosome UFMylation occurs exclusively on free 60S ribosomal subunits and present sequential cryogenic electron microscopic snapshots of the E3UFM1 engaging its substrate, uL24/RPL26. Our reconstructions indicate that E3UFM1 first binds to empty tRNA binding sites and the L1 stalk of the 60S subunit via C-terminal domains of UFL1 followed by accommodating uL24 modification more than 150 Å away. After catalyzing UFM1 transfer, E3UFM1 binds stably to its product, UFMylated 60S, forming a C-shaped clamp that extends from the tRNA binding sites to the SEC61 binding site near the polypeptide tunnel exit. We observed that E3UFM1 bound to UFMylated 60S exhibits a conformational state that is incompatible with the concomitant presence of SEC61, thus suggesting a role for E3UFM1 in modulating the interaction between the 60S and the translocon. These data provide strong support for a model in which binding of E3UFM1 to UFMylated uL24/RPL26 breaches the RTJ to enable access of RQC machinery and 26S proteasome to promote the degradation of ER-APs.
This work was supported by the NIGMS (R01GM074874) to R.R.K., by the NIH (T32 GM007276) to S.C.G., and by the Stanford Graduate Fellowship to S.C.G.