The ribosome-associated quality control factor TCF25 imposes K48 specificity on Listerin-mediated ubiquitination of nascent chains by binding and specifically orienting the acceptor ubiquitin

Polypeptides arising from interrupted translation undergo proteasomal degradation by the ribosome-associated quality control (RQC) pathway. The ASC-1 complex splits stalled ribosomes into 40S subunits and nascent chain-tRNA-associated 60S subunits (60S RNCs). 60S RNCs associate with NEMF that promotes recruitment of the RING-type E3 ubiquitin (Ub) ligase Listerin (Ltn1 in yeast), which ubiquitinates nascent chains. RING-type E3s mediate the transfer of Ub directly from the E2∼Ub conjugate, implying that the specificity of Ub linkage is determined by the given E2. Listerin is most efficient when it is paired with promiscuous Ube2D E2s. We previously found that TCF25 (Rqc1 in yeast) can impose K48 specificity on Listerin paired with Ube2D E2s. To determine the mechanism of TCF25's action, we combined functional biochemical studies and AlphaFold3 modeling and now report that TCF25 specifically interacts with the RING domain of Listerin and the acceptor ubiquitin (Ub^A) and imposes K48 specificity by orienting Ub^A such that its K48 is directly positioned to attack the thioester bond of the Ube2D1∼Ub conjugate. We also found that TCF25 itself undergoes K48-specific ubiquitination by Listerin, suggesting a mechanism for the reported upregulation of Rqc1 in the absence of Ltn1 and the observed degradation of TCF25 by the proteasome in vivo.