Abaeva Irina S, Bulakhov Alexander G, Hellen Christopher U T, Pestova Tatyana V
Department of Cell Biology, State University of New York Downstate Health Sciences University, Brooklyn, New York 11203, USA.
Department of Cell Biology, State University of New York Downstate Health Sciences University, Brooklyn, New York 11203, USA
Genes Dev. 2025 May 2;39(9-10):617-633. doi: 10.1101/gad.352389.124.
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) and imposes K48 specificity by orienting Ub 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.
由翻译中断产生的多肽通过核糖体相关质量控制（RQC）途径进行蛋白酶体降解。ASC-1复合物将停滞的核糖体分裂为40S亚基和新生链-tRNA相关的60S亚基（60S RNCs）。60S RNCs与NEMF结合，NEMF促进环状E3泛素（Ub）连接酶李斯特菌（酵母中的Ltn1）的募集，后者使新生链泛素化。环状E3直接介导Ub从E2∼Ub结合物的转移，这意味着Ub连接的特异性由特定的E2决定。李斯特菌与混杂的Ube2D E2配对时效率最高。我们之前发现TCF25（酵母中的Rqc1）可以赋予与Ube2D E2配对的李斯特菌K48特异性。为了确定TCF25的作用机制，我们结合了功能生化研究和AlphaFold3建模，现在报告TCF25与李斯特菌的环状结构域和受体泛素（Ub）特异性相互作用，并通过定向Ub使其K48直接定位以攻击Ube2D1∼Ub结合物的硫酯键来赋予K48特异性。我们还发现TCF25自身会被李斯特菌进行K48特异性泛素化，这表明了在缺乏Ltn1时报告的Rqc1上调以及体内观察到的蛋白酶体对TCF25降解的机制。
