Abstract
Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.
Highlights
Synthesis and maintenance of the functional cellular proteome are critical parts of protein homeostasis
Cells derived from ssb∆∆ and wild-type control strains were plated on YPD agar plates supplemented with aminoglycoside hygromycin B (HygB) or high concentration of NaCl (0.8 M) and grown at 30 ◦C, or plated on plain YPD agar plates and incubated at low temperature of 20 ◦C
Consistent with previously published data [52], we found that placing the FLAG tag at the C-terminus of Ltn1 does not affect catalytic activity of the ligase during ribosome-associated protein quality control (RQC), as we detected efficient ubiquitination of ribosome-bound nascent chains (RNCs) associated with 60S in gradients derived from Ltn1FLAG-expressing cells but not from cells transformed with empty vector control (Figure 2C,D and Figure A1, fractions 12–13)
Summary
Synthesis and maintenance of the functional cellular proteome are critical parts of protein homeostasis (proteostasis). Structural studies have proposed that Ssz, anchored to ribosomes by Zuo (Figure 1B, bottom), acts as a holding chaperone that directs the growing RNC from the exit tunnel towards RAC-bound Ssb [28]. Numerous biochemical studies supported by the recent selective ribosome profiling (SeRP) data provide strong evidence of cross-talk between Ssb/RAC function and protein translation through the chaperone’s participation in RNC folding [40,45]. This raises the question of whether early-stage Ssb/RAC-mediated folding and early-stage RQC are functionally connected. We provide evidence that Ssb/RAC collaborate with RQC via facilitating accommodation of the Ub ligase Ltn on ribosomes, thereby promoting efficient ubiquitination of endogenous nascent polypeptide chains
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