The ribosome-associated chaperone Zuo1 controls translation upon TORC1 inhibition.

Protein requirements of eukaryotic cells are ensured by proteostasis, which is mediated by tight control of TORC1 activity. Upon TORC1 inhibition, protein degradation is increased and protein synthesis is reduced through inhibition of translation initiation to maintain cell viability. Here, we show that the ribosome-associated complex (RAC)/Ssb chaperone system, composed of the HSP70 chaperone Ssb and its HSP40 co-chaperone Zuo1, is required to maintain proteostasis and cell viability under TORC1 inhibition in Saccharomyces cerevisiae. In the absence of Zuo1, translation does not decrease in response to the loss of TORC1 activity. A functional interaction between Zuo1 and Ssb is required for proper translational control and proteostasis maintenance upon TORC1 inhibition. Furthermore, we have shown that the rapid degradation of eIF4G following TORC1 inhibition is mediated by autophagy and is prevented in zuo1Δ cells, contributing to decreased survival in these conditions. We found that autophagy is defective in zuo1Δ cells, which impedes eIF4G degradation upon TORC1 inhibition. Our findings identify an essential role for RAC/Ssb in regulating translation in response to changes in TORC1 signalling.

Multiple phosphorylation of the Cdc48/p97 cofactor protein Shp1/p47 occurs upon cell stress in budding yeast.

The homohexameric p97 complex, composed of Cdc48 subunits in yeast, is a crucial component of protein quality control pathways including ER-associated degradation. The complex acts to segregate protein complexes in an ATP-dependent manner, requiring the engagement of cofactor proteins that determine substrate specificity. The function of different Cdc48 cofactors and how they are regulated remains relatively poorly understood. In this study, we assess the phosphorylation of Cdc48 adaptor proteins, revealing a unique and distinctive phosphorylation pattern of Shp1/p47 that changed in response to TORC1 inhibition. Site-directed mutagenesis confirmed that this pattern corresponded to phosphorylation at residues S108 and S315 of Shp1, with the double-phosphorylated form becoming predominant upon TORC1 inhibition, ER-stress, and oxidative stress. Finally, we assessed candidate kinases and phosphatases responsible for Shp1 phosphorylation and identified two regulators. We found that cells lacking the kinase Mpk1/Slt2 show reduced Shp1 phosphorylation, whereas impaired PP1 phosphatase catalytic subunit (Glc7) activity resulted in increased Shp1 phosphorylation. Overall, these findings identify a phosphoregulation of Shp1 at multiple sites by Mpk1 kinase and PP1 phosphatase upon various stresses.

Actin remodelling controls proteasome homeostasis upon stress.

When cells are stressed, bulk translation is often downregulated to reduce energy demands while stress-response proteins are simultaneously upregulated. To promote proteasome assembly and activity and maintain cell viability upon TORC1 inhibition, 19S regulatory-particle assembly chaperones (RPACs) are selectively translated. However, the molecular mechanism for such selective translational upregulation is unclear. Here, using yeast, we discover that remodelling of the actin cytoskeleton is important for RPAC translation following TORC1 inhibition. mRNA of the RPAC ADC17 is associated with actin cables and is enriched at cortical actin patches under stress, dependent upon the early endocytic protein Ede1. ede1∆ cells failed to induce RPACs and proteasome assembly upon TORC1 inhibition. Conversely, artificially tethering ADC17 mRNA to cortical actin patches enhanced its translation upon stress. These findings suggest that actin-dense structures such as cortical actin patches may serve as a translation platform for a subset of stress-induced mRNAs including regulators of proteasome homeostasis.