The heat-shock response is a complex cellular program that induces major changes in protein translation, folding and degradation to alleviate toxicity caused by protein misfolding. While heat-shock has been widely used to study proteostasis, it remained unclear how misfolded proteins are targeted for proteolysis in these conditions. We found that Rsp5 and its mammalian homologue Nedd4 are ones of the main E3-ligases responsible for the increased ubiquitination induced by heat-stress. We determined that Rsp5 ubiquitinates mainly cytosolic misfolded proteins upon heat-shock for proteasome degradation. We found that ubiquitination of heat-induced substrates requires the Hsp40 co-chaperone Ydj1 that is further associated with Rsp5 upon heat-shock. Additionally, ubiquitination is also promoted by PY Rsp5-binding motifs found primarily in the structured regions of stress-induced substrates, which can act as heat-induced degrons. Our results support a bipartite recognition mechanism combining direct and chaperone-dependent ubiquitination of misfolded cytosolic proteins by Rsp5.

Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.

Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions.

Stress granules (SGs) are stress-induced membraneless condensates that store non-translating mRNA and stalled translation initiation complexes. While metazoan SGs are dynamic compartments where proteins can rapidly exchange with their surroundings, yeast SGs seem largely static. To gain a better understanding of the yeast SGs, we identified proteins that sediment after heat-shock by mass spectrometry. Proteins that sediment upon heat-shock are biased toward a subset of abundant proteins that are significantly enriched in intrinsically disordered regions (IDRs). Heat-induced SG localization of over 80 proteins were confirmed using microscopy, including 32 proteins not previously known to localize to SGs. We found that several IDRs were sufficient to mediate SG recruitment. Moreover, the dynamic exchange of IDRs can be observed via FRAP, while other components remain immobile. Lastly, we showed that the IDR of the Ubp3 deubiquitinase was critical for yeast SG formation. This work shows that IDRs can be sufficient for SG incorporation, can remain dynamic in vitrified SGs, and can play an important role in cellular compartmentalization upon stress.