Summary The conserved transcriptional regulator Heat Shock Factor 1 (Hsf1) is a key sensor of proteotoxic and other stress in the eukaryotic cytosol, yet its regulation is poorly understood. We surveyed Hsf1 activity in a genome-wide loss-of-function library in Saccaromyces cerevisiae as well as ~78,000 double mutants and found Hsf1 activity to be modulated by highly diverse stresses. These included disruption of a ribosome-bound complex we named the Ribosome Quality Control Complex (RQC) comprising the Ltn1 E3 ubiquitin ligase, two highly conserved but poorly characterized proteins (Tae2 and Rqc1), and Cdc48 and its cofactors. Electron microscopy and biochemical analyses revealed that the RQC forms a stable complex with 60S ribosomal subunits containing stalled polypeptides and triggers their degradation. A negative feedback loop regulates the RQC and Hsf1 senses an RQC-mediated translation stress signal distinctly from other stresses. Our work reveals the range of stresses Hsf1 monitors and elucidates a conserved cotranslational protein quality control mechanism.

Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. Using the cell’s endogenous sensor (the unfolded protein response), we identified several hundred yeast genes with roles in endoplasmic reticulum folding and systematically characterized their functional interdependencies by measuring unfolded protein response levels in double mutants. This strategy revealed multiple conserved factors critical for endoplasmic reticulum folding including an intimate dependence on the later secretory pathway, a previously uncharacterized six-protein transmembrane complex, and a co-chaperone complex that delivers tail-anchored proteins to their membrane insertion machinery. The use of a quantitative reporter in a comprehensive screen followed by systematic analysis of genetic dependencies should be broadly applicable to functional dissection of complex cellular processes from yeast to man.

Cell survival under severe thermal stress requires the activity of the ClpB (Hsp104) AAA+ chaperone that solubilizes and reactivates aggregated proteins in concert with the DnaK (Hsp70) chaperone system. How protein disaggregation is achieved and whether survival is solely dependent on ClpB-mediated elimination of aggregates or also on reactivation of aggregated proteins has been unclear. We engineered a ClpB variant, BAP, which associates with the ClpP peptidase and thereby is converted into a degrading disaggregase. BAP translocates substrates through its central pore directly into ClpP for degradation. ClpB-dependent translocation is demonstrated to be an integral part of the disaggregation mechanism. Protein disaggregation by the BAP/ClpP complex remains dependent on DnaK, defining a role for DnaK at early stages of the disaggregation reaction. The activity switch of BAP to a degrading disaggregase does not support thermotolerance development, demonstrating that cell survival during severe thermal stress requires reactivation of aggregated proteins.

SUMMARY Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultra-complex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a non-canonical role for COPI, a novel protein complex (SRIC) affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

ClpB of Escherichia coli is an ATP-dependent ringforming chaperone that mediates the resolubilization of aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the Hsp100/Clp subfamily of AAA؉ proteins and is composed of an N-terminal domain and two AAA-domains that are separated by a "linker" region. Here we present a detailed structurefunction analysis of ClpB, dissecting the individual roles of ClpB domains and conserved motifs in oligomerization, ATP hydrolysis, and chaperone activity. Our results show that ClpB oligomerization is strictly dependent on the presence of the C-terminal domain of the second AAA-domain, while ATP binding to the first AAAdomains stabilized the ClpB oligomer. Analysis of mutants of conserved residues in Walker A and B and sensor 2 motifs revealed that both AAA-domains contribute to the basal ATPase activity of ClpB and communicate in a complex manner. Chaperone activity strictly depends on ClpB oligomerization and the presence of a residual ATPase activity. The N-domain is dispensable for oligomerization and for the disaggregating activity in vitro and in vivo. In contrast the presence of the linker region, although not involved in oligomerization, is essential for ClpB chaperone activity.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

The endoplasmic reticulum (ER) must target potentially toxic misfolded proteins for retrotranslocation and proteasomal degradation while avoiding destruction of productive folding intermediates. For luminal proteins, this discrimination typically depends not only on the folding status of a polypeptide, but also on its glycosylation state. Two putative sugar binding proteins, Htm1p and Yos9p, are required for degradation of misfolded glycoproteins, but the nature of the glycan degradation signal and how such signals are generated and decoded remains unclear. Here we characterize Yos9p's oligosaccharide-binding specificity and find that it recognizes glycans containing terminal alpha1,6-linked mannose residues. We also provide evidence in vivo that a terminal alpha1,6-linked mannose-containing oligosaccharide is required for degradation and that Htm1p acts upstream of Yos9p to mediate the generation of such sugars. This strategy of marking potential substrates by Htm1p and decoding the signal by Yos9p is well suited to provide a proofreading mechanism that enhances substrate specificity.