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.

Functional genomic studies in Saccharomyces cerevisiae have contributed enormously to our understanding of cellular processes. Their full potential, however, has been hampered by the limited availability of reagents to systematically study essential genes and the inability to quantify the small effects of most gene deletions on growth. Here we describe the construction of a library of hypomorphic alleles of essential genes and a high-throughput growth competition assay to measure fitness with unprecedented sensitivity. These tools dramatically increase the breadth and precision with which quantitative genetic analysis can be performed in yeast. We illustrate the value of these approaches by using genetic interactions to reveal new relationships between chromatin-modifying factors and to create a functional map of the proteasome. Finally, by measuring the fitness of strains in the yeast deletion library, we addressed an enigma regarding the apparent prevalence of gene dispensability and found that most genes do contribute to growth.A fundamental challenge of the post-genomic era is to assign functions to genes and to understand how gene networks are organized to execute diverse cellular processes. The budding yeast S. cerevisiae has served as a premier eukaryotic model system for this task [1][2][3][4][5] , and a library of strains in which each nonessential yeast gene is deleted has been an important tool for these efforts 1 . This resource has made it possible to assess the contribution of each nonessential gene to any measurable phenotype.These genomic approaches can be extended by systematically measuring genetic interactions 5 . Genetic interactions describe how the phenotype associated with compromising one gene is modulated by perturbing a second gene. Such effects can be defined quantitatively Correspondence should be addressed to J.S.W. (weissman@cmp.ucsf.edu). 6 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Methods website. HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptas the difference between the observed magnitude of a phenotype for a double mutant and that expected if the genes do not interact, as given by ε= W AB − W A × W B , where ε is the interaction score and W X refers to the phenotype seen in genetic background X (refs. 6,7 ). Theoretical considerations and empirical experience have established the utility of genetic interactions in defining functional relationships between genes 2,3,6,8 . Early studies focused on 'synthetic sick or lethal' or 'negative' interactions 3 , in which the phenotype associated with perturbing two genes simultaneously is more severe than expected given the phenotypes of the individual gene perturbations (ε < 0). More recently, interactions in which the phenotype of a double mutant is less severe than expected (ε > 0, 'alleviating' or 'positive' interactions) have proven highly valuable as they often occur between genes that have closely related functions-for example, between ...