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 ...

Protein degradation in eukaryotes usually requires multiubiquitylation and subsequent delivery of the tagged substrates to the proteasome. Recent studies suggest the involvement of the AAA ATPase CDC48, its cofactors, and other ubiquitin binding factors in protein degradation, but how these proteins work together is unclear. Here we show that these factors cooperate sequentially through protein-protein interactions and thereby escort ubiquitin-protein conjugates to the proteasome. Central to this pathway is the chaperone CDC48/p97, which coordinates substrate recruitment, E4-catalyzed multiubiquitin chain assembly, and proteasomal targeting. Concomitantly, CDC48 prevents the formation of excessive multiubiquitin chain sizes that are surplus to requirements for degradation. In yeast, this escort pathway guides a transcription factor from its activation in the cytosol to its final degradation and also mediates proteolysis at the endoplasmic reticulum by the ERAD pathway.

Transcriptionally silent chromatin localizes to the nuclear periphery, which provides a special microenvironment for gene repression. A variety of nuclear membrane proteins interact with repressed chromatin, yet the functional role of these interactions remains poorly understood. Here, we show that, in Schizosaccharomyces pombe, the nuclear membrane protein Lem2 associates with chromatin and mediates silencing and heterochromatin localization. Unexpectedly, we found that these functions can be separated and assigned to different structural domains within Lem2, excluding a simple tethering mechanism. Chromatin association and tethering of centromeres to the periphery are mediated by the N-terminal LEM (LAP2-Emerin-MAN1) domain of Lem2, whereas telomere anchoring and heterochromatin silencing require exclusively its conserved C-terminal MSC (MAN1-Src1 C-terminal) domain. Particularly, silencing by Lem2 is epistatic with the Snf2/HDAC (histone deacetylase) repressor complex SHREC at telomeres, while its necessity can be bypassed by deleting Epe1, a JmjC protein with anti-silencing activity. Furthermore, we found that loss of Lem2 reduces heterochromatin association of SHREC, which is accompanied by increased binding of Epe1. This reveals a critical function of Lem2 in coordinating these antagonistic factors at heterochromatin. The distinct silencing and localization functions mediated by Lem2 suggest that these conserved LEM-containing proteins go beyond simple tethering to play active roles in perinuclear silencing.