Mapping Pathways and Phenotypes by Systematic Gene Overexpression

Sopko, Richelle; Huang, Dongqing; Preston, Nicolle; Chua, Gordon; Papp, Balázs; Kafadar, Kimberly A.; Snyder, Mike; Oliver, Stephen G.; Cyert, Martha S.; Hughes, Timothy R.; Boone, Charles; Andrews, Brenda

doi:10.1016/j.molcel.2005.12.011

Cited by 630 publications

(783 citation statements)

References 63 publications

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“…This protein was identified via a synthetic dosage lethality screen in a pho85D and a pho80D strain and localization and phosphorylation studies confirmed that Pho85-Pho80 promotes nuclear exclusion of Crz1 via direct phosphorylation (Sopko et al 2006). Crz1 is known to be activated via dephosphorylation by the serine/threonine protein phosphatase Calcineurin, consisting of two catalytic subunits Cna1 and Cna2 and a regulatory subunit Cnb1, in response to ion stress involving Li ?…”

Section: The Protein Kinase Sch9mentioning

confidence: 99%

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

et al. 2010

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Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G 0 ) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85-Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.

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Section: The Protein Kinase Sch9mentioning

confidence: 99%

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

et al. 2010

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“…With regard to the latter, the yeast genome contains approximately 1130 essential genes, and we readily envision application of our platform to this important yeast gene set. The resulting mutant collection will provide a strong complement to existing genome-wide collections of titratable promoter alleles (Mnaimneh et al, 2004), gene deletion mutants (Winzeler and Davis, 1997;Winzeler et al, 1999) and gene overexpression strains (Sopko et al, 2006;Gelperin et al, 2005). Similarly, genome-scale mislocalization screens will augment pathway discovery tools, such as synthetic genetic array analysis (Tong et al, 2004).…”

Section: Discussionmentioning

confidence: 99%

A small molecule‐directed approach to control protein localization and function

Geda

Patury

et al. 2008

Yeast

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Protein localization is tightly linked with function, such that the subcellular distribution of a protein serves as an important control point regulating activity. Exploiting this regulatory mechanism, we present here a general approach by which protein location, and hence function, may be controlled on demand in the budding yeast. In this system a small molecule, rapamycin, is used to temporarily recruit a strong cellular address signal to the target protein, placing subcellular localization under control of the selective chemical stimulus. The kinetics of this system are rapid: rapamycin-directed nucleo-cytoplasmic transport is evident 10-12 min posttreatment and the process is reversible upon removal of rapamycin. Accordingly, we envision this platform as a promising approach for the systematic construction of conditional loss-of-function mutants. As proof of principle, we used this system to direct nuclear export of the essential heat shock transcription factor Hsf1p, thereby mimicking the cell-cycle arrest phenotype of an hsf1 temperature-sensitive mutant. Our drug-induced localization platform also provides a method by which protein localization can be uncoupled from endogenous cell signalling events, addressing the necessity or sufficiency of a given localization shift for a particular cell process. To illustrate, we directed the nuclear import of the calcineurin-dependent transcription factor Crz1p in the absence of native stimuli; this analysis directly substantiates that nuclear translocation of this protein is insufficient for its transcriptional activity. In total, this technology represents a powerful method for the generation of conditional alleles and directed mislocalization studies in yeast, with potential applicability on a genome-wide scale.

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“…Thus, Rad2p-induced cell growth arrest and consequent mitotic catastrophe would be exaggerated phenotypes that do not occur under natural growth conditions, regardless of the presence of DNA damage. It has recently been shown that overexpression arrays are useful for studying gain-of-function phenotypes of genes that cannot be examined by traditional gene deletion studies (Sopko et al, 2006). Thus, overexpression of Rad2p can elucidate the gain-of-function of Rad2p that is shown only temporarily while it is induced by DNA damage.…”

Section: Discussionmentioning

confidence: 99%

“…RAD2 is induced naturally by DNA-damaging agents (Madura and Prakash, 1986;Robinson et al, 1986), and artificial overexpression of the gene interrupts cell growth (Nicolet and Friedberg, 1987;Sopko et al, 2006). Indeed, serially cultured RAD2-overexpressing cells confer rapid growth and the rapidly growing cells express less RAD2 (Nicolet and Friedberg, 1987).…”

Section: Introductionmentioning

confidence: 99%

Mitotic catastrophe induced by overexpression of budding yeast Rad2p

Kang

Lim

et al. 2010

Yeast

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Mitotic catastrophe provokes endopolyploidy, giant cell formation and, eventually, delayed cell death. Mitotic catastrophe is induced by defective cell cycle checkpoints and by some anticancer drugs, ionizing radiation and microtubule-destabilizing agents. RAD2 is a yeast homologue of XPG, which is a human endonuclease involved in nucleotide excision repair. Here we show that Rad2p overexpression alone, in the absence of extrinsic DNA damage, causes cell growth arrest and mitotic catastrophe. Interestingly, Rad2p-induced cell growth arrest is not caused by the catalytic activity of Rad2p but rather by its C-terminal region. Cells growth-arrested by Rad2p induction do not show apoptotic phenotypes and deletion of YCA1, a yeast caspase homologue, does not affect cell growth arrest by Rad2p induction. However, Rad2p-induced cell growth arrest is released by rad9 deletion but is not affected by downstream DNA damage checkpoint genes. These observations suggest that RAD2 has a function in coordinating cell cycle regulation and damaged DNA repair.

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Mapping Pathways and Phenotypes by Systematic Gene Overexpression

Cited by 630 publications

References 63 publications

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

Life in the midst of scarcity: adaptations to nutrient availability in Saccharomyces cerevisiae

A small molecule‐directed approach to control protein localization and function

Mitotic catastrophe induced by overexpression of budding yeast Rad2p

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