Limits...
A novel genetic screen implicates Elm1 in the inactivation of the yeast transcription factor SBF.

Manderson EN, Malleshaiah M, Michnick SW - PLoS ONE (2008)

Bottom Line: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway.Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest.In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT

Background: Despite extensive large scale analyses of expression and protein-protein interactions (PPI) in the model organism Saccharomyces cerevisiae, over a thousand yeast genes remain uncharacterized. We have developed a novel strategy in yeast that directly combines genetics with proteomics in the same screen to assign function to proteins based on the observation of genetic perturbations of sentinel protein interactions (GePPI). As proof of principle of the GePPI screen, we applied it to identify proteins involved in the regulation of an important yeast cell cycle transcription factor, SBF that activates gene expression during G1 and S phase.

Methodology/principle findings: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway. We created a fluorescent protein-fragment complementation assay (PCA) to detect the interaction between Cdc28 and Swi4, which leads to the inactivation of SBF. The PCA signal was quantified by microscopy and image analysis in deletion strains corresponding to 25 candidate genes that are periodically expressed during the cell cycle and are substrates of Cdc28. We showed that the serine-threonine kinase Elm1 plays a role in the inactivation of SBF and that phosphorylation of Elm1 by Cdc28 may be a mechanism to inactivate Elm1 upon completion of mitosis.

Conclusions/significance: Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest. The ease in generating PCA assays for any protein interaction and the availability of the yeast deletion strain collection allows GePPI to be applied to any cellular network. In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease.

Show MeSH

Related in: MedlinePlus

SBF activity is increased in the Elm1 deletion strain.(A) A representative result of semi-quantitative RT-PCR analysis of the SBF-specific gene, CLN1, the MBF-specific gene, CDC45 and control gene, ACT1 in MatA, Δelm1 and elm1T551A. RT = reverse transcriptase. (B) Average expression of CLN1 and CDC45 normalized to ACT1 expression, over three different experiments. Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2198942&req=5

pone-0001500-g004: SBF activity is increased in the Elm1 deletion strain.(A) A representative result of semi-quantitative RT-PCR analysis of the SBF-specific gene, CLN1, the MBF-specific gene, CDC45 and control gene, ACT1 in MatA, Δelm1 and elm1T551A. RT = reverse transcriptase. (B) Average expression of CLN1 and CDC45 normalized to ACT1 expression, over three different experiments. Error bars represent standard deviation.

Mentions: Decreased interaction between Cdc28 and Swi4 in Δelm1 implies diminished inactivation of SBF. In support of this hypothesis, we observed increased mRNA levels of the SBF-specific gene, CLN1 in Δelm1 (P = 0.0118), but not the MBF-specific gene, CDC45 (Figure 4 A,B). There was no significant increase in CLN1 transcripts in elm1T551A in comparison to wild-type cells (P = 0.4228) (Figure 4 A,B), consistent with the finding that the Cdc28-Swi4 PCA is not perturbed in the mutant strain (Figure S3). SBF-specific targets are enriched in genes encoding proteins responsible for cell morphogenesis and budding [18], [19]. Thus, it is possible that the elongated bud phenotype of Δelm1 is at least partially due to a failure to inactivate SBF activated transcription of genes that induce budding.


A novel genetic screen implicates Elm1 in the inactivation of the yeast transcription factor SBF.

Manderson EN, Malleshaiah M, Michnick SW - PLoS ONE (2008)

SBF activity is increased in the Elm1 deletion strain.(A) A representative result of semi-quantitative RT-PCR analysis of the SBF-specific gene, CLN1, the MBF-specific gene, CDC45 and control gene, ACT1 in MatA, Δelm1 and elm1T551A. RT = reverse transcriptase. (B) Average expression of CLN1 and CDC45 normalized to ACT1 expression, over three different experiments. Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198942&req=5

pone-0001500-g004: SBF activity is increased in the Elm1 deletion strain.(A) A representative result of semi-quantitative RT-PCR analysis of the SBF-specific gene, CLN1, the MBF-specific gene, CDC45 and control gene, ACT1 in MatA, Δelm1 and elm1T551A. RT = reverse transcriptase. (B) Average expression of CLN1 and CDC45 normalized to ACT1 expression, over three different experiments. Error bars represent standard deviation.
Mentions: Decreased interaction between Cdc28 and Swi4 in Δelm1 implies diminished inactivation of SBF. In support of this hypothesis, we observed increased mRNA levels of the SBF-specific gene, CLN1 in Δelm1 (P = 0.0118), but not the MBF-specific gene, CDC45 (Figure 4 A,B). There was no significant increase in CLN1 transcripts in elm1T551A in comparison to wild-type cells (P = 0.4228) (Figure 4 A,B), consistent with the finding that the Cdc28-Swi4 PCA is not perturbed in the mutant strain (Figure S3). SBF-specific targets are enriched in genes encoding proteins responsible for cell morphogenesis and budding [18], [19]. Thus, it is possible that the elongated bud phenotype of Δelm1 is at least partially due to a failure to inactivate SBF activated transcription of genes that induce budding.

Bottom Line: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway.Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest.In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada.

ABSTRACT

Background: Despite extensive large scale analyses of expression and protein-protein interactions (PPI) in the model organism Saccharomyces cerevisiae, over a thousand yeast genes remain uncharacterized. We have developed a novel strategy in yeast that directly combines genetics with proteomics in the same screen to assign function to proteins based on the observation of genetic perturbations of sentinel protein interactions (GePPI). As proof of principle of the GePPI screen, we applied it to identify proteins involved in the regulation of an important yeast cell cycle transcription factor, SBF that activates gene expression during G1 and S phase.

Methodology/principle findings: The principle of GePPI is that if a protein is involved in a pathway of interest, deletion of the corresponding gene will result in perturbation of sentinel PPIs that report on the activity of the pathway. We created a fluorescent protein-fragment complementation assay (PCA) to detect the interaction between Cdc28 and Swi4, which leads to the inactivation of SBF. The PCA signal was quantified by microscopy and image analysis in deletion strains corresponding to 25 candidate genes that are periodically expressed during the cell cycle and are substrates of Cdc28. We showed that the serine-threonine kinase Elm1 plays a role in the inactivation of SBF and that phosphorylation of Elm1 by Cdc28 may be a mechanism to inactivate Elm1 upon completion of mitosis.

Conclusions/significance: Our findings demonstrate that GePPI is an effective strategy to directly link proteins of known or unknown function to a specific biological pathway of interest. The ease in generating PCA assays for any protein interaction and the availability of the yeast deletion strain collection allows GePPI to be applied to any cellular network. In addition, the high degree of conservation between yeast and mammalian proteins and pathways suggest GePPI could be used to generate insight into human disease.

Show MeSH
Related in: MedlinePlus