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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.

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The GePPI screening strategy to identify proteins involved in the inactivation of SBF via phosphorylation by Clb/Cdc28.(A) A schematic representation of the GePPI screening strategy. 1) A biological pathway of interest is selected and a PCA assay is created that detects one or more sentinel PPIs of this pathway in wild-type yeast. In this example, protein A activates the sentinel interaction between proteins B and C, whereas protein D inhibits the interaction via a negative feedback loop. 2) Candidate genes are selected and plasmids encoding the PCA fusion proteins for each assay are transformed into the corresponding deletion strains. 3) Transformed deletion strains are screened in 96-well plates by fluorescence microscopy and images are collected and processed using image analysis software. 4) Strains are selected for which the PCA signal is significantly decreased or increased, as this type of analysis can be easily automated for yeast without the use of counter-stains that are required to identify changes in sub-cellular localization. In this example, deletion of protein A results in a decrease in the PCA signal, whereas deletion of protein D results in an increase in signal. (B) Regulation of SBF and MBF throughout the cell cycle. Activation of SBF involves phosphorylation of the SBF-associated repressor Whi5 by Cln/Cdc28 during G1. Phosphorylation of Whi5 leads to its dissociation from SBF followed by nuclear export. Inactivation of SBF in G2/M involves phosphorylation of Swi4 by Clb/Cdc28 activity. Regulation of MBF is less well understood but phosphorylation of Swi6 by Clb6/Cdc28 followed by Swi6 nuclear export may be a mechanism of inactivation of both SBF and MBF. The sentinel interaction between Cdc28 and Swi4 is indicated by dashed box.
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pone-0001500-g001: The GePPI screening strategy to identify proteins involved in the inactivation of SBF via phosphorylation by Clb/Cdc28.(A) A schematic representation of the GePPI screening strategy. 1) A biological pathway of interest is selected and a PCA assay is created that detects one or more sentinel PPIs of this pathway in wild-type yeast. In this example, protein A activates the sentinel interaction between proteins B and C, whereas protein D inhibits the interaction via a negative feedback loop. 2) Candidate genes are selected and plasmids encoding the PCA fusion proteins for each assay are transformed into the corresponding deletion strains. 3) Transformed deletion strains are screened in 96-well plates by fluorescence microscopy and images are collected and processed using image analysis software. 4) Strains are selected for which the PCA signal is significantly decreased or increased, as this type of analysis can be easily automated for yeast without the use of counter-stains that are required to identify changes in sub-cellular localization. In this example, deletion of protein A results in a decrease in the PCA signal, whereas deletion of protein D results in an increase in signal. (B) Regulation of SBF and MBF throughout the cell cycle. Activation of SBF involves phosphorylation of the SBF-associated repressor Whi5 by Cln/Cdc28 during G1. Phosphorylation of Whi5 leads to its dissociation from SBF followed by nuclear export. Inactivation of SBF in G2/M involves phosphorylation of Swi4 by Clb/Cdc28 activity. Regulation of MBF is less well understood but phosphorylation of Swi6 by Clb6/Cdc28 followed by Swi6 nuclear export may be a mechanism of inactivation of both SBF and MBF. The sentinel interaction between Cdc28 and Swi4 is indicated by dashed box.

Mentions: We present a novel screening strategy in which genetics and proteomics are incorporated to detect genetic perturbations of protein interactions (GePPI) in order to assign function to previously uncharacterized or characterized proteins (Figure 1A). The principle is that if a protein encoded by a candidate gene plays a role in a biological pathway of interest, deletion of the gene will result in perturbation of a sentinel PPI within the pathway. The protein can be implicated in any step in the pathway upstream of the interaction measured, wherein the change propagates through the pathway resulting in a perturbation of the PPI. Alternatively, a protein can be involved in a downstream positive or negative feedback event that regulates the sentinel PPI. The sentinel PPI is detected using protein-fragment complementation assays (PCA) (Reviewed in [7]), and perturbations are measured by fluorescence microscopy and image analysis of the PCA in selected yeast deletion strains. We previously showed that fluorescent PCAs can detect spatial and/or temporal perturbations of PPIs in vivo in mammalian cells, following addition of drugs, siRNAs, or hormones [8], [9], [10], [11], [12]. Perturbations of the sentinel PPI could be due to a number of different processes including, removal of a mediator or inhibitor of the interaction, changes in the rate of protein synthesis or degradation, changes in protein localization, or post-translational modifications.


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

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

The GePPI screening strategy to identify proteins involved in the inactivation of SBF via phosphorylation by Clb/Cdc28.(A) A schematic representation of the GePPI screening strategy. 1) A biological pathway of interest is selected and a PCA assay is created that detects one or more sentinel PPIs of this pathway in wild-type yeast. In this example, protein A activates the sentinel interaction between proteins B and C, whereas protein D inhibits the interaction via a negative feedback loop. 2) Candidate genes are selected and plasmids encoding the PCA fusion proteins for each assay are transformed into the corresponding deletion strains. 3) Transformed deletion strains are screened in 96-well plates by fluorescence microscopy and images are collected and processed using image analysis software. 4) Strains are selected for which the PCA signal is significantly decreased or increased, as this type of analysis can be easily automated for yeast without the use of counter-stains that are required to identify changes in sub-cellular localization. In this example, deletion of protein A results in a decrease in the PCA signal, whereas deletion of protein D results in an increase in signal. (B) Regulation of SBF and MBF throughout the cell cycle. Activation of SBF involves phosphorylation of the SBF-associated repressor Whi5 by Cln/Cdc28 during G1. Phosphorylation of Whi5 leads to its dissociation from SBF followed by nuclear export. Inactivation of SBF in G2/M involves phosphorylation of Swi4 by Clb/Cdc28 activity. Regulation of MBF is less well understood but phosphorylation of Swi6 by Clb6/Cdc28 followed by Swi6 nuclear export may be a mechanism of inactivation of both SBF and MBF. The sentinel interaction between Cdc28 and Swi4 is indicated by dashed box.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198942&req=5

pone-0001500-g001: The GePPI screening strategy to identify proteins involved in the inactivation of SBF via phosphorylation by Clb/Cdc28.(A) A schematic representation of the GePPI screening strategy. 1) A biological pathway of interest is selected and a PCA assay is created that detects one or more sentinel PPIs of this pathway in wild-type yeast. In this example, protein A activates the sentinel interaction between proteins B and C, whereas protein D inhibits the interaction via a negative feedback loop. 2) Candidate genes are selected and plasmids encoding the PCA fusion proteins for each assay are transformed into the corresponding deletion strains. 3) Transformed deletion strains are screened in 96-well plates by fluorescence microscopy and images are collected and processed using image analysis software. 4) Strains are selected for which the PCA signal is significantly decreased or increased, as this type of analysis can be easily automated for yeast without the use of counter-stains that are required to identify changes in sub-cellular localization. In this example, deletion of protein A results in a decrease in the PCA signal, whereas deletion of protein D results in an increase in signal. (B) Regulation of SBF and MBF throughout the cell cycle. Activation of SBF involves phosphorylation of the SBF-associated repressor Whi5 by Cln/Cdc28 during G1. Phosphorylation of Whi5 leads to its dissociation from SBF followed by nuclear export. Inactivation of SBF in G2/M involves phosphorylation of Swi4 by Clb/Cdc28 activity. Regulation of MBF is less well understood but phosphorylation of Swi6 by Clb6/Cdc28 followed by Swi6 nuclear export may be a mechanism of inactivation of both SBF and MBF. The sentinel interaction between Cdc28 and Swi4 is indicated by dashed box.
Mentions: We present a novel screening strategy in which genetics and proteomics are incorporated to detect genetic perturbations of protein interactions (GePPI) in order to assign function to previously uncharacterized or characterized proteins (Figure 1A). The principle is that if a protein encoded by a candidate gene plays a role in a biological pathway of interest, deletion of the gene will result in perturbation of a sentinel PPI within the pathway. The protein can be implicated in any step in the pathway upstream of the interaction measured, wherein the change propagates through the pathway resulting in a perturbation of the PPI. Alternatively, a protein can be involved in a downstream positive or negative feedback event that regulates the sentinel PPI. The sentinel PPI is detected using protein-fragment complementation assays (PCA) (Reviewed in [7]), and perturbations are measured by fluorescence microscopy and image analysis of the PCA in selected yeast deletion strains. We previously showed that fluorescent PCAs can detect spatial and/or temporal perturbations of PPIs in vivo in mammalian cells, following addition of drugs, siRNAs, or hormones [8], [9], [10], [11], [12]. Perturbations of the sentinel PPI could be due to a number of different processes including, removal of a mediator or inhibitor of the interaction, changes in the rate of protein synthesis or degradation, changes in protein localization, or post-translational modifications.

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