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


RESULTS
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
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.
Manderson
Emily N
EN
Département de Biochimie, Université de Montréal, Montreal, Quebec, Canada.
Malleshaiah
Mohan
M
Michnick
Stephen W
SW
D008856:Microscopy, Fluorescence; D009154:Mutation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000037:antagonists & inhibitors*
C081540:SBF protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011494:Protein Kinases; C411302:ELM1 protein, S cerevisiae