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14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae
D Gelperin1, J Weigle, K Nelson
1Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106-4960, USA.
Abstract:
Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.
Insights
Deletion of the CHC1 gene in yeast causes defects, but BMH2, a 14-3-3 protein, rescues these cells. Both BMH1 and BMH2 are essential, suggesting 14-3-3 proteins are vital for yeast cell function.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Clathrin heavy-chain gene (CHC1) deletion in Saccharomyces cerevisiae leads to growth, morphology, and membrane trafficking defects, with some strains exhibiting lethality.
- Previous research identified five genes that rescue inviable CHC1-deleted yeast strains when overexpressed.
- The 14-3-3 protein family, known for diverse functions, includes abundant acidic proteins of approximately 30 kDa.
Purpose of the Study:
- To identify and characterize the function of suppressor genes involved in clathrin heavy-chain deletion phenotypes.
- To investigate the role of the 14-3-3 protein family, specifically BMH2, in yeast membrane trafficking and cellular processes.
- To explore the genetic interactions between 14-3-3 proteins and other cellular pathways like Ras signaling.
Main Methods:
- Genetic screening to identify suppressor genes of CHC1 deletion.
- Gene cloning and sequencing to identify the suppressor locus BMH2/SCD3.
- Analysis of yeast strains with single and double deletions of BMH1 and BMH2.
- Assessing the effects of overexpressing BMH genes and TPK1 on CHC1-deleted and cdc25-1 mutant yeast.
Main Results:
- The suppressor locus BMH2/SCD3 encodes a 14-3-3 protein highly similar to mammalian and other yeast isoforms.
- Single deletions of BMH1 or BMH2 show no phenotype, but their combined deletion is lethal.
- Overexpression of BMH1 or BMH2 rescues inviable CHC1-deleted yeast strains, with BMH2 being more effective.
- Combined CHC1 deletion and bmh mutations often lead to lethality, and BMH gene overexpression suppresses cdc25-1 temperature sensitivity.
- Overexpression of TPK1 (cAMP-dependent protein kinase) rescues Bmh- yeast but not CHC1-deleted yeast.
Conclusions:
- Budding yeast 14-3-3 proteins (Bmh1 and Bmh2) are essential and multifunctional.
- These proteins play critical roles in both vesicular transport and Ras signaling pathways.
- Genetic interactions highlight the involvement of 14-3-3 proteins in fundamental cellular processes beyond clathrin-mediated endocytosis.