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Updated: May 13, 2026
Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
Published on: January 26, 2017
Molecular analysis of a conditional hal3 vhs3 yeast mutant links potassium homeostasis with flocculation and
Asier González1, Carlos Casado, Silvia Petrezsélyová
1Departament de Bioquímica i Biologia Molecular and Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, 08193 Barcelona, Spain.
Abstract:
Yeast flocculation and invasive growth are processes of great interest in fundamental biology and also relevant in biotechnology and medicine. Hal3 and Vhs3 are moonlighting proteins acting in Saccharomyces cerevisiae both as inhibitors of the Ppz protein phosphatases and as components of a catalytic step in CoA biosynthesis. The double hal3 vhs3 mutant is not viable but, under semi-permissive conditions, the tetO:HAL3 vhs3 strain shows a flocculent phenotype, invasive growth and increased expression of the flocculin-encoding FLO11 gene. We show here that all these effects are caused by hyperactivation of Ppz1 as a result of depletion of its natural inhibitors. The evidence indicates that hyperactivation of Ppz1 would impair potassium transport through the Trk1/Trk2 transporters, thus resulting in a decrease in the intracellular pH and a subsequent increase in the levels of cAMP. Mutation of the TPK2 isoform of protein kinase A blocks the increase in FLO11 expression, and eliminates the flocculent and invasive phenotypes produced by depletion of Hal3 and Vhs3. Interestingly, mutation of RIM101 also significantly decreases FLO11 expression under these conditions. Cells lacking Trk1,2 display an invasive phenotype that is abolished by deletion of FLO8 or by increasing the potassium concentration in the medium. Therefore, our results support a model in which hyperactivation of Ppz phosphatases would result in alteration of potassium transport, activation of Tpk2 and signaling to the FLO11 promoter by means of the Flo8 transcription factor, thus modulating flocculation and invasive growth. This model highlights an unsuspected link between potassium homeostasis and these important morphogenetic events.
Insights
Yeast cell flocculation and invasive growth are linked to potassium transport. Hyperactivation of Ppz phosphatases disrupts this, affecting cell morphology and gene expression via protein kinase A and Flo8.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Yeast flocculation and invasive growth are crucial biological processes with biotechnological and medical relevance.
- Hal3 and Vhs3 are moonlighting proteins in Saccharomyces cerevisiae, inhibiting Ppz protein phosphatases and aiding CoA biosynthesis.
- A double hal3 vhs3 mutant is non-viable, but specific conditions reveal phenotypes like flocculation and invasive growth.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the flocculent and invasive growth phenotypes observed in yeast strains with depleted Hal3 and Vhs3.
- To elucidate the role of Ppz phosphatases, potassium transport, and protein kinase A in regulating these morphogenetic events.
- To establish a link between potassium homeostasis and yeast cell morphology.
Main Methods:
- Genetic manipulation of Saccharomyces cerevisiae strains, including creating double mutants and conditional expression systems (tetO:HAL3).
- Analysis of gene expression, specifically FLO11, using techniques sensitive to genetic alterations.
- Phenotypic analysis of yeast cells, focusing on flocculation, invasive growth, intracellular pH, and cAMP levels.
Main Results:
- Depletion of Hal3 and Vhs3 leads to hyperactivation of Ppz1, causing flocculation, invasive growth, and increased FLO11 expression.
- Hyperactivated Ppz1 impairs potassium transport via Trk1/Trk2, decreasing intracellular pH and increasing cAMP levels.
- Mutation of TPK2 (a protein kinase A isoform) or RIM101, and deletion of FLO8, significantly affects FLO11 expression and the observed phenotypes.
Conclusions:
- Hyperactivation of Ppz phosphatases disrupts potassium homeostasis, activating Tpk2 signaling.
- This signaling pathway, involving Flo8, modulates the FLO11 promoter, thereby regulating yeast flocculation and invasive growth.
- The study reveals a novel connection between potassium balance and key yeast morphogenetic processes.
