Title

Adaptation to Membrane Stress in Saccharomyces Cerevisiae: Identification of Novel Proteins and Mechanisms

Date of Award

5-2013

Degree Type

Dissertation

Embargo Date

5-24-2013

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Advisor(s)

Scott E. Erdman

Keywords

ergosterol, membrane, mitochondria, PDR, S. cerevisiae, S. pombe

Subject Categories

Cell Biology | Genetics

Abstract

Sensing and effectively responding to cellular stresses are fundamental processes for all living cells. In fungi, membrane stress is especially relevant because many clinically used antifungal agents target ergosterol, a fungal specific sterol necessary for the integrity of the plasma membrane. We hypothesized that there existed genes and processes involved in tolerance to membrane stress in fungi that remained to be discovered. Plants produce numerous and diverse secondary metabolites as a mechanism of defense against microorganisms. Many plant secondary metabolites have antifungal activity. However, the mechanism of activity for most plant secondary metabolites is unknown. Triterpene Glycosides (TTGs) are a structurally and functionally diverse group of molecules, many of which have antifungal activity. The antifungal activity of these molecules is thought to be due to the formation and aggregation of TTG-ergosterol complexes in fungal plasma membranes and the resulting disruption of plasma membrane integrity, but additional mechanisms of activity are also possible.

The mechanism of activity and potential mechanisms of resistance to particular TTGs isolated from agria cacti (agria TTGs) were investigated in two complementary genome-wide screens using the yeast Saccharomyces cerevisiae as a model. In a high-copy suppression screen of the agria TTG sensitive phenotype of the sip3∆ strain, both novel and known genes and mechanisms contributing to membrane stress tolerance were found. Specifically, vesicular trafficking and Pleiotropic Drug Resistance (PDR) mediated changes in lipid composition were found to be mechanisms contributing to agria TTG resistance. Genes involved in ergosterol metabolism (PDR16), sphingolipid metabolism (LAG1), vesicular trafficking (AGE1) and calcium homeostasis (PMC1) increase tolerance of yeast cells to agria TTGs when present in high-copy number.

The sip3∆ high-copy suppression screen also uncovered two related PDR target genes of unknown function that increase tolerance of yeast cells to membrane stress. These two genes are members of a small family of three PDR target genes, PDR19, PDR20 and PDR21, that share a seven residue region of homology. The analysis of the growth phenotypes of the three possible double mutants and the triple mutant reveal that these genes are functionally related and protect cells from membrane stress. Interestingly, the deletion of these genes results in resistance to ketoconazole, an inhibitor of ergosterol biosynthesis. Consistent with a role in membrane integrity, lack of these genes results in increased membrane permeability as measured by a Rhodamine 6G (R6G) uptake assay.

In the initial genome-wide screen of haploid non-essential gene deletion strains in S. cerevisiae, the deletion of SIP3 and its paralog YSP1 resulted in sensitivity to agria TTGs. We decided to investigate the functions of these genes in more detail. We discovered that both these genes have similar growth phenotypes and are required for adaptation to membrane, weak acid and calcium stresses. The deletion of these genes also results in resistance to ketoconazole. We also found an unexpected, suppressive genetic interaction between these genes under some growth conditions. Additionally, SIP3 and YSP1 influence membrane permeability as measured by the R6G uptake assay.

Ysp1 family proteins have a conserved predicted domain organization of adjacent BAR and PH domains (BAR-PH domain) at the amino terminus and a transmembrane helix at the carboxy terminus. We investigated the function of the Schizosaccharomyces pombe Ysp1 protein as well as its putative BAR-PH and transmembrane domains. The deletion of the S. pombe YSP1 homolog (ysp1+) results in a growth defect on the membrane active agents amphotericin B and digitonin, but not other antifungal agents, indicating that the S. pombe Ysp1 protein has a more restricted role in tolerance to membrane stress compared to the S. cerevisiae Ysp1 protein. The S. pombe Ysp1 protein is also required for optimal survival of cells exposed to a high dose of amphotericin B. The predicted BAR-PH and transmembrane domains of the S. pombe Ysp1 protein are both required for adaptation to membrane stress, as deletion of these domains results in a growth defect on media containing digitonin. Finally, the mutation of conserved, basic residues in the S. pombe ysp1+ BAR-PH domain results in growth defects under membrane stress conditions, indicating that conserved residues of the BAR-PH domain of the S. pombe Ysp1 protein are important to its function.

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