PEP3 overexpression shortens lag phase but does not alter growth rate in Saccharomyces cerevisiae exposed to acetic acid stress
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In fungi, two recognized mechanisms contribute to pH homeostasis: the plasma membrane proton-pumping ATPase that exports excess protons and the vacuolar proton-pumping ATPase (V-ATPase) that mediates vacuolar proton uptake. Here, we report that overexpression of PEP3 which encodes a component of the HOPS and CORVET complexes involved in vacuolar biogenesis, shortened lag phase in Saccharomyces cerevisiae exposed to acetic acid stress. By confocal microscopy, PEP3-overexpressing cells stained with the vacuolar membrane-specific dye, FM4-64 had more fragmented vacuoles than the wild-type control. The stained overexpression mutant was also found to exhibit about 3.6-fold more FM4-64 fluorescence than the wild-type control as determined by flow cytometry. While the vacuolar pH of the wild-type strain grown in the presence of 80 mM acetic acid was significantly higher than in the absence of added acid, no significant difference was observed in vacuolar pH of the overexpression strain grown either in the presence or absence of 80 mM acetic acid. Based on an indirect growth assay, the PEP3-overexpression strain exhibited higher V-ATPase activity. We hypothesize that PEP3 overexpression provides protection from acid stress by increasing vacuolar surface area and V-ATPase activity and, hence, proton-sequestering capacity.
KeywordsSaccharomyces cerevisiae Yeast Acetic acid PEP3 V-ATPase HOPS CORVET Vacuole STM1 PEP5
We thank Severino Zara for help in screening the overexpression library; Van Anh Vu for technical assistance; Brett Tyler and Viviana Perez for help with western blotting; Tom Stevens, Patricia Kane, and Christian Ungermann for helpful discussions; and Jennifer Lorang for providing FM4-64. This work was supported in part by grant no. 2010-65504-0345 from the United States Department of Agriculture National Institute of Food and Agriculture (USDA-NIFA) program to A.T.B. and M.H.P. and from the United States National Institutes of Health (NIH) grant no. R15GM104876 to J.P.-V. The flow cytometry analysis was performed in the Cell Image and Analysis Facilities Core of the Oregon State University Environmental Health Sciences Center supported in part by grant no. P30ES000210-42 from the National Institute of Environmental Health Sciences (NIEHS), United States National Institutes of Health.
Conflict of interest
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