Wasko, BrianStephens, Brian2024-07-022024-07-022022-052022-05-12May 2022https://hdl.handle.net/10657.1/4960The P-type proton pump, Pma1p and the vacuolar H+-ATPase (V-ATPase) proton pump play significant roles in balancing pH homeostasis in eukaryotic cells. In budding yeast, the PMA1 gene is critical for yeast survival, whereas mutations compromising V-ATPase activity cause conditional lethality. The yeast VMA21 gene encodes for the chaperone protein Vma21 that is required for complete assembly of the multisubunit V-ATPase complex. The loss of vacuolar acidity due to deficiency of V-ATPase activity has been linked to disrupted growth phenotypes. Yeast vma21 deficient mutants exhibit sensitivity to cold temperatures, CaCl2 and acetic acid. This sensitivity can be suppressed by a Pma1-G158S mutation suggesting an interdependence between Pma1p and the V-ATPase. In this study, we address how Pma1-G158S mutation affects wildtype and V-ATPase deficient cells in response to changes in extracellular conditions. Using CRISPR/Cas9 gene editing technology, we performed site-directed mutagenesis of G158 in an attempt to analyze structure-function relationships at this amino acid position. Our observations suggest that mutations at this position may regulate aging phenotype and cellular dysfunctions in yeast Saccharomyces cerevisiae and multicellular eukaryotes.  application/pdfenDNA damageVacuolar aciditySaccharomyces cerevisiaeProton pumpVacuolar ATPasepH homeostasisThesis2024-07-02