Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cellular Biology
W. Scott Moye-Rowley
Pdr5 and Yor1 are two ATP-binding cassette transporters regulated by the pleiotropic drug resistance (PDR) network in the yeast Saccharomyces cerevisiae. Recent work from another group demonstrated that a pdr5Δ yor1 strain confers remarkable resistance to a sphingolipid intermediate, phytosphingosine (PHS), which was surprising as loss of these transporters normally leads to elevated drug sensitivity.
PHS is toxic to the cell at high levels due to mislocalization of nutrient permeases, such as the high affinity tryptophan transporter, Tat2. Although the above study suggested that this resistance was due to increased expression of Rsb1, a known mediator of PHS tolerance, this was not reproducible in our hands and we sought to identify other determinants for this phenotype. The work presented here demonstrates that the pdr5Δ yor1 strain exhibits delayed turnover of Tat2 and an increase in tryptophan uptake, which we postulate is due to changes membrane asymmetry resulting in decreased endocytosis. Conversely, cells lacking Rsb1 showed a decrease in tryptophan import and increased Tat2 turnover, independent of endogenous PHS levels. Rsb1 has a predicted 7 transmembrane (7TM) topology, which argues against the idea that Rsb1 functions directly in PHS transport, as there are currently no known transporters with this topology. These data suggest that Rsb1 and Pdr5/Yor1 function in regulation of endocytosis of Tat2, and possibly other membrane proteins.
Ethyl methanesulfonate mutagenesis of the pdr5Δ yor1 strain and a candidate gene approach were alternative methods used to identify mediators of PHS tolerance in this strain. Inconsistent results from PHS selection led to the discovery that the pdr5Δ yor1 strain was also robustly resistant to the sphingolipid biosynthesis inhibitor, Aureobasidin A (AbA), which was subsequently used for analysis. These approaches revealed several genes, including Gda1, Mss4, and Ypk1 that are important for AbA tolerance in the pdr5Δ yor1 strain. Many of these determinants play a role in cell wall integrity, suggesting that loss of Pdr5 and Yor1 may lead to activated cell wall integrity pathways resulting in altered cell wall structure.
xi, 117 pages
Includes bibliographical references (pages 111-117).
Copyright 2010 Soraya Johnson