Control of the protein and lipid content of the plasma membrane by ATP-binding cassette transporter proteins in S. Cerevisiae

Pdr5 and Yor1 are two ATP-binding cassette transporters regulated by the pleiotropic drug resistance (PDR) network in the yeast Saccharomyces cerevisiae. Recent work demonstrated that a pdr5 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 pdr5 yor1 strain exhibits delayed turnover of Tat2 and an increase in tryptophan uptake, which we postulate is due to changes in asymmetric phospholipid distribution of the plasma membrane, 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. I discovered that pdr5 yor1 strains were also highly tolerant of the sphingolipid biosynthesis inhibitor, Aureobasidin A (AbA). AbA represents a novel phenotype for pdr5 yor1 strains and was used as a tool to find genes that are involved in the response to loss of Pdr5 and Yor1. Ethyl methanesulfonate