Ubiquitin-mediated degradation of drug transporter proteins from the yeast plasma membrane

We study plasma membrane transporter proteins involved in resistance to antifungal drugs to understand the underlying molecular mechanisms. The budding yeast, Saccharomyces cerevisiae, is used as an experimental model, and our approaches combine biochemistry, genetics and cell physiology. Our research focuses on a yeast homolog of human MDR proteins, Pdr5, and on a transporter of the Rta family only found in fungi. We are interested in the posttranslational ubiquitin conjugation, which marks these transporters for degradation by the vacuolar proteases or the cytosolic 26S proteasome. Our goal is to identify new proteins that are responsible for addressing Pdr5p and Rta1 to the vacuole or the proteasome, and to determine the sequence of the transporter that directs it to either proteolytic pathway.

Resistance to antifungal drugs

The development of azole resistance in S. cerevisiae was found to correlate with the expression and activity of Pdr5p, an ATP-dependent pump that extrudes the drug from cells before it can exert its effects. Compared to the mammalian MDR protein, the yeast ABC protein shows a reversal sequential order of the membrane and ATP-binding domains. Expression of the PDR5 gene is under the control of a complex network of well-characterized transcription factors. However, the physiological conditions leading to PDR5 transactivation remain to be determined. Rta1 is a plasma membrane transporter of the MSF family. Gene expression is upregulated during hypoxia, which leads to increased tolerance to aminocholesterol, an inhibitor or ergosterol biosynthesis. Pulse –chase analysis has shown that Rta1 is a short-lived protein, in contrast to Pdr5.

ER-associated degradation of ABC-transporter Pdr5

Proteins of the plasma membrane are synthesised on cytosolic ribosome and transported to the endoplasmic reticulum (ER). This compartment is endowed with a quality control system which monitors newly synthesised proteins for proper chain folding or modifications. We have found that the Pdr5 variant having a leucine residue at position 183 replaced by proline is retrotranslocated to the cytosol where it is ubiquitylated and eliminated by the 26S proteasome. Delivery of the mutated Pdr5 form to the proteasome requires a ubiquitin-dependent segregase consisting of Cdc48, Ufd1 and Npl4. Cdc48p, named p97 in mammals, has two ATP-binding domains, suggesting that a nucleotide-dependent conformational switch may apply tension to ubiquitylated protein and allow membrane extraction and/or polypeptide unfolding.

Identification of new delivery factors

Five proteins of unknown function have been shown to interact with Cdc48p in a two-hybrid assay. We are currently analysing their subcellular localisation using GFP fusion and fluorescent microscopy, their role in ubiquitin-dependent degradation and resistance to arsenite or DNA damaging agents.