Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidation of its mechanisms via genetic and molecular biological investigations. Here we report the isolation of yeast mutants defective in autophagocytosis (aut mutants), using a rapid colony screening procedure.

P.Piper and Y.Mahé contributed equally to this workExposure of Saccharomyces cerevisiae to sorbic acid strongly induces two plasma membrane proteins, one of which is identified in this study as the ATP-binding cassette (ABC) transporter Pdr12. In the absence of weak acid stress, yeast cells grown at pH 7.0 express extremely low Pdr12 levels. However, sorbate treatment causes a dramatic induction of Pdr12 in the plasma membrane. Pdr12 is essential for the adaptation of yeast to growth under weak acid stress, since Δpdr12 mutants are hypersensitive at low pH to the food preservatives sorbic, benzoic and propionic acids, as well as high acetate levels. Moreover, active benzoate efflux is severely impaired in Δpdr12 cells. Hence, Pdr12 confers weak acid resistance by mediating energydependent extrusion of water-soluble carboxylate anions. The normal physiological function of Pdr12 is perhaps to protect against the potential toxicity of weak organic acids secreted by competitor organisms, acids that will accumulate to inhibitory levels in cells at low pH. This is the first demonstration that regulated expression of a eukaryotic ABC transporter mediates weak organic acid resistance development, the cause of widespread food spoilage by yeasts. The data also have important biotechnological implications, as they suggest that the inhibition of this transporter could be a strategy for preventing food spoilage.

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Degradation of misfolded or tightly regulated proteins in the endoplasmic reticulum (ER) is performed by the cytosolic ubiquitin-proteasome system and therefore requires their prior transport back to the cytosol. Here, we report on the extraction and degradation mechanism of a polytopic membrane protein. Rapid proteasomal degradation of a mutated form of the ATPbinding cassette transporter Pdr5 retained in the ER is initialized at the lumenal face of the ER membrane. Using different antibodies directed against the cytosolic tails or a lumenal loop of the transmembrane protein, it could be demonstrated that the turnover of Pdr5* demands the concerted action of both the Sec61 translocon and the ubiquitin-proteasome system. We observed a stabilization of the entire molecule within the ER membrane in yeast mutants characterized by a reduced translocation capacity or by functionally attenuated proteasomes. Moreover, no degradation intermediates were detected in any of the mutants that impede degradation of Pdr5*. Therefore, initial steps are rate-limiting for cleavage and mutations that impede downstream events prevent initiation of the process.Our data suggest that ER degradation is a mechanistically highly integrated process, requiring the combined operation of components of the degradation system acting at the lumenal face of the ER membrane, the Sec61 translocon, and the ubiquitin-proteasome system.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast An important type of multidrug resistance (MDR) in mammalian tumor cells and cultured cells (19) is caused by gene amplification and subsequent overexpression of the P-glycoprotein (Mdr1) and MDR-associated protein (MRP) (11, 57) transport proteins, both of which are members of the ubiquitous ATP-binding cassette (ABC) protein superfamily (23, 32). P-glycoprotein and MRP are integral plasma membrane proteins that function as ATP-dependent efflux pumps for a variety of structurally unrelated cytotoxic compounds. Elevated P-glycoprotein levels permit tumor cells to survive cytotoxic drug regimens and thus represent a major impediment to curative cancer chemotherapy (9,19,57).The first ABC transporter protein to be identified in the yeast Saccharomyces cerevisiae was the Ste6 a-factor pheromone transporter (31,35). Although Ste6 and mammalian P-glycoprotein are structurally closely related, overexpression of Ste6 is not associated with MDR. However, MDR in S. cerevisiae is a well-documented phenomenon (33), and there is compelling evidence that MDR in S. cerevisiae could be subject to tight transcriptional control by PDR (pleiotropic drug resistance) genes encoding transcriptional regulators. For instance, transcription factors such as Pdr1 (2), Yap1/Snq3 (22, 37), Pdr3 (14, 26), and, putatively, Pdr7 and Pdr9 (15) may form a highly complicated network implicated in MDR development (3), similar to P-glycoprotein-or MRP-mediated MDR in mammalian cells. Mutations in PDR genes are often associated with MDR, since they are thought to control the expression of individual drug pumps, some of which were shown to be members of the ABC family of multidrug transporters. For example, two highly homologous yeast ABC transporter genes, SNQ2 (50) and PDR5/STS1/YDR1/LEM1 (4,8,24,29), hereafter called PDR5, were recently shown to represent functional and structural homologs of mammalian Mdr1 because their overexpression in yeast cells is linked to MDR development.The transcription of PDR5 is under control of the transcription-regulatory proteins Pdr1 and Pdr3 (14,26). Genetic experiments have shown that pdr1-mediated cycloheximide resistance requires the presence of PDR5 and that PDR5 mRNA levels were elevated in a drug-resistant pdr1-3 mutant (36). By contrast, in ⌬pdr1 ⌬pdr3 double mutants, PDR5 mRNA synthesis is completely abolished (26). The Pdr5 drug pump was found mainly in membrane preparations enriched for plasma membrane vesicles (4, 13) isolated from pdr1 mutants, whereas previous studies from crude fractionation experiments of wildtype yeast cells located epitope-tagged Pdr5 to both the plasma membrane and intracellular membranes (8).Overexpression of PDR5 and SNQ2 leads to resistance against a variety of structurally unrelated cytotoxic compounds, including mycotoxins, cycloheximide, staurosporine, cerulenin, 4-nitroquinoline-N-oxide, and sulfomethuron methyl (4,8,24,50). However, each transporter mediates resistance to only a distinct subset of d...