Yeast ATP-binding Cassette Transporter Research Articles

Protein interactions of Gts1p of Saccharomyces cerevisiae throughout a region similar to a cytoplasmic portion of some ATP-binding cassette transporters.

The GTS1 gene product, Gts1p, has pleiotropic effects on the timing of budding, cell size, heat tolerance, sporulation and the lifespan of the yeast Saccharomyces cerevisiae. In this study, we found (using the yeast two-hybrid system) that Gts1p forms homodimers throughout the 18-amino acid region 296-313 which has considerable similarity to a region downstream of the Walker nucleotide-binding motif A of some ATP-binding cassette (ABC) transporters. The region contains two aspartic acid residues at 301 and 310 preceded by hydrophobic amino acid residues, and Gts1p with an Asp310 to Ala substitution showed considerably reduced homodimerization, as shown by the two-hybrid assay. Overexpression of the point-mutated Gts1p did not efficiently induce the Gts1p-related phenotypes described above, suggesting that the homodimerization of Gts1p is required for it to function in vivo. The C-terminal cytoplasmic domain of the yeast ABC transporters Mdl1p (multidrug resistance-like transporter) and Ycf1p (yeast cadmium factor or glutathione S-conjugate pump) bound to Gts1p in the two-hybrid system, and the heterodimerization activity of the Gts1p with the Asp301 to Ala substitution was more affected than the Gts1p with the Asp310 to Ala substitution. Overexpression of GTS1 considerably reduced, and disruption of GTS1 slightly decreased, cellular resistance to cycloheximide, cadmium, cisplatin and 1-chloro-2,4-dinitrophenol, which (except for cycloheximide) are all substrates of Ycf1p. These results suggest that Gts1p interacts with some ABC transporters through the binding site overlapping that of homodimerization and modulates their activity.

A fourth gene from the Candida albicans CDR family of ABC transporters.

Using primers derived from a region of the Candida albicans CDR1 (Candida drug resistance) gene that is conserved in other ABC (ATP-binding cassette) transporters, a DNA fragment from a previously unknown CDR gene was obtained by polymerase chain reaction (PCR). After screening a C. albicans genomic library with this fragment as a probe, the complete CDR4 gene was isolated and sequenced. CDR4 codes for a putative ABC transporter of 1490 amino acids with a high degree of homology to Cdr1p, Cdr2p and Cdr3p from C. albicans (62, 59 and 57% amino acid sequence identity, respectively). Cdr4p has a predicted structure typical for cluster I. 1 of yeast ABC transporters, characterized by two homologous halves, each comprising an N-terminal hydrophilic domain with consensus sequences for ATP binding and a C-terminal hydrophobic domain with six transmembrane helices. In contrast to the CDR1/CDR2 genes, the genetic structure of the CDR4 gene was conserved in 59 C. albicans isolates from six different patients. Northern hybridization analysis showed that the CDR4 gene was expressed in most isolates, but no correlation between CDR4 mRNA levels and the degree of fluconazole resistance of the isolates was found. In addition, a C. albicans mutant in which both copies of the CDR4 gene were disrupted by insertional mutagenesis was not hypersusceptible to fluconazole as compared to the parent strain. Unlike CDR1 and CDR2, CDR4 does not, therefore, seem to be involved in fluconazole resistance in C. albicans.

Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter.

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.

An FK506-sensitive Transporter Selectively Decreases Intracellular Levels and Potency of Steroid Hormones

Steroid hormones bind and activate intracellular receptors that are ligand-regulated transcription factors. Mammalian steroid receptors can confer hormone-dependent transcriptional enhancement when expressed in yeast, thereby enabling the genetic identification of nonreceptor proteins that function in the hormone signal transduction pathway. Pdr5p (Lem1/Sts1/Ydr1p), a yeast ATP-binding cassette transporter, selectively decreases the intracellular levels of particular steroid hormones, indicating that active processes can affect the passage of steroids across biological membranes. In yeast, the immunosuppressive drug FK506 inhibited Pdr5p, thereby potentiating activation of the glucocorticoid receptor by dexamethasone, a ligand that is exported by Pdr5p. In mammalian L929 cells but not in HeLa cells, FK506 potentiated dexamethasone responsiveness and increased dexamethasone accumulation, without altering the hormone-binding properties of the glucocorticoid receptor. We suggest that an FK506-sensitive transporter in L929 cells selectively decreases intracellular hormone levels and, consequently, the potency of particular steroids. Thus, steroid transporters may modulate, in a cell-specific manner, an initial step in signaling, the availability of hormone to the receptor.

The ATP-binding cassette multidrug transporter Snq2 of Saccharomyces cerevisiae: a novel target for the transcription factors Pdr1 and Pdr3.

Pleiotropic drug resistance (PDR) in the yeast Saccharomyces cerevisiae can arise from overexpression of ATP-binding cassette (ABC) efflux pumps such as Pdr5 and Snq2. Mutations in the transcription factor genes PDR1 and PDR3 are also associated with PDR. We show here that a pdr1-3 mutant exhibits a PDR phenotype, including elevated resistance to the mutagen 4-nitroquinoline-N-oxide, a known substrate for Snq2 but not for Pdr5. Northern analysis and immunoblotting demonstrated that the SNQ2 gene is 10-fold overexpressed in a pdr1-3 gain-of-function mutant strain, whereas Snq2 expression is severely reduced in a delta pdr1 deletion strain, and almost abolished in a delta pdr1 delta pdr3 double disruptant when compared to the PDR1 strain. However, expression of the Ste6 a-factor pheromone transporter, another yeast ABC transporter not associated with PDR, is unaffected in pdr1-3 mutant cells and in strains carrying delta pdr1, delta pdr3, or delta pdr1 delta pdr3 deletions. Finally, DNA footprint analysis revealed that the SNQ2 promoter contains three binding sites for Pdr3. Our results identify SNQ2 as a novel target for both Pdr1 and Pdr3, and demonstrate that the PDR phenotype of a pdr1-3 mutant strain results from overexpression of more than one ABC drug-efflux pump.

Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance.

We have cloned a yeast gene that confers a multidrug resistance phenotype on Saccharomyces cerevisiae when present in multiple copies. The STS1 (for Sporidesmin Toxicity Suppressor) gene encodes a 1511-residue protein whose predicted structural organization is characterized by 12 alpha-helical membrane segments and two domains containing consensus sites for ATP binding, indicating that STS1 is a new yeast ATP-binding cassette (ABC) transporter. A chromosomal deletion of STS1 leads to viable delta sts1 cells of both mating types, suggesting that STS1 is not essential for cell growth. However, delta sts1 cells exhibit supersensitivity to sporidesmin and to other structurally unrelated drugs such as cycloheximide. Conversely, overexpression of STS1 leads to increased resistance to the same drugs. Although Northern analysis showed that STS1 mRNA is present in all yeast cell types, its drastically reduced level in alpha-factor-arrested cells indicates that expression of STS1 is regulated by mating pheromones. Subcellular fractionation and immunoblotting using monoclonal antibodies, which recognize a fully functional epitope-tagged Sts1 protein, showed that Sts1 is a 175-kDa membrane protein localized mainly to intracellular membranes.