Abstract
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.
Highlights
From the Wepartment of Molecular Genetics, University and Biocenter of Vienna, A-1030 Vienna, Austria and Research Ltd., Crown Research Institute of Pastoral Agricultural Research, Hamilton 3123, New Zealand
Furresistancpehenotype on Saccharomycecserevisiae ther, overexpression of LtpgpA from Leishmania is responsible whenpresent in multiplecopies.The STSl (forSpo- for methotrexate and heavy metal resistance[5, 6], and P2asridesmin Toxicity fuppressor) gene encodes a 1511-remsio-dium pfMdr has been implicated in chloroquine resistance due protezn whose predicted structural organizationis of the malarial parasite [7].Likewise, bacterial erythromycin characterized by 12 a-helical membrane segments and resistance in Staphylococcus is caused by MsrA overexpression two domains containing consensus sites forATP bind- (81, and theABC protein DrrAB of Streptomyces appears tobe ing, indicating that STSl is a new yeast ATP-binding cassette (ABC) transporter
We describe the isolation and characterization of the S. cerevisiae S T S l gene that encodes a membrane protein with extensive homology to otherABC proteins
Summary
The recently cloned SNQ2 gene present in all yeast cell types, its drastically reduced encodes a n ABC protein whose expression is associated with a level in a-factor-arrested cells indicates that expressioMn DR phenotype [15].In some cases, like that of the putative of STSl is regulatedby mating pheromones. ABC transporters (for ATP-binding cassette)constitute a gether, numerous yeast genes may comprise a distinct gene novel and rapidlygrowing superfamily of membrane transport family implicated in MDR and drug hypersensitivityphenomproteins that are found operating from microorganisms to man ena [13, 16]. Individual membersof such a gene family may be Membrane fractions was carried by an established procedure [34]using minor modificationsas described elsewhere [10].Briefly,a 12,000x g P1
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