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A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

ABSTRACT: Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identified a unique region within lumenal loop 6 of Ycf1p, designated the loop 6 insertion (L6(ins)), which appears to be necessary and sufficient for proteolytic cleavage, since L6(ins) can promote processing when moved to new locations in Ycf1p or into a related transporter, Bpt1p. Surprisingly, mutational results indicate that proteolytic processing is not essential for Ycf1p transport activity. Instead, the L6(ins) appears to regulate substrate specificity of Ycf1p, since certain mutations in this region lower cellular cadmium resistance with a concomitant gain in arsenite resistance. Although some of these L6(ins) mutations block processing, there is no correlation between processing and substrate specificity. The activity profiles of the Ycf1p L6(ins) mutants are dramatically affected by the strain background in which they are expressed, raising the possibility that another cellular component may functionally impact Ycf1p activity. A candidate component may be a new full-length MRP-type transporter (NFT1), reported in the Saccharomyces Genome Database as two adjacent open reading frames, YKR103w and YKR104w, but which we show here is present in most Saccharomyces strains as a single open reading frame.

SUBMITTER: Mason DL

PROVIDER: S-EPMC161439 | biostudies-literature | 2003 Jun

REPOSITORIES: biostudies-literature

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A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

Mason Deborah L DL Mallampalli Monica P MP Huyer Gregory G Michaelis Susan S

Eukaryotic cell 20030601 3

Ycf1p, a member of the yeast multidrug resistance-associated protein (MRP) subfamily of ATP-binding cassette proteins, is a vacuolar membrane transporter that confers resistance to a variety of toxic substances such as cadmium and arsenite. Ycf1p undergoes a PEP4-dependent processing event to yield N- and C-terminal cleavage products that remain associated with one another. In the present study, we sought to determine whether proteolytic cleavage is required for Ycf1p activity. We have identifie ...[more]

PMID: 12796304

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Project description:This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. The A. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine ?-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

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A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

Publications

A region within a lumenal loop of Saccharomyces cerevisiae Ycf1p directs proteolytic processing and substrate specificity.

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