Abstract
The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4 allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression of SIT4 leads to an elevation in resistance to paromomycin and to lesser extent tolerance to sorbic acid, but it has no detectable effect on resistance to 4-NQO. These observations suggest that the Sit4 protein phosphatase has a broad role in modulating multidrug resistance in K. lactis. Expression or activity of a membrane transporter specific for paromomycin and the ABC pumps responsible for 4-NQO and sorbic acid would be positively regulated by Sit4p. In contrast, the function of a Pdr5-type transporter responsible for ketoconazole and econazole extrusion, and probably also for efflux of oligomycin and antimycin, is likely to be negatively regulated by the phosphatase. Drug resistance of sit4 mutants was shown to be mediated by ABC transporters as efflux of the anionic fluorescent dye rhodamine 6G, a substrate for the Pdr5-type pump, is markedly increased in sit4 mutants in an energy-dependent and FK506-sensitive manner.
Highlights
The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis
This screening procedure was based on observations that the majority of oligomycin-resistant mutants in S. cerevisiae and K. lactis occur in the mitochondrial DNA-encoded subunits 6 and 9 of the ATP synthase F0 complex [62,63,64,65]
K. lactis strains PM6-7A/oli4 and PM6-7A/oli18 are resistant to 1.0 g/ml oligomycin on GlyYP medium compared with the wild-type parent PM6-7A whose growth is completely inhibited by the drug (Fig. 1)
Summary
84 This study This study involved in the regulation of the efflux activity of the drug transporter [40, 41]. Recent mutational analysis showed that a mutant P-gp, with the putative phosphorylation sites for protein kinase C within the linker region changed to non-phosphorylatable alanine residues, or to aspartic acid residues to mimic permanently dephosphorylated serine residues, is still functionally active to diminish drug accumulation within cells [44, 45] This suggests that phosphorylation by protein kinase C may not play a role in regulating drug transport of P-gp. It was found that the Sit protein has a broad role in regulating MDR by altering the expression or activity of different membrane drug transporters To our knowledge this is the first protein phosphatase genetically identified that is involved in modulation of multidrug resistance
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