Positive and Negative Control of Multidrug Resistance by the Sit4 Protein Phosphatase in Kluyveromyces lactis

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.The occurrence of multidrug resistance (MDR) 1 is one of the main obstacles to the successful treatment of cancer. When treated with chemotherapeutic drugs, many cancer cells develop resistance to a variety of structurally and functionally unrelated compounds (for review, see Ref. 1). In most cases, MDR is mediated by an increased expression of the integral membrane multidrug transporters (reviewed in Refs. 2-4). These membrane proteins, known as ATP-binding cassette (ABC) transporters, function in an ATP-dependent way probably by increasing drug efflux and consequently lowering their intracellular accumulation. In a similar manner, many pathogenic microorganisms such as Candida albicans and Plasmodium falciparum can use the ABC transporter-mediated drug efflux mechanism to evade chemotherapy (5-8). However, despite the rapidly growing number of ABC transporters identified in various organisms, little is known about how activities of the drug transporters are modulated and how aberrant regulation of the expression of ABC transporter genes contributes to the acquisition of MDR in vivo.The ABC transporter-mediated drug efflux mechanism is evolutionarily conserved and occurs in a variety of living organisms ranging from bacteria to humans. An example is a recent work demonstrating that the Lactococcus lactis ABC transporter LmrA is able to confer MDR in human cells (9). The recently completed genome sequencing project of Saccharomyces cerevisiae has revealed the presence of as many as 29 proteins belonging to the ubiquitous ABC superfamily (10) that transport a wide range of chemical compounds (11,12). The yeast ABC proteins so far characterized, such as Pdr5, Snq2, Ycf1 and Yor1, confer MDR with physiological and biochemical properties very similar to the human MDR1-en...

Elucidation of Cellular Mechanisms Involved in Experimental Paromomycin Resistance in Leishmania donovani

“…ABC transporter genes are regulated by protein phosphatase, which may activate expression of the transporter genes or may modulate the drug-pumping activity or substrate specificity of the transporters. It was demonstrated that increased expression of Sit4 protein phosphatase conferred PMM resistance in Kluyveromyces lactis, mediated by ABC transporters (25). Here we observed a marked increase in the expression of protein phosphatase 2A (a Sit4 protein phosphatase homologue in Leishmania) in paromomycin-resistant L. donovani, compared with its sensitive counterpart.…”

“…Additionally, protein phosphatase 2A, which has a probable role in activating the expression of these transporters (25), was found to be highly upregulated (4.47 Ϯ 0.71-fold) in PMM-R parasites (Fig. 3).…”

Activity of the Kluyveromyces lactis Pdr5 Multidrug Transporter Is Modulated by the Sit4 Protein Phosphatase

“…In previous work it was indicated that the Sit4 protein phosphatase of K. lactis plays a critical role in controlling drug transport (9). This finding makes K. lactis an attractive model system for investigating the novel regulatory mechanism of MDR for several reasons.…”

“…SIT4 has been found to have a broad role in regulating MDR (9). It negatively regulates the resistance of cells to oligomycin, antimycin, ketoconazole, and econazole and positively modulates tolerance to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide.…”

Yap1 overproduction restores arsenite resistance to the ABC transporter deficient mutantycf1by activatingACR3expression

“…Such a pathway could involve another transporter in yeast, which probably has a weaker affinity to transport arsenite. This is a likely possibility as many transporters have a broad range of substrate specificity, for example, Yor1 (Chen et al 2000).…”