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• W1965788116 abstract "The nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression ofSIT4 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 nuclear gene encoding the Sit4 protein phosphatase was identified in the budding yeast Kluyveromyces lactis. K. lactis cells carrying a disrupted sit4allele are resistant to oligomycin, antimycin, ketoconazole, and econazole but hypersensitive to paromomycin, sorbic acid, and 4-nitroquinoline-N-oxide (4-NQO). Overexpression ofSIT4 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. multidrug resistance ATP-binding cassette transporter complete glycerol medium minimal glucose medium complete glucose medium 4-nitroquinoline-N-oxide phosphate-buffered saline P-glycoprotein 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.Simon S.M. Schindler M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3497-3504Crossref PubMed Scopus (419) Google Scholar). In most cases, MDR is mediated by an increased expression of the integral membrane multidrug transporters (reviewed in Refs. 2.Cole S.P. Deeley R.G. BioEssays. 1998; 20: 931-940Crossref PubMed Scopus (331) Google Scholar, 3.Gottesman M.M. Hrycyna C.A. Schoenlein P.V. Germann U.A. Pastan I. Annu. Rev. Genet. 1995; 29: 607-649Crossref PubMed Scopus (458) Google Scholar, 4.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). 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.Cowman A.F. Foote S.J. Int. J. Parasitol. 1990; 20: 503-513Crossref PubMed Scopus (62) Google Scholar, 6.Foote S.J. Thompson J.K. Cowman A.F. Kemp D.J. Cell. 1989; 57: 921-930Abstract Full Text PDF PubMed Scopus (515) Google Scholar, 7.Prasad R. De Wergifosse P. Goffeau A. Balzi E. Curr. Genet. 1995; 27: 320-329Crossref PubMed Scopus (398) Google Scholar, 8.Sanglard D. Kuchler K. Ischer F. Pagani J.L. Monod M. Bille J. Antimicrob. Agents Chemother. 1995; 39: 2378-2386Crossref PubMed Scopus (703) Google Scholar). 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 theLactococcus lactis ABC transporter LmrA is able to confer MDR in human cells (9.Van Veen H.W. Callaghan R. Soceneantu L. Sardini A. Konings W.N. Higgins C.F. Nature. 1998; 291: 291-295Crossref Scopus (216) Google Scholar). 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.Decottignies A. Goffeau A. Nat. Genet. 1997; 15: 137-145Crossref PubMed Scopus (391) Google Scholar) that transport a wide range of chemical compounds (11.Kolaczkowski M. Kolaczowska A. Luczynski J. Witek S. Goffeau A. Microb. Drug Resist. 1998; 4: 143-158Crossref PubMed Scopus (194) Google Scholar,12.Kolaczkowski M. van der Rest M. Cybularz-Kolaczkowska A. Soumillion J.P. Konings W.N. Goffeau A. J. Biol. Chem. 1996; 271: 31543-31548Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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-encoded P-glycoprotein (P-gp) and to Mrp1, which is known as a multidrug resistance associated protein (13.Balzi E. Wang M. Leterme S. van Dyck L. Goffeau A. J. Biol. Chem. 1994; 269: 2206-2214Abstract Full Text PDF PubMed Google Scholar, 14.Bissinger P.H. Kuchler K. J. Biol. Chem. 1994; 269: 4180-4186Abstract Full Text PDF PubMed Google Scholar, 15.Cui Z. Hirata D. Tsuchiya E. Osada H. Miyakawa T. J. Biol. Chem. 1996; 271: 14712-147126Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16.Hirata D. Yano K. Miyahara K. Miyakawa T. Curr. Genet. 1994; 26: 285-294Crossref PubMed Scopus (133) Google Scholar, 17.Katzmann D.J. Hallström T.C. Voet M. Wysock W. Golin J. Volckaert G. Moye-Rowley W.S. Mol. Cell. Biol. 1995; 15: 6875-6883Crossref PubMed Scopus (199) Google Scholar, 18.Li Z.S. Szczypka M. Lu Y.P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 19.Rebbeor J.F. Connolly G.C. Dumont M.E. Ballatori N. J. Biol. Chem. 1998; 273: 33449-33454Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20.Servos J. Haase E. Brendel M. Mol. Gen. Genet. 1993; 236: 214-218Crossref PubMed Scopus (171) Google Scholar, 21.Szczpka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar, 22.Wemmie J.A. Szczypka M.S. Thiele D.J. Moye-Rowley W.S. J. Biol. Chem. 1994; 269: 32592-32597Abstract Full Text PDF PubMed Google Scholar). Functional similarities between yeast and human ABC transporters have also been supported by a number of studies showing that expression of human P-gp and Mrp1 confers drug resistance in yeast (23.Kuchler K. Thorner J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2302-2306Crossref PubMed Scopus (88) Google Scholar, 24.Raymond M. Ruetz S. Thomas D.Y. Gros P. Mol. Cell. Biol. 1994; 14: 277-286Crossref PubMed Google Scholar, 25.Ruetz S. Brault M. Kast C. Hemenway C. Heitman J. Grant C.E. Cole S.P. Deeley R.G. Gros P. J. Biol. Chem. 1996; 271: 4154-4160Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 26.Tommasini R. Evers R. Vogt E. Mornet C. Zaman G.J. Achinkel A.H. Borst P. Martinoia E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6743-6748Crossref PubMed Scopus (155) Google Scholar, 27.Ueda K. Shimabuku A.M. Konishi H. Fujii Y. Takebe S. Nishi K. Yoshida M. Beppu T. Komano T. FEBS Lett. 1993; 330: 179-282Crossref Scopus (31) Google Scholar).Extensive efforts have been directed to understanding the regulation of ABC transporter activity at both the transcriptional and post-translational levels. Three transcriptional activators of the Cys6 zinc finger type have been genetically identified inS. cerevisiae (28.Balzi E. Chen W. Ulaszewski S. Capieaux E. Goffeau A. J. Biol. Chem. 1987; 262: 16871-16879Abstract Full Text PDF PubMed Google Scholar, 29.Cui Z. Shiraki T. Hirata D. Miyakawa T. Mol. Microbiol. 1998; 29: 1307-1315Crossref PubMed Scopus (56) Google Scholar, 30.Delaveau T. Delahodde A. Carvajal E. Subik J. Jacq C. Mol. Gen. Genet. 1994; 244: 501-511Crossref PubMed Scopus (176) Google Scholar, 31.Dexter D. Moye-Rowley W.S. Wu A.-L. Golin J. Genetics. 1994; 136: 505-515Crossref PubMed Google Scholar, 32.Subik J. Ulaszewki S. Goffeau A. Curr. Genet. 1986; 10: 665-670Crossref PubMed Scopus (53) Google Scholar). The Pdr1 and Pdr3 proteins control the transcriptional levels of PDR5, SNQ2, YOR1,PDR10, and PDR15 by direct binding to DNA in the promoter of the target genes (33.Decottignies A. Lambert L. Catty P. Degand H. Epping E.A. Moye-Rowley W.S. Balzi E. Goffeau A. J. Biol. Chem. 1995; 270: 18150-18157Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 34.Hallström T.C. Moye-Rowley W.S. J. Biol. Chem. 1998; 273: 2098-2104Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 35.Katzmann D.J. Burnett P.E. Golin J. Mahé Y. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 4653-4661Crossref PubMed Scopus (180) Google Scholar, 36.Mahé Y. Parle-McDermott A. Delahodde A. Lamprecht A. Kuchler K. Mol. Microbiol. 1996; 20: 109-117Crossref PubMed Scopus (103) Google Scholar, 37.Meyers S. Schauer W. Balzi E. Wagner M. Goffeau A. Golin J. Curr. Genet. 1992; 21: 431-436Crossref PubMed Scopus (124) Google Scholar). The Yrr1 protein is involved in the regulation of SNQ2 (29.Cui Z. Shiraki T. Hirata D. Miyakawa T. Mol. Microbiol. 1998; 29: 1307-1315Crossref PubMed Scopus (56) Google Scholar). However, the mechanism of the regulatory pathway upstream of PDR1, PDR3, andYRR1 remains elusive. Two recent studies have revealed a functional link of the Pdr1 and Pdr3 transcriptional factors to the yeast homologues of the stress-dependent transcriptional factor AP1 and the heat shock protein Hsp70 (38.Hallström T.C. Katzmann D.J. Torres R.J. Sharp W.J. Moye-Rowley W.S. Mol. Cell. Biol. 1998; 18: 1147-1155Crossref PubMed Scopus (69) Google Scholar, 39.Wendler F. Bergler H. Prutej K. Jungwirth H. Zisser G. Kuchler K. Högenauer G. J. Biol. Chem. 1997; 272: 27091-27098Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). These observations suggest the presence of a signaling pathway upstream of Pdr1 and Pdr3 in the cellular response to drug stress.At a post-translational level, attention has been directed to a possible role of protein phosphorylation/dephosphorylation in the modulation of ABC transporter activity. Because the human P-gp is phosphorylated in vivo, an approach is to develop chemosensitizers that inhibit P-gp function at the level of phosphorylation and reverse the MDR phenotype in tumor cells. Early studies have demonstrated that a change in the state of phosphorylation of P-gp has been associated with differences in relative drug resistance of mammalian cells, suggesting that the phosphorylation/dephosphorylation mechanisms may be involved in the regulation of the efflux activity of the drug transporter (40.Center M.S. Biochem. Biophys. Res. Commun. 1983; 115: 159-166Crossref PubMed Scopus (45) Google Scholar, 41.Hamada H. Hagiwara K.I. Nakajima T. Tsuruo T. Cancer Res. 1987; 47: 2860-2865PubMed Google Scholar). More recently, similar results were obtained with the human Mrp1 transporter. By using protein kinase inhibitors, it has been shown that phosphorylation of the serine residues of Mrp1, probably by protein kinase C, plays an important role in modulating drug accumulation in resistant cells (42.Gekeler V. Boer R. Ise W. Sanders K.H. Schachtele C. Beck J. Biochem. Biophys. Res. Commun. 1995; 206: 119-126Crossref PubMed Scopus (68) Google Scholar, 43.Ma L.-D. Krishnamachary N. Center M.S. Biochemistry. 1995; 34: 3338-3343Crossref PubMed Scopus (63) Google Scholar). Biochemical and genetic studies identified four serine residues in a basic domain of the linker region of the human P-gp that are accessible and recognized as major targets for phosphorylation by protein kinase C or protein kinase A. However, 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.Germann U.A. Chambers T.C. Ambudkar S.V. Licht T. Cardarelli C.O. Pastan I. Gottesman M.M. J. Biol. Chem. 1996; 271: 1708-1716Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 45.Goodfellow H.R. Sardini A. Ruetz S. Callaghan R. Gros P. McNaughton P.A. Higgins C.F. J. Biol. Chem. 1996; 271: 13668-13674Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This suggests that phosphorylation by protein kinase C may not play a role in regulating drug transport of P-gp. Whether the phosphorylation of an acidic domain of the linker region is involved in the modulation of drug transport activity remains to be established (46.Glavy J.S. Horwitz S.B. Orr G.A. J. Biol. Chem. 1997; 272: 5909-5914Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). By contrast, in S. cerevisiae, a phosphorylation site has been proposed in the Ycf1 protein (21.Szczpka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar), an orthologue of the human Mrp1 transporter located on vacuolar membrane (18.Li Z.S. Szczypka M. Lu Y.P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 19.Rebbeor J.F. Connolly G.C. Dumont M.E. Ballatori N. J. Biol. Chem. 1998; 273: 33449-33454Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 21.Szczpka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar). With Ycf1, substitution of a serine to alanine residue in the potential protein kinase A phosphorylation site in a central region of Ycf1 renders the protein non-functional.In this study we identified nuclear mutations conferring MDR in the budding yeast Kluyveromyces lactis. We find that the gene defined by the mutations encodes a protein phosphatase belonging to the Sit4/PPV/PP6 family. It was found that the Sit4 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.DISCUSSIONThe Sit4 protein is a type 2A-related protein phosphatase (75.Stark M.J.R. Black S. Sneddon A.A. Andrews P.D. FEMS Microbiol. Lett. 1994; 117: 121-130Crossref PubMed Scopus (12) Google Scholar) that has been originally identified in S. cerevisiae. Specific mutations in the ScSIT4 gene restore transcription of the HIS4 gene in the absence of thetrans-acting DNA binding factors GCN4, BAS1, and BAS2 that are normally required for HIS4 expression (66.Arndt K.T. Styles C.A. Fink G.R. Cell. 1989; 56: 527-537Abstract Full Text PDF PubMed Scopus (177) Google Scholar). Loss of theSIT4 gene product was proposed to cause aberrant transcriptional activity of the RNA polymerase II because of accumulation of the phosphorylated form of an unknown transcription factor. Subsequent studies have shown that the Sit4 protein phosphatase is involved in cell cycle progression and bud formation (72.Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (271) Google Scholar, 76.Fernandez-Sarabia M.J. Sutton A. Zhong T. Arndt K.T. Genes Dev. 1992; 6: 2417-2428Crossref PubMed Scopus (115) Google Scholar), in control of glycogen synthase and phosphorylase activities (77.Posas F. Clotet J. Arino J. FEBS Lett. 1991; 279: 341-345Crossref PubMed Scopus (28) Google Scholar), and in the ceramide-induced and Tor-signaling pathways (78.Nickels J.T. Broach J.R. Genes Dev. 1996; 10: 382-394Crossref PubMed Scopus (176) Google Scholar, 79.Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (439) Google Scholar, 80.Jiang Y. Broach J.R. EMBO J. 1999; 18: 2782-2792Crossref PubMed Scopus (273) Google Scholar). The Sit4 function is evolutionarily conserved as S. cerevisiae sit4mutants can be complemented by the SIT4 homologues fromD. melanogaster and human cells (70.Mann D.J. Dombrádi V. Cohen P.T.W. EMBO J. 1993; 12: 4833-4842Crossref PubMed Scopus (45) Google Scholar, 71.Bastians H. Ponstingl H. J. Cell Sci. 1996; 109: 2865-2874Crossref PubMed Google Scholar).Reported in this study is the isolation of a SIT4 homologue from the budding yeast K. lactis. Several novel functional aspects of the Sit4 protein phosphatase in K. lactis are described. First, K. lactis SIT4 is not essential as disruption of the gene in several strains did not cause non-viability. In S. cerevisiae, null mutants are viable only in a genetic background with the SSD-v1 version of the polymorphicSSD1 gene (72.Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (271) Google Scholar). Second, K. lactis sit4 mutants are clearly impaired in respiratory function. Respiratory growth ofsit4 mutants can be totally abolished when cells are grown at 37 or 19 °C. Although a growth defect on non-fermentable carbon sources was observed in the S. cerevisiae transcriptional suppressor sit4 mutants (66.Arndt K.T. Styles C.A. Fink G.R. Cell. 1989; 56: 527-537Abstract Full Text PDF PubMed Scopus (177) Google Scholar), a SIT4-disrupted strain with a SSD1-v1 background is respiratory-competent (72.Sutton A. Immanuel D. Arndt K.T. Mol. Cell. Biol. 1991; 11: 2133-2148Crossref PubMed Scopus (271) Google Scholar). Third, we find that sit4 mutants of K. lactis have an increased formation of specific nuclear mutations on exposure to ethidium bromide that permits the recovery of cells lacking mitochondrial DNA, suggesting that Sit4p may affect susceptibility to a mutagenic agent such as ethidium bromide. 5G. D. Clark-Walker, unpublished data. Finally, Sit4p has an important role in regulating MDR.In S. cerevisiae, two classes of multidrug resistance genes have been genetically identified. The first class of genes are ones encoding membrane ATP-binding cassette (ABC) transporters such asPDR5/STS1/YDR1, SNQ2, YCF1, YOR1, andPDR12 that mediate drug efflux out of cells (13.Balzi E. Wang M. Leterme S. van Dyck L. Goffeau A. J. Biol. Chem. 1994; 269: 2206-2214Abstract Full Text PDF PubMed Google Scholar, 14.Bissinger P.H. Kuchler K. J. Biol. Chem. 1994; 269: 4180-4186Abstract Full Text PDF PubMed Google Scholar, 15.Cui Z. Hirata D. Tsuchiya E. Osada H. Miyakawa T. J. Biol. Chem. 1996; 271: 14712-147126Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16.Hirata D. Yano K. Miyahara K. Miyakawa T. Curr. Genet. 1994; 26: 285-294Crossref PubMed Scopus (133) Google Scholar, 17.Katzmann D.J. Hallström T.C. Voet M. Wysock W. Golin J. Volckaert G. Moye-Rowley W.S. Mol. Cell. Biol. 1995; 15: 6875-6883Crossref PubMed Scopus (199) Google Scholar, 18.Li Z.S. Szczypka M. Lu Y.P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 19.Rebbeor J.F. Connolly G.C. Dumont M.E. Ballatori N. J. Biol. Chem. 1998; 273: 33449-33454Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 20.Servos J. Haase E. Brendel M. Mol. Gen. Genet. 1993; 236: 214-218Crossref PubMed Scopus (171) Google Scholar, 21.Szczpka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar, 22.Wemmie J.A. Szczypka M.S. Thiele D.J. Moye-Rowley W.S. J. Biol. Chem. 1994; 269: 32592-32597Abstract Full Text PDF PubMed Google Scholar, 47.Piper P. Mahé Y. Thompson S. Pandjaitan R. Holyoak C. Egner R. Mühlbauer M. Coote P. Kuchler K. EMBO J. 1998; 17: 4257-4265Crossref PubMed Scopus (283) Google Scholar). The second class of genes are those acting as transcriptional activators for the ABC transporter genes. Among the well characterized transcriptional regulators are PDR1 and PDR3, which control the transcriptional levels of PDR5,YOR1, PDR10, and PDR15 (28.Balzi E. Chen W. Ulaszewski S. Capieaux E. Goffeau A. J. Biol. Chem. 1987; 262: 16871-16879Abstract Full Text PDF PubMed Google Scholar, 30.Delaveau T. Delahodde A. Carvajal E. Subik J. Jacq C. Mol. Gen. Genet. 1994; 244: 501-511Crossref PubMed Scopus (176) Google Scholar, 35.Katzmann D.J. Burnett P.E. Golin J. Mahé Y. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 4653-4661Crossref PubMed Scopus (180) Google Scholar,37.Meyers S. Schauer W. Balzi E. Wagner M. Goffeau A. Golin J. Curr. Genet. 1992; 21: 431-436Crossref PubMed Scopus (124) Google Scholar, 81.Wolfger H. Mahé Y. Parle-McDermott A. Delahodde A. Kuchler K. FEBS Lett. 1997; 418: 269-274Crossref PubMed Scopus (82) Google Scholar). Thus, the simplest interpretation for the MDR phenotype associated with K. lactis sit4 mutants is that the Sit4 protein phosphatase has a regulatory role on either of the two types of genes mentioned above, which ultimately modify the drug efflux capacity of the membrane ABC transporters.K. lactis sit4 mutants display altered sensitivity to a wide range of mechanistically unrelated drugs. These drugs include the mitochondrial inhibitors oligomycin, antimycin A, and paromomycin that target to the ATP synthase, the bc 1 complex of the respiratory chain, and mitochondrial ribosomes, respectively, the antifungal drugs ketoconazole and econazole, the antimicrobial preservative sorbic acid, and the mutagen 4-NQO. Efflux of most of these compounds has been shown to require specific ABC transporters in yeast. In S. cerevisiae, efflux of the antifungal drugs ketoconazole and econazole4 is mediated by the Pdr5 transporter (8.Sanglard D. Kuchler K. Ischer F. Pagani J.L. Monod M. Bille J. Antimicrob. Agents Chemother. 1995; 39: 2378-2386Crossref PubMed Scopus (703) Google Scholar, 73.Egner R. Rosenthal F.E. Kralli A. Sanglard D. Kuchler K. Mol. Biol. Cell. 1998; 9: 523-543Crossref PubMed Scopus (134) Google Scholar); oligomycin is transported by Yor1 (17.Katzmann D.J. Hallström T.C. Voet M. Wysock W. Golin J. Volckaert G. Moye-Rowley W.S. Mol. Cell. Biol. 1995; 15: 6875-6883Crossref PubMed Scopus (199) Google Scholar); the detoxification of sorbic acid requires the Pdr12 pump (47.Piper P. Mahé Y. Thompson S. Pandjaitan R. Holyoak C. Egner R. Mühlbauer M. Coote P. Kuchler K. EMBO J. 1998; 17: 4257-4265Crossref PubMed Scopus (283) Google Scholar), whereas 4-NQO is a specific substrate for the ABC protein Snq2 (20.Servos J. Haase E. Brendel M. Mol. Gen. Genet. 1993; 236: 214-218Crossref PubMed Scopus (171) Google Scholar). Transporters for antimycin A and paromomycin have not yet been assigned.In K. lactis sit4 mutants, at least four distinct types of transporters appear to be affected. Recent studies have revealed that a single pump is responsible for transport of ketoconazole, econazole, oligomycin, and antimycin.2 This transporter, most likely corresponding to the Pdr5 homologue in K. lactis, is negatively regulated by Sit4p. Rhodamine 6G could also be transported by the same protein. In contrast, a membrane transporter, responsible for detoxification of paromomycin, is positively regulated by Sit4p.sit4 mutants are hypersensitive to paromomycin, and overexpression of SIT4 causes a drastic elevation of resistance to the antibiotic. In addition, two other transporters, required for resistance to sorbic acid and 4-NQO, are also positively regulated by Sit4p. Based on the differential responses to overexpression of SIT4, these two transporters, together with that for paromomycin, are functionally distinct. In contrast to the paromomycin transporter, the function of the pump for sorbic acid is only slightly augmented in cells overexpressing SIT4,whereas the detoxifying capacity of the pump for 4-NQO is not affected by increasing SIT4 dosage. By analogy to S. cerevisiae, the pumps responsible for sorbic acid and 4-NQO might be related to the Pdr12 and Snq2 transporters of S. cerevisiae (20.Servos J. Haase E. Brendel M. Mol. Gen. Genet. 1993; 236: 214-218Crossref PubMed Scopus (171) Google Scholar, 47.Piper P. Mahé Y. Thompson S. Pandjaitan R. Holyoak C. Egner R. Mühlbauer M. Coote P. Kuchler K. EMBO J. 1998; 17: 4257-4265Crossref PubMed Scopus (283) Google Scholar).Strong support for a role of Sit4p in modulating MDR by altering the function of membrane transporters and ultimately the intracellular drug accumulation comes from the drug efflux assay using the fluorescent dye rhodamine 6G. In K. lactis, we have shown that an ABC transporter, likely to be of the Pdr5-type, promotes the efflux of rhodamine 6G in an energy-dependent and FK506-sensitive manner. When sit4 mutants were examined, accumulation of rhodamine 6G was indeed significantly decreased compared with wild-type cells. As the strong rhodamine 6G efflux in sit4 cells is sensitive to inhibition by the Pdr5-specific drug FK506, we can conclude that the function of a Pdr5-type pump is up-regulated. These results are in accord with the elevated resistance of sit4mutants to the antifungal drugs ketoconazole and econazole, which share the same transporter with rhodamine 6G in S. cerevisiae. Another line of evidence supporting the involvement of Sit4p in regulating expression/activity of membrane ABC transporters is our preliminary study showing altered protein levels of ABC transporters insit4 mutants. Immunoblot analysis using antibodies againstS. cerevisiae proteins has revealed that the accumulation of Pdr12- and Snq2-like transporters in K. lactis is in fact decreased by severalfold in sit4 cells,4 which is in agreement with the down-regulation pattern of resistance for sorbic acid and 4-NQO in sit4 mutants.As discussed above, the Sit4 phosphatase is involved in the regulation of different types of membrane transporters in both a positive and a negative manner. Mechanistically, control by the Sit4 phosphatase can intervene at different levels as follows: 1) Sit4p could modulate the function of the transcriptional factors that activate expression of particular ABC transporter genes; 2) Sit4p may control membrane targeting and turnover of the drug transporters; and 3) Sit4p could also directly regulate the drug pumping activity or substrate specificity of the membrane transporters by dephosphorylating a phospho-Ser/Thr residue(s). In S. cerevisiae, although most multidrug-resistant mutants are confined to mutations in transcriptional factors, some ABC transporters such as Pdr5 are ubiquitinated (82.Egner R. Kuchler K. FEBS Lett. 1996; 378: 177-181Crossref PubMed Scopus (102) Google Scholar) and subjected to vacuolar degradation (83.Egner R. Mahé Y. Pandjaitan R. Kuchler K. Mol. Cell. Biol. 1995; 15: 5879-5887Crossref PubMed Scopus (114) Google Scholar). It has also been documented that phosphorylation in a hypothetical modular domain of the yeast Ycf1 transporter is important for drug resistance (21.Szczpka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar). Each type of ABC transporter may thus adopt a different regulatory pattern. In K. lactis, because both positive and negative regulation are observed in sit4 mutants, it can be imagined that more than one pathway operates to achieve the regulation of individual transporters by Sit4p. Elucidation of the regulatory mechanisms would first require the isolation and characterization ofK. lactis ABC transporter genes, which is currently underway in our laboratories. 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.Simon S.M. Schindler M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3497-3504Crossref PubMed Scopus (419) Google Scholar). In most cases, MDR is mediated by an increased expression of the integral membrane multidrug transporters (reviewed in Refs. 2.Cole S.P. Deeley R.G. BioEssays. 1998; 20: 931-940Crossref PubMed Scopus (331) Google Scholar, 3.Gottesman M.M. Hrycyna C.A. Schoenlein P.V. Germann U.A. Pastan I. Annu. Rev. Genet. 1995; 29: 607-649Crossref PubMed Scopus (458) Google Scholar, 4.Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3346) Google Scholar). 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" @default.
• W1965788116 created "2016-06-24" @default.
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• W1965788116 date "2000-05-01" @default.
• W1965788116 modified "2023-10-01" @default.
• W1965788116 title "Positive and Negative Control of Multidrug Resistance by the Sit4 Protein Phosphatase in Kluyveromyces lactis" @default.
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• W1965788116 doi "https://doi.org/10.1074/jbc.275.20.14865" @default.
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