FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073343350> ?p ?o ?g. }

• W2073343350 endingPage "12447" @default.
• W2073343350 startingPage "12438" @default.
• W2073343350 abstract "The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of ∼40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Δ cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein. The Na+/H+ antiporter Nha1p of Saccharomyces cerevisiae plays an important role in maintaining intracellular pH and Na+ homeostasis. Nha1p has a two-domain structure composed of integral membrane and hydrophilic tail regions. Overexpression of a peptide of ∼40 residues (C1+C2 domains) that is localized in the juxtamembrane area of its cytoplasmic tail caused cell growth retardation in highly saline conditions, possibly by decreasing Na+/H+ antiporter activity. A multicopy suppressor gene of this growth retardation was identified from a yeast genome library. The clone encodes a novel membrane protein denoted as COS3 in the genome data base. Overexpression or deletion of COS3 increases or decreases salinity-resistant cell growth, respectively. However, in nha1Δ cells, overexpression of COS3 alone did not suppress the growth retardation. Cos3p and a hydrophilic portion of Cos3p interact with the C1+C2 peptide in vitro, and Cos3p is co-precipitated with Nha1p from yeast cell extracts. Cos3p-GFP mainly resides at the vacuole, but overexpression of Nha1p caused a portion of the Cos3p-GFP proteins to shift to the cytoplasmic membrane. These observations suggest that Cos3p is a novel membrane protein that can enhance salinity-resistant cell growth by interacting with the C1+C2 domain of Nha1p and thereby possibly activating the antiporter activity of this protein. Na+ concentrations and the intracellular pH are maintained at certain levels in all living cells ranging from prokaryotes to eukaryotes (1Padan E. Schuldiner S. J. Bioenerg. Biomembr. 1993; 25: 647-669PubMed Google Scholar, 2Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1185: 129-151Crossref PubMed Scopus (143) Google Scholar, 3Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). The proliferation of eukaryotic cells also requires that the cytoplasmic pH is shifted toward alkalinity (4Johnson J.D. Epel D. Nature. 1976; 262: 661-664Crossref PubMed Scopus (384) Google Scholar, 5Winkler M.M. Grainger J.L. Nature. 1978; 273: 536-538Crossref PubMed Scopus (67) Google Scholar, 6Houle J.G. Wasserman W.J. Dev. Biol. 1983; 97: 302-312Crossref PubMed Scopus (47) Google Scholar, 7Rubin H. J. Cell Physiol. 1973; 82: 231-238Crossref PubMed Scopus (39) Google Scholar). Ion transporters in the cytoplasmic and endocytic vesicular membranes play important roles in the mechanisms that regulate these Na+ concentrations and pH levels. In particular, Na+/H+ antiporters play a central role in all species ranging from bacteria to humans in maintaining the intracellular homeostasis of H+ and Na+ ions (1Padan E. Schuldiner S. J. Bioenerg. Biomembr. 1993; 25: 647-669PubMed Google Scholar, 2Padan E. Schuldiner S. Biochim. Biophys. Acta. 1994; 1185: 129-151Crossref PubMed Scopus (143) Google Scholar, 3Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar). Supporting this is that many Na+/H+ antiporters (NHAs), 1The abbreviations used are: NHAs, Na+/H+ antiporters, NHEs, Na+/H+ exchangers; ORF, open reading frame; GFP, green fluorescent protein; UTR, untranslated region; GST, glutathione S-transferase; PMSF, phenylmethylsulfonyl fluoride; MES, 4-morpholineethanesulfonic acid; Ni-NTA, nickel-nitrilotriacetic acid; DDM, n-dodecyl β-d-maltoside. which are also called Na+/H+ exchangers (NHEs), occur ubiquitously in bacteria, yeast, plants, and animals (3Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, 8Counillon L. Pouyssegur J. J. Biol. Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 9Blumwald E. Curr. Opin. Cell Biol. 2000; 12: 431-434Crossref PubMed Scopus (748) Google Scholar, 10Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (290) Google Scholar, 11Jia Z.P. McCullough N. Martel R. Hemmingsen S. Young P.G. EMBO J. 1992; 11: 1631-1640Crossref PubMed Scopus (159) Google Scholar). All antiporters that have been identified to date are integral membrane proteins with the same function, but their primary structures are quite diverse (3Orlowski J. Grinstein S. J. Biol. Chem. 1997; 272: 22373-22376Abstract Full Text Full Text PDF PubMed Scopus (520) Google Scholar, 8Counillon L. Pouyssegur J. J. Biol. Chem. 2000; 275: 1-4Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 9Blumwald E. Curr. Opin. Cell Biol. 2000; 12: 431-434Crossref PubMed Scopus (748) Google Scholar, 10Padan E. Venturi M. Gerchman Y. Dover N. Biochim. Biophys. Acta. 2001; 1505: 144-157Crossref PubMed Scopus (290) Google Scholar, 11Jia Z.P. McCullough N. Martel R. Hemmingsen S. Young P.G. EMBO J. 1992; 11: 1631-1640Crossref PubMed Scopus (159) Google Scholar). Eight NHE isoforms have been reported in mammals (12Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 13Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 14Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 17Goyal S. Vanden Heuvel G. Aronson P.S. Am. J. Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (181) Google Scholar), and their structure-function relationships have been studied extensively (12Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 13Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 14Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 17Goyal S. Vanden Heuvel G. Aronson P.S. Am. J. Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (181) Google Scholar). Hydropathy plot analysis of these mammalian NHE isoforms has revealed that they consist of an integral membrane region and a hydrophilic cytoplasmic tail region (12Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 13Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 14Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 17Goyal S. Vanden Heuvel G. Aronson P.S. Am. J. Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (181) Google Scholar). The integral membrane region is required for the ion transport across the membrane and is highly conserved (12Sardet C. Franchi A. Pouyssegur J. Cell. 1989; 56: 271-280Abstract Full Text PDF PubMed Scopus (671) Google Scholar, 13Orlowski J. Kandasamy R.A. Shull G.E. J. Biol. Chem. 1992; 267: 9331-9339Abstract Full Text PDF PubMed Google Scholar, 14Baird N.R. Orlowski J. Szabo E.Z. Zaun H.C. Schultheis P.J. Menon A.G. Shull G.E. J. Biol. Chem. 1999; 274: 4377-4382Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 15Numata M. Petrecca K. Lake N. Orlowski J. J. Biol. Chem. 1998; 273: 6951-6959Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 16Numata M. Orlowski J. J. Biol. Chem. 2001; 276: 17387-17394Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar, 17Goyal S. Vanden Heuvel G. Aronson P.S. Am. J. Physiol. 2003; 284: F467-F473Crossref PubMed Scopus (181) Google Scholar). In contrast, the hydrophilic cytoplasmic regions (tail region) are structurally diverse. Accumulating evidence suggests that isoform-specific elements in the tail region mediate interactions with other proteins (18Bertrand B. Wakabayashi S. Ikeda T. Pouyssegur J. Shigekawa M. J. Biol. Chem. 1994; 269: 13703-13709Abstract Full Text PDF PubMed Google Scholar, 19Lin X. Barber D.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12631-12636Crossref PubMed Scopus (151) Google Scholar, 20Matsumoto M. Miyake Y. Nagita M. Inoue H. Shitakubo D. Takemoto K. Ohtsuka C. Murakami H. Nakamura N. Kanazawa H. J. Biochem. (Tokyo). 2001; 130: 217-225Crossref PubMed Scopus (44) Google Scholar, 21Weinman E.J. Steplock D. Wang Y. Shenolikar S. J. Clin. Investig. 1995; 95: 2143-2149Crossref PubMed Scopus (310) Google Scholar, 22Denker S.P. Huang D.C. Orlowski J. Furthmayr H. Barber D.L. Mol. Cell. 2000; 6: 1425-1436Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar, 23Lehoux S. Abe J. Florian J.A. Berk B.C. J. Biol. Chem. 2001; 276: 15794-15800Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 24Mailander J. Muller-Esterl W. Dedio J. FEBS Lett. 2001; 507: 331-335Crossref PubMed Scopus (60) Google Scholar, 25Pang T. Su X. Wakabayashi S. Shigekawa M. J. Biol. Chem. 2001; 276: 17367-17372Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and regulate the antiporter activity. However, the molecular details of these regulatory interactions remain to be determined. The yeast Na+/H+ antiporter SOD2 in Schizosaccharomyces pombe was originally discovered as a factor that makes the cell growth resistant to high NaCl levels in the medium (11Jia Z.P. McCullough N. Martel R. Hemmingsen S. Young P.G. EMBO J. 1992; 11: 1631-1640Crossref PubMed Scopus (159) Google Scholar). The SOD2 isoform denoted as NHA1 also exists in other yeasts (26Watanabe Y. Miwa S. Tamai Y. Yeast. 1995; 11: 829-838Crossref PubMed Scopus (76) Google Scholar, 27Soong T.W. Youg T.F. Ramanan N. Wang Y. Microbiology. 2000; 146: 1035-1044Crossref PubMed Scopus (35) Google Scholar, 28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar) and fungal species (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar), including Saccharomyces cerevisiae (29Prior C. Potier S. Souciet J.L. Sychrova H. FEBS Lett. 1996; 387: 89-93Crossref PubMed Scopus (151) Google Scholar), and it has been shown to antiport Na+ for H+ (30Banuelos M.A. Sychrova H. Bleykasten-Grosshans C. Souciet J.L. Potier S. Microbiology. 1998; 144: 2749-2758Crossref PubMed Scopus (200) Google Scholar, 31Sychrova H. Ramirez J. Pena A. FEMS Microbiol. Lett. 1999; 171: 167-172Crossref PubMed Scopus (64) Google Scholar). As with the mammalian NHE family, the Nha1p family of proteins bear a highly conserved integral membrane region and a structurally diverse cytoplasmic tail region (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar). Nha1p-mediated export of Na+ is coupled to the proton motive force created by the H+-translocating ATPase on the cytoplasmic membrane (31Sychrova H. Ramirez J. Pena A. FEMS Microbiol. Lett. 1999; 171: 167-172Crossref PubMed Scopus (64) Google Scholar). Similarly, S. cerevisiae has another Na+/H+ antiporter denoted Nhx1p (32Nass R. Cunningham K.W. Rao R. J. Biol. Chem. 1997; 272: 26145-26152Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) that is localized at endocytic vesicular membranes (33Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and that drives Na+ transport across the membranes by means of a proton gradient that is probably formed by a V-type ATPase (33Nass R. Rao R. J. Biol. Chem. 1998; 273: 21054-21060Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 34Gaxiola R.A. Rao R. Sherman A. Grisafi P. Alper S.L. Fink G.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1480-1485Crossref PubMed Scopus (519) Google Scholar, 35Quintero F.J. Blatt M.R. Pardo J.M. FEBS Lett. 2000; 471: 224-228Crossref PubMed Scopus (152) Google Scholar). Although the overall primary sequences of the cytoplasmic tail regions of the various Nha1p proteins vary among various yeast species, we have reported that this region bears six small but distinct conserved domains that we have denoted C1 to C6 (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar). We have also shown that these conserved domains probably play similar functions in yeast and other fungi (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar). The C1, C2, and C3 domains consist of 16, 23, and 15 residues, respectively, and are located at the juxtamembrane area of the cytoplasmic tail region. Deletion of these domains decreases salinity-resistant growth, which suggests that these domains are important for growth during highly saline conditions, possibly because they influence the antiporter activity of Nha1p (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar). However, the precise mechanisms by which these domains confer salinity resistance have not yet been clarified. In this study, we analyzed the role that these domains play in the high salinity-resistant growth of cells. We found that the C1+C2 domains bind to a novel membrane protein and that this binding event enhances salinity-resistant cell growth, possibly by increasing the antiporter activity of Nha1p. Escherichia coli and Yeast Strains and Cell Culture—The S. cerevisiae strains G19 (36Banuelos M.A. Klein R.D. Alexander-Bowman S.J. Rodriguez-Navarro A. EMBO J. 1995; 14: 3021-3027Crossref PubMed Scopus (118) Google Scholar), SK5 (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar), and FOY41 constructed in this study are derivatives of W303-1B (MATα leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100) (36Banuelos M.A. Klein R.D. Alexander-Bowman S.J. Rodriguez-Navarro A. EMBO J. 1995; 14: 3021-3027Crossref PubMed Scopus (118) Google Scholar). The ENA gene of G19 (ena1Δ::HIS3::ena4Δ, a kind gift from Dr. A. Rodriguez-Navarro, Ciudad University) has been deleted (36Banuelos M.A. Klein R.D. Alexander-Bowman S.J. Rodriguez-Navarro A. EMBO J. 1995; 14: 3021-3027Crossref PubMed Scopus (118) Google Scholar). SK5 (ena1Δ::HIS3::ena4Δ nha1Δ::LEU2) (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar) and FOY (ena1Δ::HIS3::ena4Δ cos3Δ::TRP1) are both derived from G19 and lack the NHA1 and COS3 genes, respectively. These strains were routinely grown in rich medium (YPD) (37Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar) containing 1% yeast extract, 2% peptone, and 2% glucose or in minimal medium (SD) (37Sherman F. Methods Enzymol. 1991; 194: 3-21Crossref PubMed Scopus (2545) Google Scholar) containing 0.67% yeast nitrogen base and 2% glucose supplemented with the appropriate amino acids. Escherichia coli JM109 (38Messing J. Vieira J. Gene (Amst.). 1982; 19: 269-276Crossref PubMed Scopus (1718) Google Scholar) and BL21 were used to propagate the plasmids and to express various proteins. E. coli cells were cultured in L broth (39Kanazawa H. Miki T. Tamura F. Yura T. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1126-1130Crossref PubMed Scopus (73) Google Scholar) at 37 °C with an appropriate antibiotic for the selection of transformants, as described previously (39Kanazawa H. Miki T. Tamura F. Yura T. Futai M. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 1126-1130Crossref PubMed Scopus (73) Google Scholar). Solid media contained 1.5% agar. Screening for a Multicopy Suppressor of Growth Retardation in Highly Saline Conditions—G19 cells transformed with a plasmid that expresses the C1+C2 domain peptide (pKT10-NHA1(C1+C2)) were further transfected by a multicopy genomic library (kindly provided by Drs. Yoh Wada and Masamitsu Futai, Osaka University) derived from YEp13 (40Broach J.R. Strathern J.N. Hicks J.B. Gene (Amst.). 1979; 8: 121-133Crossref PubMed Scopus (672) Google Scholar). The transformed G19 cells were then plated on pH 5.5 SD plates that had been supplemented with 0.4 m NaCl and lacked Leu. After 4 days of incubation at 37 °C, leu+ cells that appeared on the selecting plate were isolated as candidate cells bearing a suppressor of high salinity-induced growth retardation. The restriction sites in the plasmids recovered from the candidate yeast cells were determined. The recovered plasmids were then reintroduced into the same genetic background. Plasmids—Plasmids 4-3-1 and 4-3-2 were constructed from the isolated library clone 4-3 as follows. A ClaI-SacI 6.2-kb fragment was isolated from 4-3 by digestion with ClaI and SacI, and this was ligated into pRS424 (41Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), a multicopy yeast expression vector, to produce 4-3-1. 4-3-2 was constructed by the self-ligation of the 4-3-1 fragment resulting from digestion with the BglII restriction enzyme. The plasmids pKT10-NHA1(C1+C2)-GFP, pKT10-NHA1(C1)-GFP, and pKT10-NHA1(C2)-GFP contain DNA fragments encoding GFP-tagged parts of the hydrophilic domain (residues 434-523, 434-449, and 453-523, respectively) of Nha1p. pKT10-COS3Loop-FLAG and pKT10-Cos3pTail-FLAG contain DNA fragments encoding FLAG-tagged parts of the hydrophilic domain (residues 94-224 and 297-379, respectively) of Cos3p. All of these GFP- or FLAG-tagged plasmids were constructed by amplification by PCR with primer oligonucleotides (Table I, lines 3-10) and cosmid 2A16 (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar) plasmid 4-3 DNA as the template, followed by digestion with KpnI and SphI. The DNAs were then joined together with the SphI-SalI FLAG adaptor (Table I, lines 1 and 2) at the unique KpnI and SalI sites of plasmid pKT10 (42Tanaka K. Nakafuku M. Tamanoi F. Kaziro Y. Matsumoto K. Toh-e A. Mol. Cell. Biol. 1990; 10: 4303-4313Crossref PubMed Scopus (241) Google Scholar), a multicopy yeast expression vector with a glyceraldehyde-3-phosphate dehydrogenase promoter and terminator.Table IOligonucleotide DNAs used in this studyOligonucleotide DNAsSequence (5′–3′)Description1. FLAG-FwaFw and Rv indicate primers for forward and reverse primers in PCR, respectivelyCAGGACTATAAAGACGACGACGACAAATGAGFLAG adaptor (SphI-SalI)2. FLAG-RvaFw and Rv indicate primers for forward and reverse primers in PCR, respectivelyTCGACTCATTTGTCGTCGTCGTCTTTATAGTCCTGCATGFLAG adaptor (SphI-SalI)3. NHA1C1-FwGCGGTACCTCCTCAGTTGCAATCATAACbRestriction site of KpnI is underlinedNHA1(C1-C2)/NHA1(C1)4. NHA1C2-RvACATGCATGCCAGTATCCATACGATGCAATGcRestriction site of SphI underlinedNHA1(C1-C2)/NHA1(C2)5. NHA1C1-RvACATGCATGCCTGTAGTGAATGTTTTGGTbRestriction site of KpnI is underlinedNHA1(C1)6. NHA1C2-FwGCGGTACCTCATGGATGCAAAGGTTGCcRestriction site of SphI underlinedNHA1(C2)7. COS3CL-FwGCGGTACCTCTCGTAAGCGTTCCTTATbRestriction site of KpnI is underlinedCOS3-Loop8. COS3CL-RvACATGCATGCCAAATCGGTAAGCTTCCTTGcRestriction site of SphI underlinedCOS3-Loop9. COS3CT-FwGCGGTACCAATGAGCAAGAAAGTGGTGbRestriction site of KpnI is underlinedCOS3-Tail10. COS3CT-RvACATGCATGCCCACTAAGAGCACCTCACTcRestriction site of SphI underlinedCOS3-Tail11. COS3-SphI-FwCCATCCAGCATGCAATACAGTGACcRestriction site of SphI underlined5′-UTR + COS3 ORF12. COS3-XhoI-RvCCG CTCGAGCACTAAGAGCACCTCACTGCdRestriction site of XhoI is underlined5′-UTR + COS3 ORF13. COS3-XhoI-FwCCGCTCGAGTAGAGATAAATACAACTTTTTCdRestriction site of XhoI is underlined3′-UTR14. COS3-XbaI-RvGACCCTCTAGAGTGAACAAATTTTTGGeRestriction site of XbaI is underlined3′-UTR15. GFP-XhoI-FwCCGCTCGAGGTGAGCAAGGCCGAGGAGCcRestriction site of SphI underlinedGFP (XhoI-XhoI)16. GFP-XhoI-RvCCGCTCGAGCTTGTACAGCTCGTCCATGCdRestriction site of XhoI is underlinedGFP (XhoI-XhoI)17. COS3ORF-FwCGGAATTCATGAAAGAGAATGAACfRestriction site of EcoRI is underlinedCOS3 ORF18. COS3ORF-RvACATGCATGCCCACTAAGAGCACCTCACTGcRestriction site of SphI underlinedCOS3 ORF19. GFP-SphI-FwACATGCATGCCCATGCTGAGCAAGGGCGAGGcRestriction site of SphI underlinedGFP (SphI-SalI)20. GFP-SalI-RvCCGTCGACTTACTTGTACAGCTCGTCCgRestriction site of SalI is underlinedGFP (SphI-SalI)21. SK6TACTGGTACCATGGCTATCTGGGAGCAAbRestriction site of KpnI is underlinedNHA1/NHA1-His22. SK31TCTGCATGCCCTTATTGAGACCAcRestriction site of SphI underlinedNHA1-His23. SK40TATTGTCGACTTACTTATTGAGACCAAGCgRestriction site of SalI is underlinedNHA124. His6-FwCATCATCATCATCATCATTAATGHis6 adaptor (SphI-Sal)25. His6-RvTCGACATTAATGATGATGATGATGATGCATGHis6 adaptor (SphI-Sal)a Fw and Rv indicate primers for forward and reverse primers in PCR, respectivelyb Restriction site of KpnI is underlinedc Restriction site of SphI underlinedd Restriction site of XhoI is underlinede Restriction site of XbaI is underlinedf Restriction site of EcoRI is underlinedg Restriction site of SalI is underlined Open table in a new tab To express the Cos3p-GFP fusion protein (43Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Science. 1994; 263: 802-805Crossref PubMed Scopus (5539) Google Scholar), pRS316-COS3-GFP was constructed as follows. The ClaI-SacI 6.3-kb fragment of plasmid 4-3-1, which encodes the COS3 open reading frame (ORF), was cloned into the CEN plasmid pRS316 (41Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The plasmid thus created was named pRS316-COS3. Two DNA fragments containing the 0.5-kb 3′-UTR of COS3 or the COS3 ORF plus the 1.7-kb 5′-UTR were amplified with the appropriate primers shown in Table I (lines 11-14) and then digested with SphI and XhoI or XhoI and XbaI, respectively. The 2.8-kb SphI-XhoI and the 0.5-kb XhoI-XbaI fragments thus created were inserted together into the SphI and XbaI restriction sites of the pUC18 vector to generate the plasmid pUC18-COS3. The 0.7-kb DNA fragment encoding GFP that was amplified with primers containing an XhoI restriction site sequence (Table I, lines 15 and 16) was inserted into pUC18-COS3 after digestion with XhoI. The 4.2-kb SphI-XbaI fragment encoding the COS3-GFP fusion that was derived from the GFP-bearing pUC18-COS3 plasmid was used to replace a 3.5-kb XbaI-SphI fragment from pRS316-COS3 to construct pRS316-COS3-GFP. pKT10-COS3-GFP was created by amplification of the entire COS3 ORF with appropriate primers (Table I, lines 17 and 18), followed by digestion with EcoRI and SphI and insertion into the EcoRI and SalI restriction sites of pKT10 together with the 0.7-kb GFP fragment that had been amplified from pEGFP-N3 (Clontech) with primers containing SphI and SalI sites (Table I, lines 19 and 20). Plasmids p520-NHA1 and the His6-tagged p520-NHA1-His were constructed to express the full-length NHA1 in yeast under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter in p520 (a kind gift from Dr. Yoshinobu Kaneko, Osaka University), which is a multicopy yeast expression vector that is trp+. DNA fragments encoding the full-length NHA1 protein were amplified with suitable primers (Table I, lines 21-23) using the cosmid 2A16 (28Kamauchi S. Mitsui K. Ujike S. Haga M. Nakamura N. Inoue H. Sakajo S. Ueda M. Tanaka A. Kanazawa H. J. Biochem. (Tokyo). 2002; 131: 821-831Crossref PubMed Scopus (33) Google Scholar) as the template and then digested with KpnI and SalI or KpnI and SphI. To construct p520-NHA1-His, the KpnI-SphI fragment was inserted together with the SphI-SalI His6 tag adaptor (Table I, lines 24 and 25) between the KpnI and SalI restriction sites of the p520 vector. To construct p520-NHA1, the KpnI-SalI fragment was inserted between the KpnI and SalI restriction sites of the p520 vector. Disruption of the COS3 Locus in the G19 Strain—The 1.8-kb SphI-BglII and the 2.2-kb HincII-SacI fragments that correspond to the upstream and downstream halves of the COS3 ORF, respectively, and that were derived from the 4-3 plasmid, were inserted between the unique SphI and BglII sites and SmaI and SacI sites, respectively, of the pJJ281 plasmid (44Jones J.S. Prakash L. Yeast. 1990; 6: 363-366Crossref PubMed Scopus (327) Google Scholar) that contains the TRP1 marker to create the targeting sequence of COS3 disruption. The 4.9-kb SphI-SacI targeting fragment thus created was introduced into the G19 strain, and TRP+ clones were selected by growth on SD plates lacking tryptophan. The replacement was verified by PCR analysis. Detection of Intracellular Localization of Cos3p-GFP by Fluorescence Microscopy—SK5 or G19 cells expressing Cos3p-GFP fusion proteins were grown in SD medium at 30 °C to the logarithmic growth phase and then examined by fluorescence microscopy (Olympus BX51) with appropriate filter sets (narrow band 1B excitation cube). Fractionation of the Whole Cell Proteins and Immunoblot Analysis— Cells were grown to the logarithmic growth phase, collected by centrifugation, resuspended in lysis buffer (20 mm Tris-HCl, pH 7.6, 150 mm NaCl, 1 mm PMSF, protease inhibitor mixture (Roche Diagnostics)), and then disrupted with glass beads at 4 °C. After removing unbroken cells and debris, the cleared cell extracts were centrifuged at 100,000 × g for 1 h to generate the pellet (P100) and supernatant (S100) fractions containing the membrane and soluble proteins, respectively. For the extraction experiments, the P100 fraction was incubated for 30 min on ice with 1 m NaCl or 1% Triton X-100 and then spun at 100,000 × g to give the supernatant and the pellet fractions. Each fraction was resuspended in an equal volume of SDS sample buffer. The fractions were subjected to SDS-PAGE and transferred to a membrane filter (GVHP, Millipore) (45Miki J. Fujiwara K. Tsuda M. Tsuchiya T. Kanazawa H. J. Biol. Chem. 1990; 265: 21567-21572Abstract Full Text PDF PubMed Google Scholar). The membranes were treated with anti-GFP serum (Molecular Probes) or anti-Nha1p polyclonal antibody prepared as described below. Immunoreactive bands were visualized by means of the enhanced chemiluminescence method (Amersham Biosciences) (46Gillespie P.G. Hudspeth A.J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2563-2567Crossref PubMed Scopus (102) Google Scholar), as described previously (45Miki J. Fujiwara K. Tsuda M. Tsuchiya T. Kanazawa H. J. Biol. Chem. 1990; 265: 21567-21572Abstract Full Text PDF PubMed Google Scholar). Fractionation of Membranes by Sucrose Density Gradient Centrifugation—Fractionation of yeast cell extract by centrifugation through a sucrose step gradient was performed according to the procedure published previously (47Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar). Briefly, 300 A600 units of mid-log phase cells were harvested by centrifugation, washed twice with water, and resuspended in 5 ml of 100 mm Tris, pH 9.4, and 10 mm dithiothreitol. After 10 min of incubation at 30 °C, the cells were harvested by centrifugation and resuspended in 10 ml of spheroplast medium (1 m sorbitol in SD medium). Zymolyase 100T (Seikagaku Corp., Tokyo," @default.
• W2073343350 created "2016-06-24" @default.
• W2073343350 creator A5034547906 @default.
• W2073343350 creator A5034949085 @default.
• W2073343350 creator A5041641898 @default.
• W2073343350 creator A5054306304 @default.
• W2073343350 creator A5059482063 @default.
• W2073343350 creator A5091739635 @default.
• W2073343350 date "2004-03-01" @default.
• W2073343350 modified "2023-09-29" @default.
• W2073343350 title "A Novel Membrane Protein Capable of Binding the Na+/H+ Antiporter (Nha1p) Enhances the Salinity-resistant Cell Growth of Saccharomyces cerevisiae" @default.
• W2073343350 cites W1508132604 @default.
• W2073343350 cites W1553426129 @default.
• W2073343350 cites W1554578780 @default.
• W2073343350 cites W1577463474 @default.
• W2073343350 cites W159884039 @default.
• W2073343350 cites W1603658332 @default.
• W2073343350 cites W16789104 @default.
• W2073343350 cites W1747506719 @default.
• W2073343350 cites W1775749144 @default.
• W2073343350 cites W1951759266 @default.
• W2073343350 cites W1965057959 @default.
• W2073343350 cites W1968889088 @default.
• W2073343350 cites W1970705112 @default.
• W2073343350 cites W1971584001 @default.
• W2073343350 cites W1973530388 @default.
• W2073343350 cites W1975304761 @default.
• W2073343350 cites W1976965279 @default.
• W2073343350 cites W1979486759 @default.
• W2073343350 cites W1982621562 @default.
• W2073343350 cites W1989641787 @default.
• W2073343350 cites W1990829962 @default.
• W2073343350 cites W1997200285 @default.
• W2073343350 cites W1998911006 @default.
• W2073343350 cites W2000298437 @default.
• W2073343350 cites W2001815208 @default.
• W2073343350 cites W2007651870 @default.
• W2073343350 cites W2009776346 @default.
• W2073343350 cites W2015564029 @default.
• W2073343350 cites W2023415094 @default.
• W2073343350 cites W2024436009 @default.
• W2073343350 cites W2025100148 @default.
• W2073343350 cites W2031094547 @default.
• W2073343350 cites W2032579724 @default.
• W2073343350 cites W2033279834 @default.
• W2073343350 cites W2040298705 @default.
• W2073343350 cites W2041508066 @default.
• W2073343350 cites W2043282701 @default.
• W2073343350 cites W2046239497 @default.
• W2073343350 cites W2050611086 @default.
• W2073343350 cites W2051419932 @default.
• W2073343350 cites W2053004133 @default.
• W2073343350 cites W2054468433 @default.
• W2073343350 cites W2059699772 @default.
• W2073343350 cites W2065723356 @default.
• W2073343350 cites W2068037334 @default.
• W2073343350 cites W2069939390 @default.
• W2073343350 cites W2081230957 @default.
• W2073343350 cites W2084944074 @default.
• W2073343350 cites W2092211446 @default.
• W2073343350 cites W2097039879 @default.
• W2073343350 cites W2101175098 @default.
• W2073343350 cites W2143337684 @default.
• W2073343350 cites W2143371694 @default.
• W2073343350 cites W2151639549 @default.
• W2073343350 cites W2156442455 @default.
• W2073343350 cites W2169333620 @default.
• W2073343350 cites W4240065971 @default.
• W2073343350 cites W98250993 @default.
• W2073343350 doi "https://doi.org/10.1074/jbc.m310806200" @default.
• W2073343350 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14718542" @default.
• W2073343350 hasPublicationYear "2004" @default.
• W2073343350 type Work @default.
• W2073343350 sameAs 2073343350 @default.
• W2073343350 citedByCount "19" @default.
• W2073343350 countsByYear W20733433502014 @default.
• W2073343350 countsByYear W20733433502016 @default.
• W2073343350 countsByYear W20733433502017 @default.
• W2073343350 crossrefType "journal-article" @default.
• W2073343350 hasAuthorship W2073343350A5034547906 @default.
• W2073343350 hasAuthorship W2073343350A5034949085 @default.
• W2073343350 hasAuthorship W2073343350A5041641898 @default.
• W2073343350 hasAuthorship W2073343350A5054306304 @default.
• W2073343350 hasAuthorship W2073343350A5059482063 @default.
• W2073343350 hasAuthorship W2073343350A5091739635 @default.
• W2073343350 hasBestOaLocation W20733433501 @default.
• W2073343350 hasConcept C12554922 @default.
• W2073343350 hasConcept C129513315 @default.
• W2073343350 hasConcept C185592680 @default.
• W2073343350 hasConcept C18903297 @default.
• W2073343350 hasConcept C2777576037 @default.
• W2073343350 hasConcept C2779222958 @default.
• W2073343350 hasConcept C41625074 @default.
• W2073343350 hasConcept C55493867 @default.
• W2073343350 hasConcept C57181701 @default.
• W2073343350 hasConcept C62112901 @default.
• W2073343350 hasConcept C86803240 @default.
• W2073343350 hasConcept C95444343 @default.

• next