Evidence for a Salt Bridge between Transmembrane Segments 5 and 6 of the Yeast Plasma-membrane H+-ATPase
1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês
10.1074/jbc.273.51.34328
ISSN1083-351X
AutoresSoma Sen Gupta, Natalie DeWitt, Kenneth E. Allen, Carolyn W. Slayman,
Tópico(s)Enzyme Structure and Function
ResumoThe plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998)J. Biol. Chem. 273, 17411–17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide. The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998)J. Biol. Chem. 273, 17411–17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide. 2-(N-morpholino)ethanesulfonic acid endoplasmic reticulum. The past few years have seen steady progress toward understanding the structure and function of P2-type cation-transporting ATPases, including the plasma-membrane H+-ATPases ofSaccharomyces cerevisiae and Neurospora crassaand the Na+,K+-, H+,K+-, and Ca2+-ATPases of mammalian cells (1Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar). In particular, it now seems clear that the 100-kDa ATPase polypeptides are embedded in the lipid bilayer by 10 transmembrane segments, four at the amino-terminal end and six at the carboxyl-terminal end of the molecule. Evidence for this view came initially from a combination of indirect approaches including hydropathy analysis, gene fusions (2Smith D.L. Tao T. Maguire M.E. J. Biol. Chem. 1993; 268: 22469-22479Abstract Full Text PDF PubMed Google Scholar), tryptic digestion (3Besancon M. Shin J.M. Mercier F. Munson K. Miller M. Hersey S. Sachs G. Biochemistry. 1993; 32: 2345-2355Crossref PubMed Scopus (128) Google Scholar), andin vitro translation of hydrophobic segments (4Bamberg K. Sachs G. J. Biol. Chem. 1994; 269: 16909-16919Abstract Full Text PDF PubMed Google Scholar, 5Bayle D. Weeks D. Sachs G. J. Biol. Chem. 1995; 270: 25678-25684Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 6Lin J. Addison R. J. Biol. Chem. 1995; 270: 6942-6948Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 7Shin J.M. Besancon M. Bamberg K. Sachs G. Ann. N. Y. Acad. Sci. 1997; 834: 65-76Crossref PubMed Scopus (33) Google Scholar). More recently, cryo-electron microscopy of two-dimensional crystals at 8-Å resolution has provided direct images of 10 membrane-spanning α-helices in the plasma-membrane H+-ATPase of N. crassa (8Auer M. Scarborough G.A. Kuhlbrandt W. Nature. 1998; 392: 840-843Crossref PubMed Scopus (184) Google Scholar) and the sarcoplasmic reticulum Ca2+-ATPase (9Zhang P. Toyoshima C. Yonekura K. Green N.M. Stokes D.L. Nature. 1998; 392: 835-839Crossref PubMed Scopus (265) Google Scholar). Among the various membrane segments, there is particular interest in M5 and M6, which are generally connected by a hydrophilic loop of only five or six amino acid residues and thus are likely to form a hairpin in the membrane. In the mammalian P2-ATPases, mutagenesis studies have identified amino acid residues within M5 and M6 that appear to play a direct role in cation translocation, including Glu-771 (M5) and Asn-796, Thr-799, and Asp-800 (M6) of the sarcoplasmic reticulum Ca2+-ATPase (10Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (468) Google Scholar, 11Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 6262-6267Abstract Full Text PDF PubMed Google Scholar) and Glu-779 (M5) and Asp-804 and Asp-808 (M6) of the Na+,K+-ATPase (12Van Huysse J.W. Kuntzweiler T.A. Lingrel J.B. FEBS Lett. 1996; 389: 179-185Crossref PubMed Scopus (15) Google Scholar, 13Arguello J.M. Peluffo R.D. Feng J. Lingrel J.B. Berlin J.R. J. Biol. Chem. 1996; 271: 24610-24616Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 14Nielsen J.M. Pedersen P.A. Karlish S.J.D. Jorgensen P.L. Biochem. 1998; 37: 1961-1968Crossref PubMed Scopus (77) Google Scholar). It was therefore intriguing when Lutsenko et al. (15Lutsenko S. Anderko R. Kaplan J.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7936-7940Crossref PubMed Scopus (96) Google Scholar) detected a change in the state of the M5-M6 hairpin, depending upon the presence or absence of the transported cation. This finding was based on previous work by Shainskaya and Karlish (16Shainskaya A. Karlish S.J.D. J. Biol. Chem. 1994; 269: 10780-10789Abstract Full Text PDF PubMed Google Scholar), who demonstrated that most of the extramembranous regions of the Na+,K+-ATPase could be removed by proteolytic digestion in the presence of K+ or Rb+, leaving a preparation still capable of occluding K+. Subsequently, Lutsenko et al. (15Lutsenko S. Anderko R. Kaplan J.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7936-7940Crossref PubMed Scopus (96) Google Scholar) showed that proteolytic digestion in the absence of K+ was accompanied by the disappearance of M5 and M6 from the membrane, and went on to speculate that the hairpin may move in and out of the bilayer as a normal part of the ATPase reaction cycle. Based on recent evidence from site-directed mutagenesis, M5 and M6 are also structurally and functionally important in the Pma1 H+-ATPase of S. cerevisiae. Several residues in both membrane segments are required for normal biogenesis of the ATPase, and others play a role in the conformational changes that accompany the reaction cycle (17Dutra M.B. Ambesi A. Slayman C.W. J. Biol. Chem. 1998; 273: 17411-17417Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 18Padmanabha K.P. Pardo J.P. Petrov V.V. Sen Gupta S. Slayman C.W. Folia Microbiol. 1997; 42: 245-249Crossref PubMed Scopus (4) Google Scholar). Asp-730, located near the middle of M6, appears to be especially critical, since replacement by Asn or Val leads to a complete failure of newly synthesized ATPase to reach the secretory vesicles responsible for delivering it to the plasma membrane (18Padmanabha K.P. Pardo J.P. Petrov V.V. Sen Gupta S. Slayman C.W. Folia Microbiol. 1997; 42: 245-249Crossref PubMed Scopus (4) Google Scholar). To investigate this finding in greater detail, we have examined the effect of the D730N and D730V mutations on the folding and subcellular localization of the ATPase, and have gone on to search for compensatory mutations in M5. The results point to the presence of a salt bridge between Arg-695 in M5 and Asp-730 in M6, the first clearcut example of such an interaction in any of the P-type ATPases. S. cerevisiae strains SY4 (MATa; ura3-52; leu2-3, 112;his 4-619; sec 6-4ts;GAL2;pma1::YIpGAL-PMA1::URA3) and NY605 (MAT a; ura3-52; leu2-3, 112;GAL2) were used in these studies. SY4 has been described in detail by Nakamoto et al. (19Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar), and the sec6-4ts mutation by Schekman and Novick (20Schekman R. Novick P.J Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism & Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 361-398Google Scholar). A 519-base pairBglII-SalI fragment of the PMA1 gene (21Serrano R. Kielland-Brandt M.C. Fink G.R. Nature. 1986; 319: 689-693Crossref PubMed Scopus (570) Google Scholar), subcloned into a modified Bluescript vector (Stratagene, La Jolla, CA), was used for mutagenesis. Mutations were introduced with the ChameleonTM Double Stranded Site-directed Mutagenesis Kit (Stratagene, La Jolla, CA) and verified by automated DNA sequencing. After the BglII-SalI fragment was subcloned into plasmid pPMA1.2 (19Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar), the 3.77-kilobaseHindIII-SacI fragment containing the entire ATPase gene was moved into the expression vector YCp2HSE to bring the gene under heat-shock control. Plasmids were transformed into SY4 cells by the method of Ito et al. (22Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). SY4 cells were grown to mid-exponential phase (A 600 ∼ 1) at 23 °C in minimal medium supplemented with 2% (w/v) galactose. The cells were then shifted to minimal medium containing 2% (w/v) glucose for 3 h, and subsequently transferred to 39 °C for an additional 2 h. The cells were harvested, washed, and lysed, and secretory vesicles were isolated by sucrose density gradient centrifugation (23Ambesi A. Allen K.E. Slayman C.W. Anal. Biochem. 1997; 251: 127-129Crossref PubMed Scopus (26) Google Scholar). To assay the level of Pma1 protein expressed in secretory vesicles, isolated vesicles (5–20 μg) were subjected to SDS-gel electrophoresis followed by immunoblotting (19Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar) with polyclonal antiserum raised against the closely related Pma1 ATPase of N. crassa (24Hager K.M. Mandala S.M. Davenport J.W. Speicher D.W. Benz Jr., E.J. Slayman C.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7693-7697Crossref PubMed Scopus (112) Google Scholar); control experiments with partially proteolyzed preparations have shown that the antiserum recognizes epitopes scattered throughout the 100-kDa polypeptide. 1K. H. Hager, unpublished data. Expression levels were calculated relative to a wild-type control using a PhosphorImager programmed with ImageQuant Software Version 3.3 (Molecular Dynamics, Sunnyvale, CA) (25Petrov V.V. Slayman C.W. J. Biol. Chem. 1995; 270: 28535-28540Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To determine the total amount of ATPase synthesized by the cell, SY4 cells were shifted from galactose medium at 23 °C to glucose medium at 39 °C as described above, and labeled for varying lengths of time (15, 30, 60, and 90 min) with [35S]methionine (26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Total membranes were isolated and immunoprecipitated with anti-Pma1 antibody (24Hager K.M. Mandala S.M. Davenport J.W. Speicher D.W. Benz Jr., E.J. Slayman C.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 7693-7697Crossref PubMed Scopus (112) Google Scholar), and after SDS-polyacrylamide gel electrophoresis, the gels were fixed, incubated in 1 m sodium salicylate (30 min at 23 °C), dried, and exposed to Hyperfilm-MP (Amersham, Arlington Heights, IL). To determine the subcellular localization of mutant ATPases, NY605 cells were transformed with a centromeric plasmid carrying the PMA1 gene that had been tagged with c-Myc epitope in a position corresponding to the NH2 terminus of the ATPase and placed under control of theGAL1 promoter (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Cells were grown in 4% (w/v) raffinose, transferred to 2% (w/v) galactose and 0.5% (w/v) raffinose, and after 4 h, immunofluorescence microscopy was carried out by the method of Redding et al. (28Redding K. Holcomb C. Fuller R.S. J. Cell Biol. 1991; 113: 527-538Crossref PubMed Scopus (200) Google Scholar) as modified by DeWitt et al. (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Two different primary antibodies were used: Myc monoclonal 9E10.2 from ascites fluid (provided by H. Dohlman), diluted 1:100; and Kar2 polyclonal antibody (provided by M. Rose), diluted 1:5000 (29Rose M.D. Misra L.M. Vogel J.P. Cell. 1989; 57: 1211-1222Abstract Full Text PDF PubMed Scopus (519) Google Scholar). Cells were observed with a Bio-Rad MRC-600 Scanning Confocal Microscope (Melville, NY) using dual channel filters for simultaneous viewing of Texas Red and fluorescein isothiocyanate fluorochromes and a slit width set to provide an optical slice less than 1 μm. Images were collected and processed as described previously (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). 35S-Labeled total membranes were diluted to 1 mg/ml in 1 mm EGTA-Tris, pH 7.5 (without protease inhibitors). Membranes (5 μg) were added to 10 μl of 20 mm Tris, 5 mm MgCl2, pH 7.0, and after a 2-min preincubation at 30 °C, tosyl-phenylalanyl chloromethyl ketone-trypsin (Worthington Biochemical Corp., Freehold, NJ) was added to give the desired trypsin:protein ratio in a final volume of 20 μl. After incubation for 0 to 10 min at 30 °C, the reaction was stopped by the addition of 20 μl of 2 mmdiisopropyl fluorophosphate. Reaction products were analyzed by immunoprecipitation (25Petrov V.V. Slayman C.W. J. Biol. Chem. 1995; 270: 28535-28540Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), followed by SDS-polyacrylamide gel electrophoresis and fluorography. NY605 cells transformed with GAL-pma1 plasmids were grown at 30 °C in synthetic medium lacking uracil and containing 2% (w/v) glucose (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The cells were then diluted to 1 A 600/ml in sterile deionized water and plated in 5-μl droplets onto synthetic medium, lacking uracil and containing either 2% (w/v) glucose (chromosomal PMA1 gene expressed) or 2% (w/v) galactose (both chromosomal and plasmid-borne genes expressed). The plates were incubated at 30 °C for 48 h and photographed. ATP hydrolysis was assayed at 30 °C in buffer containing 50 mmMES,2 pH 5.7, 5 mm NaN3, 5 mm Na2ATP, 10 mm MgCl2, and an ATP regenerating system composed of 5 mm phosphoenolpyruvate and 50 μg/ml pyruvate kinase, as described by Ambesi et al. (30Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The specific activity was measured as the difference between hydrolysis in the presence and absence of 100 μm orthovanadate. For the determination of K m values, the Na2ATP concentration was varied from 0.15 to 5 mm and the actual concentration of MgATP was calculated by the method of Fabiato and Fabiato (31Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar). For the determination of K i values, the concentration of orthovanadate was varied from 0 to 100 μm. The pH optimum was determined by varying the pH from 5.0 to 8.0 with Tris base. Proton transport was assayed as the initial rate of acridine orange fluorescence quenching in 0.6 m sorbitol, 0.1 m KCl, 20 mm HEPES/KOH, pH 6.7, Na2ATP (0.3–3.0 mm), and MgCl2 (5 mm excess over ATP concentration), as described by Ambesi et al. (30Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Parallel measurements were made of ATP hydrolysis under the same conditions. Protein concentrations were measured by the method of Lowry et al. (32Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar), as modified by Ambesi et al. (30Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The starting point for this study was the finding that certain mutations of Asp-730 cause a virtual arrest of ATPase biogenesis. In the experiment of Fig.1 A, D730E, D730N, and D730V were expressed in the secretory vesicle system of Nakamoto et al. (19Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar), which uses a temperature-sensitive allele of thesec6 gene to block the last step in the delivery of newly synthesized proteins to the plasma membrane. Shifting the cells from 23 to 39 °C led to the accumulation of secretory vesicles, which were isolated (23Ambesi A. Allen K.E. Slayman C.W. Anal. Biochem. 1997; 251: 127-129Crossref PubMed Scopus (26) Google Scholar) and assayed by immunoblotting with anti-Pma1 polyclonal antibody. As shown in Fig. 1 A, an appreciable amount of ATPase carrying the conservative D730E mutation reached the vesicles, but neither D730N nor D730V could be detected there by the antibody. To verify that the D730N and D730V polypeptides were synthesized and to explore their stability, the cells were incubated with [35S]methionine for varying lengths of time (15, 30, 60, and 90 min) at 39 °C. A total membrane fraction was then isolated, and the ATPase was immunoprecipitated with polyclonal anti-Pma1 antiserum. Both the wild-type (Fig. 1 B) and D730E polypeptides (Fig. 1 C) appeared as prominently labeled 100-kDa bands by 15 min. The wild-type polypeptide remained stable over the entire labeling time course and D730E was nearly as stable, with only traces of a lower molecular weight band appearing over time. By contrast, D730N (Fig. 1 D) was readily visible at 15 min but decreased markedly at 60 and 90 min, and D730V (Fig. 1 E) became labeled more slowly, reaching a maximum at 60 min and tapering off again at 90 min. Thus, the D730N and D730V polypeptides were clearly made, but they appeared to be less stable than the wild-type and D730E ATPases. To pinpoint the subcellular compartment in which the mutant ATPases were arrested, the D730N and D730V genes were tagged with c-Myc epitope, placed under control of the GAL1promoter on a centromeric plasmid, and transformed into wild-type strain NY605, which lacks the sec6ts mutation and should allow newly synthesized ATPase to move all the way to the plasma membrane. The cells were grown on raffinose, shifted to galactose to induce expression of the plasmid-borne gene, and examined by immunofluorescence confocal microscopy. In the experiment of Fig.2, double labeling was carried out with c-Myc antibody to detect the epitope-tagged ATPase and Kar2 antibody to serve as a marker for the endoplasmic reticulum (ER) (29Rose M.D. Misra L.M. Vogel J.P. Cell. 1989; 57: 1211-1222Abstract Full Text PDF PubMed Scopus (519) Google Scholar). As expected, control cells expressing wild-type ATPase showed c-Myc labeling (red) at the cell surface, while Kar2 labeling (green) appeared in structures surrounding the nucleus and at the cell periphery, typical of the yeast ER (33Preuss D. Mulholland J. Kaiser C.A. Orlean P. Albright C. Rose M.D. Robbins P.W. Botstein D. Yeast. 1991; 7: 891-911Crossref PubMed Scopus (146) Google Scholar). The c-Myc and Kar2 patterns were largely distinct from one another as illustrated by the merged image in the top righthand panel, except for a slight overlap (yellow) where the peripheral ER was in close juxtaposition to the plasma membrane. By contrast, in cells expressing the D730N and D730V ATPases (middle and bottom panels), the c-Myc and Kar2 patterns could be superimposed with one another in prominent ER-like structures. In a recent study of Ala substitutions throughout the phosphorylation domain of the Pma1 ATPase, a close correlation was observed between protein misfolding (as assayed by limited trypsinolysis) and retention in the ER (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). To examine the folding states of the D730N and D730V ATPases,35S-labeled total membranes were incubated at a trypsin:protein ratio of 1:20 for varying amounts of time and then immunoprecipitated with anti-Pma1 antibody (Fig.3). As expected, the 100-kDa wild-type polypeptide was relatively little affected by trypsin under these conditions, most of it remaining intact after 20 min of digestion (WT). By contrast, D730N was largely degraded (D730N) and D730V was barely detectable (D730V) after only 0.5 min of digestion. Previous work has shown that the wild-type Pma1 ATPase can be digested by higher concentrations of trypsin but that it can be protected by ligands such as MgADP, MgATP, and vanadate, producing distinctive patterns of fragments that correspond to the E1 and E2 conformational states of the enzyme (e.g.Refs. 30Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and 34Perlin D.S. Brown C.L. J. Biol. Chem. 1987; 262: 6788-6794Abstract Full Text PDF PubMed Google Scholar). These patterns are illustrated in Fig.4 A, where the wild-type ATPase was treated with trypsin (1:4) for 10 min. A conspicuous 97-kDa fragment remained in the presence of 20 mm MgADP (ADP) or 20 mm MgATP (ATP), and fragments of 97 and 80 kDa, in the presence of 100 μm vanadate (VO4). To ask whether the D730N and D730V ATPases might be similarly protected (Fig.4, B and C), the trypsin:protein ratio was decreased to 1:50 to give a comparable amount of digestion in the absence of ligands. Under these conditions, there was no sign of protection by vanadate (VO4), but bands of 100, 90, and 80 kDa could be seen in the presence of MgADP (ADP) and MgATP (ATP). Thus, unlike mutants bearing substitutions at the catalytic phosphorylation site of the ATPase (D378N and D378V; Ref. 26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), D730N and D730V appeared able to bind adenine nucleotides, even though they were less well protected than the wild-type enzyme and their overall conformation was clearly abnormal. Consistent with the fact that D730N and D730V could be distinguished from D378N and D378V (26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) in the ligand protection assay, they also appeared less severely affected in a genetic test. This set of experiments made use of NY605 cells that had been transformed with centromeric plasmids carrying each of the four mutant alleles under control of the GAL1 promoter (27DeWitt N.D. Tourinho dos Santos F. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 21744-21751Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar); thus, only the chromosomal wild-type gene was expressed on glucose-containing medium, while the wild-type and mutant genes were co-expressed on galactose-containing medium. When the test was carried out using minimal medium, there was no difference among the four mutants; all behaved in a dominant negative fashion, completely inhibiting growth in the presence of galactose (not shown). But when the test was performed with a rich synthetic medium, D730N and D730V were recessive, allowing growth on galactose (Fig. 5). Two other mutants (R695A and H701A; see below) behaved similarly on synthetic medium, while D378A, D378N, and D378V still acted as dominant negatives. In considering the structural defects that result when Asp-730 is replaced with a neutral amino acid such as Asn or Val, one possible explanation is that the negative charge of the Asp residue may interact with a nearby positive charge to stabilize the ATPase during folding and biogenesis. If so, a logical place to look for the positive charge is in M5, which is connected to M6 by a short loop of only five amino acids. Indeed, M5 contains two such residues, Arg-695 and His-701, which have themselves been shown to be required for proper biogenesis. In a recent study, when Dutra et al. (17Dutra M.B. Ambesi A. Slayman C.W. J. Biol. Chem. 1998; 273: 17411-17417Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) substituted either the Arg or His by Ala, the ATPase became highly sensitive to trypsin and was unable to reach the secretory vesicles. To look for a possible interaction between M5 and M6, double mutants were constructed in which the positive and negative charges were swapped (R695D/D730R; H701D/D730H) or eliminated altogether (R695A/D730A; H701A/D730A). The double mutants and corresponding single mutants were transformed into strain SY4 on a centromeric plasmid under control of the heat-shock promoter, as described in the first section under “Results,” and after the cells were shifted from galactose medium at 23 °C to glucose medium at 39 °C, secretory vesicles were isolated and assayed for ATPase expression and ATP hydrolysis. As summarized in Table I, the double substitutions H701D/D730H and H701A/D730A, like all of the single substitutions of Arg-695, His-701, and Asp-730, gave undetectable amounts of ATPase in the secretory vesicles. Strikingly, however, the R695D/D730R and R695A/D730A ATPases reached the vesicles at 50 and 85% of the level seen in the wild-type control (Table I Fig.6), and after correction for the level of expression, were capable of nearly normal ATP hydrolysis (84 and 135%; Table I).Table IEffect of Arg-695, His-701, and Asp-730 mutations on expression in secretory vesicles, ATP hydrolysis, and H+ transportMutationExpressionaCalculated from yields of mutant and wild-type ATPase protein per mg of total secretory vesicle protein as determined by quantitative immunoblotting. Values are the mean of two determinations (single mutants) or six determinations (double mutants), with an average standard error of 15%.ATP hydrolysisbVanadate-sensitive ATP hydrolysis was measured as described under “Experimental Procedures.” Values are the mean of two to six determinations with an average standard error of 10%. One unit is defined as 1 μmol of P1/min.Proton transportcThe initial rate of acridine orange fluorescence quenching (H+-transport) was determined as described under “Experimental Procedures.” A unit is defined as 1% of total fluorescence quenching/min. Values represent the mean of at least three determinations with a standard error less than 20%.UncorrectedCorrected%UncorrectedCorrected%%units/mgunits/mgWild-type1005.395.39100850850100Vector10.08—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—eProton transport was not detectable.—eProton transport was not detectable.—eProton transport was not detectable.Single mutantsR695AfData from Dutra et al. (17).140.09—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—eProton transport was not detectable.—eProton transport was not detectable.—eProton transport was not detectable.R695D80.20—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—eProton transport was not detectable.—eProton transport was not detectable.—eProton transport was not detectable.H701AfData from Dutra et al. (17).150.12—dCorrections were not made for mutants with measured ATP hydrolysis below 10% of the wild-type value.—dCorrections were not made for mutants with
Referência(s)