Packing Interactions between Transmembrane Helices Alter Ion Selectivity of the Yeast Golgi Ca2+/Mn2+-ATPase PMR1
2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês
10.1074/jbc.m306166200
ISSN1083-351X
AutoresDebjani Mandal, Samuel Rulli, Rajini Rao,
Tópico(s)Fungal and yeast genetics research
ResumoPMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions. PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions. The Secretory Pathway Ca2+-ATPases (SPCAs) 1The abbreviations used are: SPCA, secretory pathway Ca2+-ATPase; SERCA, sarcoendoplasmic reticulum Ca2+-ATPase; PMCA, plasma membrane Ca2+-ATPase; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis; GFP, green fluorescent protein; MES, 4-morpholineethanesulfonic acid. are an emerging family of Mn2+- and Ca2+-transporting P-type ATPases localized to the Golgi apparatus (1Wuytack F. Raeymaekers L. Missiaen L. Eur. J. Physiol. 2003; 446: 148-153Crossref PubMed Scopus (95) Google Scholar, 2Ton V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Members of this family have been identified in the genomes of diverse organisms, including Saccharomyces cerevisiae and other yeasts (PMR1), Caenorhabditis elegans (PMR1), Drosophila melanogaster (Q9VNR2), and Homo sapiens (ATP2C1). S. cerevisiae PMR1 has distinct inhibitor and transport characteristics, which clearly separate it from the well-studied sarcoendoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+-ATPases (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 4Wei Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). Perhaps the most interesting transport characteristic of SPCA pumps is their unique ability to transport Mn2+ ions with high affinity (1Wuytack F. Raeymaekers L. Missiaen L. Eur. J. Physiol. 2003; 446: 148-153Crossref PubMed Scopus (95) Google Scholar, 2Ton V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Haploinsufficiency of the human ATP2C1 gene for SPCA1 results in Hailey Hailey disease, characterized by acantholysis of the epidermis (5Hu Z. Bonifas J.M. Beech J. Bench G. Shigihara T. Ogawa H. Ikeda S. Mauro T. Epstein E.H. Nat. Genet. 2000; 24: 61-65Crossref PubMed Scopus (446) Google Scholar, 6Sudbrak R. Brown J. Dobson-Stone C. Carter S. Ramser J. White J. Healy E. Dissanayake M. Larregue M. Perrussel M. Lehrach H. Munro C.S. Strachan T. Burge S. Hovnanian A. Monaco A.P. Hum. Mol. Genet. 2000; 9: 1131-1140Crossref PubMed Scopus (262) Google Scholar). Members of the P-ATPase superfamily share common structural and mechanistic properties yet have strikingly different ion specificities that underlie their individual physiological roles. In the reaction cycle of the P-ATPases, the E1 conformation of the pump binds ATP and the cation(s) to be transported with high affinity, followed by transfer of the γ-phosphoryl group of ATP to an invariant aspartate to form an aspartyl-phosphate reaction intermediate. A major change in enzyme conformation to the E2 form ensues, with a large reduction in ion binding affinity and reorientation of the binding pocket resulting in vectorial ion transport (7East J.M. Mol. Membr. Biol. 2000; 17: 189-200Crossref PubMed Scopus (85) Google Scholar). The x-ray crystal structure of SERCA in the E1 conformation, at 2.6-Å resolution, provides a structural definition for two high affinity Ca2+ binding sites enclosed by the transmembrane domains M4, M5, M6, and M8 (8). More recently, a second structure of the enzyme locked in the E2 conformation, was resolved by x-ray crystallography to 3.1 Å (9Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). These structures revealed large domain movements within the cytoplasmic regions as well as twisting and elongation of transmembrane helices involved in cation interactions presumably during transition from the E1 to E2 conformations. The repertoire of cations transported by the P-ATPases includes, but is not limited to, H+, K+, Na+, Ca2+, Cu2+, Zn2+, and Mg2+. To understand the molecular basis for ion specificity, we have used phenotypic screening in yeast to identify mutations that alter selectivity for Ca2+ and Mn2+ in PMR1. Growth of the Δpmr1 null strain is hypersensitive to divalent cation-chelating agents such as BAPTA and EGTA, due to depletion of Ca2+ and Mn2+ from the secretory pathway where they serve essential functions in protein processing, sorting, and glycosylation (10Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J.M. Moir D.T. Cell. 1989; 58: 133-145Abstract Full Text PDF PubMed Scopus (436) Google Scholar, 11Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar, 12Dürr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar). BAPTA toxicity can be overcome by the addition of either Ca2+ or Mn2+ to the medium, indicating that these two ions play largely surrogate roles in supporting growth (13Loukin S. Kung C. J. Cell Biol. 1995; 131: 1025-1037Crossref PubMed Scopus (54) Google Scholar). However, because Ca2+ is present in ∼100-fold excess over Mn2+ in standard yeast media and because Mn2+ is efficiently removed at low chelator concentrations, the observed growth inhibition of pmr1 mutants by BAPTA represents a titration of available Ca2+. Thus, we have previously demonstrated an excellent correlation between loss of Ca2+ transport activity in PMR1 mutants and growth sensitivity to BAPTA (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Loss of Mn2+ transport by PMR1 mutants is known to confer hypersensitivity to Mn2+ toxicity (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 15Lapinskas P.J. Cunningham K.W. Liu X. Fink G.R. Culotta V. Mol. Cell Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). Although Mn2+ is an essential trace element, excess Mn2+ is toxic and must be sequestered and removed via the secretory pathway by PMR1 (15Lapinskas P.J. Cunningham K.W. Liu X. Fink G.R. Culotta V. Mol. Cell Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar). Taken together, growth sensitivities to BAPTA and Mn2+ are rapid and effective means to screen pmr1 mutants for loss of ion transport. Differential sensitivity to BAPTA and Mn2+ is potentially indicative of a change in ion selectivity in the mutant, which can then be verified by biochemical characterization. In a previous study we have described the effects of mutations at Gln-783, positioned at the cytoplasmic interface of transmembrane helix M6 (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Bulky residues (Leu, Glu, and Thr) could effectively substitute for Gln in the transport of both Ca2+ and Mn2+ ions. Conversely, introduction of small polar side chains (Ser, Asn, and Cys) at this site led to complete loss of transport, whereas an Ala substitution was unique in conferring a striking and selective loss of Mn2+ transport. Evidence for a loss of Mn2+ selectivity in the Q783A mutant came from the observed loss of (i) Mn2+-tolerant growth, (ii) Mn2+ inhibition of 45Ca2+ transport, (iii) Mn2+-dependent ATP hydrolysis and phosphoenzyme formation, and (iv) Mn2+ inhibition of phosphoenzyme formation from inorganic phosphate. In contrast, BAPTA tolerance, 45Ca2+ transport, and Ca2+-dependent ATP hydrolysis and phosphoenzyme formation were normal in this mutant (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Ab initio modeling of transmembrane helices M4 and M6 of PMR1 placed Gln-783 (M6) in close proximity to Val-335 (M4) and led us to propose that mutations at position 783 might alter ion selectivity by interfering with helix packing. In this work, we examine additional residues in the vicinity of Gln-783 and specifically test the hypothesis that the interaction between Gln-783 and Val-335 is critical for Mn2+ transport. Media, Strains, and Plasmids—Yeast were grown in minimal medium containing yeast nitrogen base (6.7 g/liter, Difco), glucose (2%), and supplements as needed. Strain K616 (Δpmr1Δpmc1Δcnb1 (17Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (365) Google Scholar)) was used as host; this strain has no endogenous Ca2+-ATPase activity (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Mutations were introduced in the low copy plasmid YCpHR4 and screened for changes in phenotype (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This plasmid carries the PMR1 gene behind a tandem repeat of the yeast heat shock element and directs the expression of low levels of PMR1 in cells cultured at 25 °C. For biochemical assays, the mutations were moved into the multicopy plasmid YEpHisPMR1 from which N-terminal His9-tagged PMR1 was expressed at moderate levels from the PGK promoter (4Wei Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). This was done by cloning a unique 3.3-kbp BamHI to SacI fragment of the PMR1 gene containing the desired mutation(s) from YCpHR4 into the same sites on YEpHisPMR1. An N-terminal GFP-tagged version of PMR1 (pSR850) was constructed by cutting pYepHis-PMR1 (the amino-tagged His9 version of PMR1) with MluI and SacI and ligated in-frame into plasmid pRA101, which is a 2μ expression plasmid expressing GFP under control of the PGK promoter. Mutant proteins were constructed by amplifying the open reading frame of mutant PMR1 from the YCpHR4 vector using a 5′ primer that contained an MluI site and a 3′ primer that contained a SacI site. The amplified product was purified form was gel-purified, cut with MluI and SacI, and ligated into MluI- and SacI-digested pSR850. Sequencing was done to confirm the existence of the intended mutation in the final GFP-tagged construct. Mutagenesis—In most cases, site-directed mutations in M2, M4, M5, and M6 were generated in either a 1.4-kb HindIII-EcoRI, 1-kb BamHI-EcoRI fragment of PMR1 (M2 or M4) or in a 0.9-kbp EcoRI-PstI fragment (M5 or M6) subcloned into pBluescript, sequenced to confirm the mutation, and moved to pYCPHR4 expression plasmid as previously described (14). In some cases, the QuikChange site-directed mutagenesis kit (Stratagene) was used to introduce mutations. For random mutations at Val-335, one primer containing random bases at codon 335 was used in conjunction with a complementary primer to generate mutations at this site using the modified cyclical polymerase chain reaction approach of Gama and Breitwieser (18Gama L. Breitwieser G.E. BioTechniques. 1999; 5: 814-816Crossref Scopus (25) Google Scholar). The fragment was completely sequenced to identify the one or more base substitutions in codon 335 and to rule out unwanted errors that may have been incorporated during in vitro synthesis. The fragment was then used to replace the corresponding DNA in YCpHR4. Double mutations at 335 were combined with mutations at residue 783 by subcloning the corresponding fragments into the YCp plasmid. The identity of mutations in the YCp expression plasmid was confirmed to rule out the possibility of unwanted cloning artifacts or wild type sequences. Phenotype Screens—Growth assays were performed in 96-well plates containing 175 μl of supplemented YNB medium. 2–5 μl of saturated seed culture was incubated at 25 or 30 °C for 2 days. MnCl2 or BAPTA was added to the growth medium at the final concentrations indicated; BAPTA-containing medium was buffered to pH 6.0 with 100 mm MES/HCl. Growth was assessed by measuring absorbance at 600 nm on a Molecular Devices SpectraMax 340 plate reader. Unless otherwise noted, readings were normalized to control samples in which MnCl2 or BAPTA were omitted. Enzyme Purification and Biochemical Assays—Solubilization and purification of wild type or mutant (His)9-Pmr1 using nickel-nitrilotriacetic acid-agarose (Qiagen) has been described in detail (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Hydrolysis of [γ-32P]ATP (3000 Ci/mmol, Amersham Biosciences) by purified (His)9-Pmr1 (1 μg) was assayed at 25 °C in buffer containing 50 mm Hepes/Tris, pH 7.0, 100 mm KCl, 1 mm MgCl2, and 50 μm [γ-32P]ATP (500–600 cpm/pmol). CaCl2 or MnCl2 was added and buffered with EGTA to give free cation concentrations that were determined using the WinMaxChelator computer program. Activated charcoal (Sigma) was used to separate free phosphate from ATP, as described earlier (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). 54Manganese Accumulation—Yeast from 2-day-old saturated cultures were inoculated into 500 μl of minimal media with nutrient supplements to yield an initial A 600 of 0.1 for n = 3 wells in a 24-well tissue culture plate. Approximately 1 nm54MnCl (PerkinElmer Life Sciences, 7600 mCi/mg, ∼1.2 × 106 counts/well) was added per well, and yeast were grown at room temperature and assayed for 54Mn2+ accumulation 24 h post-inoculation by collection onto Millipore 0.45-μm HAWP filters using vacuum filtration. Filters were washed with 10 ml of wash buffer (10 mm HEPES-KOH, pH 7.4, 150 mm KCl), dissolved in 1 ml of dimethylformamide, and counted in 10 ml of scintillation fluid. Yeast growth was measured after 10-fold dilution by absorbance at 600 nm on Genesys 5 spectrometer (Spectronic). Homology Modeling of PMR1—Homology modeling of S. cerevisiae PMR1 (Accession NP_011348) in the E1 conformation was done using Swiss-PDB viewer (version 3.7b2) by "fitting the raw sequence" of PMR1 to 1EUL.pdb, which is the 2.6-Å resolution crystal structure of Oryctolagus cuniculus SERCA1a (accession P04191) in the Ca2+-bound E1 state (8Toyoshima C. Nakasako M. Nomura H. Ogawa H. Nature. 2000; 405: 647-655Crossref PubMed Scopus (1619) Google Scholar). Similarly, a homology model of PMR1 in the E2 conformation was generated using atomic coordinates from 1IWO.pdb for the E2-like SERCA structure (9Toyoshima C. Nomura H. Nature. 2002; 418: 605-611Crossref PubMed Scopus (809) Google Scholar). The default alignment settings in Swiss-PDB viewer were used for the initial alignment followed by manual optimization of the alignment to maximize homology between M4 and M5–M7 as shown in Fig. 5. The resulting homology model of PMR1 is based on the Cα positions of 1EUL.pdb and 1IWO.pdb. The single Ca2+ ion shown in the model was incorporated by merging the Ca2+ ion from site II of the SERCA1a into the aligned regions of PMR1. The images in Fig. 6 were generated by using the rendering program Pov-Ray.Fig. 6E1 and E2 conformations of PMR1 showing transmembrane helices M4, M5, and M6. Homology models of PMR1 were based on the crystal structures of the E1 and E2 conformations of SERCA1a and the alignment in Fig. 6 ("Experimental Procedures"). Helices are shown perpendicular to the plane of the membrane (A and B) or in cross-section from the cytoplasmic side of the membrane (C and D). Residues Gln-783 (orange) and Val-335 (purple) are shown in space-filled form. A calcium ion (yellow sphere) occupying Site II in the E1 conformation has been included. Amino acid side chains are colored green, red, and yellow based on the effect of substitutions reported in this and previous work (Table I and Ref. 14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Residues contributing to the calcium binding site in the E1 structure are displaced in E2. Note the movements in the cytoplasmic (lower) halves of M4 and M6 that bring Val-335 and Gln-783 in proximity in the E2 conformation.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Scanning Mutagenesis of Side Chains Proximal to Gln-783— The unique ion selectivity defect of mutant Q783A prompted a scanning mutagenesis survey of neighboring residues, within a 7-Å radius, based on the E1 crystal structure of the homologous SERCA1 pump. Specifically, we targeted residues Gln-134, Glu-135, Tyr-136, and Arg-137 of M2, and Phe-738 in M5 that could potentially interact with the Gln side chain at position 783. Each of these residues was replaced with Ala and Ile. Because Gln-783 lies near the cytoplasmic interface of M5, we also performed alanine-scanning mutagenesis of residues in the cytoplasmic half of transmembrane segments M4, M5, and M6. Mutants were expressed from a low copy plasmid in a yeast host strain lacking endogenous Ca2+ pumps (K616 (3Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar)) and screened for complementation of BAPTA and Mn2+-hypersensitive phenotypes. Previously, we have assayed ion transport in purified Golgi vesicles and ATP hydrolysis in purified enzyme preparations and demonstrated that these properties correlate with growth sensitivity of mutant strains to BAPTA and Mn2+ toxicity (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Table I depicts K 0.5 values for BAPTA and Mn2+ for mutants, expressed as percentage of wild type. Substitutions at Gln-134, Glu-135, and Tyr-136 in transmembrane segment M2 had no appreciable effect on PMR1 activity, as judged by their normal or near-normal phenotypes. Only Arg-137 appeared to be sensitive to substitution, and surprisingly, the Ala substitution was more deleterious than the bulkier Met. One mutation in M4, L341A, showed complete loss of function when expressed in low copy (Table I) but was similar to wild type when expressed from a multicopy plasmid (not shown). This mutation is therefore likely to have some reduction in turnover that can be compensated by overexpression. Substitution of the evolutionarily conserved Phe-738 at the cytoplasmic interface of M5 by either Ile or Ala led to a complete loss in ability to complement the pmr1 null phenotypes, from single copy (Table I) or multicopy (not shown) expression plasmids, indicating a critical role for this residue. We had reported earlier that substitution of another residue in M5, Gln-742, with alanine led to a loss of function (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). To investigate the importance of this residue further, we performed random mutagenesis at position 742 and screened the resulting substitutions for BAPTA and Mn2+ sensitivity (Fig. 1, A and B). Of nine different substitutions tested (Arg, Lys, Glu, Thr, Leu, Pro, Ser, Ala, and Cys), only the closely related side-chain Glu could partially replace Gln at this site, whereas all other substitutions led to loss of function.Table ISummary of phenotype screens on PMR1 mutationsFig. 1Phenotypic effects of substitutions at Gln-742 on BAPTA and Mn2+ tolerance. The host strain K616, carrying null alleles of endogenous Ca2+ pumps (Delta PMR1), was transformed with low copy plasmid expressing wild type or mutant PMR1 carrying the indicated amino acid substitutions at position 742. Mutant Q742X has a termination codon at position 742. Cultures were grown in media supplemented with BAPTA (A) or Mn2+ (B). Growth (A 600) is expressed as a percentage of control (no BAPTA or Mn2+ added).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In M6, mutant L785A, also near the cytoplasmic interface, exhibited a partial loss of function as judged by partial complementation of BAPTA and Mn2+ sensitivities in low copy (Table I), and full complementation following high copy expression (not shown). Not surprisingly, mutant G779T located in a conserved motif immediately adjacent to the calcium-binding residue Asp-778 was also inactive. Interestingly, mutant G779A was more sensitive to Mn2+ toxicity, relative to BAPTA (Table I), suggestive of a change in the relative affinity for Ca2+ and Mn2+ ions in this mutant. Similarly, analysis of phosphoenzyme formation in the human SPCA1 homologue has revealed that the Hailey Hailey disease mutant G309C in M4 fails to bind Mn2+ while retaining reduced Ca2+ binding affinity (19Fairclough R.J. Dode L. Vanoevelen J. Andersen J.P. Missiaen L. Raeymaekers L. Wuytack F. Hovnanian A. J. Biol. Chem. 2003; 278: 24721-24730Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). G309C is located immediately proximal to the conserved Glu in the ion binding pocket. It should be noted that most substitutions result in largely similar changes in BAPTA and Mn2+ sensitivities (for example, E788A in Table I). This study revealed several potential ion selectivity mutants, including V335A in M4, M777A and P781A in M6, that are worthy of further investigation. Of these, a detailed analysis of Val-335 is reported in this work. In summary, the scanning mutagenesis identified several hydrophobic residues near the cytoplasmic interface of M4, M5, and M6 that appear to be critical for ion transport. In addition, the requirement for a carboxyl oxygen (from Gln or Glu) suggests that residue Gln-742 may be participate in ion binding. In an earlier study, we had identified several polar or charged residues distributed in the middle tiers of transmembrane helices M4–M8 that might participate in ion binding. The results from this and our previous mutagenesis studies give an increasingly comprehensive picture of the importance of individual residues along the transport pathway. Subcellular Localization of GFP-tagged PMR1 Mutants—We have previously demonstrated that some loss-of-function PMR1 mutants are retained in the endoplasmic reticulum, where they appear hypersensitive to limited proteolysis by trypsin, indicative of structural or folding defects (14Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). It was therefore important to determine if mutations reported in this study showed normal biogenesis and trafficking to the Golgi. All mutants showing partial or complete loss of function, were tagged with GFP, and examined by confocal fluorescence microscopy. The mutants showed a punctate distribution typical of yeast Golgi bodies and were essentially identical to wild type (Fig. 2). Thus, loss of function appears to be related to a catalytic, rather than a biogenesis defect. Substitutions at Val-335 Mimic or Suppress the Mn2 + Transport Defect of Mutation Q783A—Based on ab initio modeling of M4 and M6 helices, we had suggested earlier that Gln-783 in M6 formed a hydrophobic contact with Val-335 in M4 (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). To test this hypothesis, we generated a series of substitutions at position 335 of PMR1 and analyzed the effect of the mutations by screening for BAPTA and Mn2+ toxicity. We found that Cys was not tolerated at 335, whereas residues with large side chains (Thr, Leu, and Ile) were similar to wild type (not shown). These findings were reminiscent of our mutational analysis of residue 783 (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Interestingly, mutant V335G showed a significant and reproducible difference in response to BAPTA and Mn2+ toxicity, indicative of a change in ion selectivity (Fig. 3). A similar trend was observed for the V335A mutation (Table I). Next, we tested whether introduction of the bulkier Ile side chain at residue 335 (V335I) could compensate for loss of side-chain volume in the Q783A mutant. We found that the double mutant V335I/Q783A showed a striking recovery in Mn2+-tolerant growth, relative to that of the single mutant Q783A (Fig. 3). In contrast, mutations in Gln-134, Glu-135, Tyr-136, and Arg-137, also within interacting distance, were ineffective in restoring the Mn2+-sensitive phenotype of mutant Q783A (not shown). To verify these findings, we measured cation dependence of ATP hydrolysis. Mutants V335G and V335I/Q783A were tagged at the N terminus with a 9xHis epitope and purified by nickel-nitrilotriacetic acid chromatography ("Experimental Procedures"). ATP hydrolysis was assayed as described earlier (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar), and the apparent K m for Ca2+ and Mn2+ was determined. We have shown earlier that ATPase activity of His-tagged wild type PMR1 is strictly dependent on the presence of calcium (Km 0.07 μm) or manganese (Km 0.02 μm) ions (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Although the apparent affinity for Ca2+ was not significantly different from wild type, the V335G mutant showed an 8-fold loss in affinity for Mn2+, consistent with the prediction from the phenotype screen (Table II). Previously, we had shown that Ca2+-dependent ATPase activity in the Q783A mutant was nearly identical to wild type (0.06 μm), whereas Mn2+-ATPase was too low to analyze (16Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar). Table II shows that the apparent K m for Mn2+ in the V335I/Q783A double mutant was similar to wild type, demonstrating an effective correction of the Mn2+ transport defect. Thus, engineering a large reduction in side-chain volume at residue 335 (V335G) mimics the Q783A substitution, whereas an increase in side-chain volume (V335I) compensates for the Q783A mutation, consistent with a packing interaction between M4 and M6 at these sites.Table IICation dependence of ATP hydrolysisPMR1Mn2+ KMCa2+ KMμmWild type0.02 ± 0.0060.07 ± 0.001Q783ANDaND, not detectable; Mn2+-stimulated ATPase activity was too low to measure (16).0.06 ± 0.001V335G0.16 ± 0.020.12 ± 0.05V335I/Q783A0.02 ± 0.0070.09 ± 0.02a ND, not detectable; Mn2+-stimulated
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