Phenotypic Screening of Mutations in Pmr1, the Yeast Secretory Pathway Ca2+/Mn2+-ATPase, Reveals Residues Critical for Ion Selectivity and Transport

Abstract

Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca2+and Mn2+ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca2+ pumps and screened for loss of Ca2+ and Mn2+ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and Mn2+ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn2+ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn774 and Asp778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca2+ and Mn2+ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps. Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca2+and Mn2+ ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca2+ pumps and screened for loss of Ca2+ and Mn2+ transport on the basis of hypersensitivity to 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and Mn2+ toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn2+ toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including 45Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable 45Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn774 and Asp778 in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca2+ and Mn2+ ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps. 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid polyacrylamide gel electrophoresis sarco/endoplasmic reticulum Ca2+-ATPase endoplasmic reticulum Ion pumps belonging to the family of P-type ATPases occur in all cells, where they drive transmembrane ion gradients of up to 10,000-fold (reviewed in Ref. 1Moller J.V. Juul B. le Maire M. Biochim. Biophys. Acta. 1996; 1286: 1-51Crossref PubMed Scopus (660) Google Scholar). Well known members of the family include the Na+/K+-ATPase of animal cells, H+/ATPase of fungi and plants, and various Ca2+-ATPases found in plasma membrane and endomembrane compartments. Individual pumps have evolved distinct cation selectivities to fulfill a variety of different physiological functions including Ca2+ homeostasis, acid (H+) extrusion, generation of Na+ and K+electrochemical gradients, and the detoxification of soft metals (Cu2+, Cd2+, and Zn2+). Despite their differences in cation selectivity, [P]ATPases share many structural and mechanistic similarites. Remarkably, the electron micrograph structures (8 Å) of the fungal H+-ATPase and the sarcoplasmic reticulum Ca2+-ATPase are virtually superimposable within the dimensions of the membrane (2Zhang P. Toyoshima C. Yonekura K. Green N.M. Stokes D.L. Nature. 1998; 392: 835-839Crossref PubMed Scopus (265) Google Scholar, 3Auer M. Scarborough G.A. Kuhlbrandt W. Nature. 1998; 392: 840-843Crossref PubMed Scopus (185) Google Scholar). In both cases, densities corresponding to ten transmembrane helices are clearly visible, of which three are clustered to enclose a well defined pore, plausibly the pathway for ion translocation, extending from the cytoplasmic to the lumenal/extracellular space. A variety of experimental evidence points to three helices, transmembrane segments M4, M5, and M6, that are most likely to be involved in transport. For example, in the sarcoplasmic reticulum Ca2+-ATPase, mutagenesis of residues in M4, M5, and M6 prevent cation binding and inactivate transport (reviewed in Ref. 4MacLennan D.H. Rice W.J. Odermatt A. Ann. N. Y. Acad. Sci. 1997; 834: 175-185Crossref PubMed Scopus (21) Google Scholar), and cysteine residues engineered into M4 and M6 can be disulfide-linked (5Rice W.J. Green N.M. MacLennan D.H. J. Biol. Chem. 1997; 272: 31412-31419Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The proximity of M5 and M6 may be inferred from studies on the fungal H+-ATPase showing the presence of a salt bridge between residues in these two helices (6Sen Gupta S. DeWitt N.D. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 34328-34334Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Defining the residues that compose the ion-binding site(s) is a first step toward understanding how vectorial transport occurs. Extensive mutagenesis studies have contributed to the emerging molecular picture of the transport site in which oxygen-containing side chains coordinate one or more cations. Thus, in SERCA, residues Glu309 (M4), Glu771 (M5), and Asn796, Thr799, and Asp800 (M6), are required for binding two Ca2+ ions (reviewed in Ref. 4MacLennan D.H. Rice W.J. Odermatt A. Ann. N. Y. Acad. Sci. 1997; 834: 175-185Crossref PubMed Scopus (21) Google Scholar). Similar studies in the Na+/K+-ATPase have revealed the important role of residues Ser775 (M5), Asp804, and Asp808 (M6) in K+ and possibly Na+, binding (reviewed in Ref. 7Lingrel J.B. Croyle M.L. Woo A.L. Arguello J.M. Acta Physiol. Scand. 1998; 163: 69-77Google Scholar). Interestingly, Asn796 and Asp800 of SERCA occupy equivalent positions to Asp804 and Asp808 in Na+/K+-ATPase, suggesting that the architecture of the ion-binding site may be similar in the two enzymes. Additional studies on other ion pumps will be key to determining whether this similarity extends throughout the family. The [P]ATPases offer a striking paradigm for the evolutionary development of ion selectivity; yet the molecular basis for selectivity remains one of the fundamental unanswered questions in the field. From studies on other classes of transport proteins, it is clear that selectivity is determined by the local environment around key residues (8Hama H. Wilson T.H. J. Biol. Chem. 1994; 269: 1063-1067Abstract Full Text PDF PubMed Google Scholar, 9Galzi J.-L. Devillers-Thiery A. Hussy N. Bertrand S. Changeux J.-P. Bertrand D. Nature. 1992; 359: 500-505Crossref PubMed Scopus (345) Google Scholar). However, it is difficult to identify, from conventional site-directed mutagenesis alone, which residues are important for distinguishing between different ions, such as Ca2+ and Na+. Despite having completely nonoverlapping ion selectivities, four of five residues important for Ca2+binding in SERCA are conserved in equivalent positions in the Na+/K+-ATPase sequence so that these residuescannot be considered to define ion selectivity. Here we report on the mutagenesis of every oxygen-containing side chain within membrane segments M4–M8 of Pmr1, the yeast secretory pathway/Golgi ion pump. Previous studies from our lab (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 11Wei Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar), as well as others (12Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar, 13Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar, 14Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar), have suggested that Pmr1 mediates the high affinity transport of Ca2+ and Mn2+ into the secretory pathway for a variety of secretory functions, including protein sorting, processing, and glycosylation. A novel aspect of this study is the phenotypic screening of yeast mutants to identify residues that are critical for Ca2+ and Mn2+ transport and selectivity. We show that this approach greatly simplifies the analysis of mutants and can be used to screen large numbers of mutants in future random mutagenesis studies. We expect that this approach can be extended to the study of heterologous ion pumps expressed in yeast, including Ca2+-ATPases from animal and plant systems (15Liang F. Cunningham K.W. Harper J.F. Sze H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8579-8584Crossref PubMed Scopus (134) Google Scholar,16Degand I. Catty P. Talla E. Thines-Sempoux D. d'Exaerde A. Goffeau A. Ghislain M. Mol. Microbiol. 1999; 31: 545-556Crossref PubMed Scopus (36) Google Scholar). Yeast strains were grown in defined media containing yeast nitrogen base (6.7 g/liter; Difco), dextrose (2%), and supplements as needed. PMR1-containing plasmids were introduced into strain K616 (17Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (365) Google Scholar), which carries null alleles of calcineurin B (CNB1) and two Ca2+-ATPases (PMR1 and PMC1), resulting in low basal Ca2+ pump activity, as described previously (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Growth assays were performed by inoculating 100 μl of YNB medium in a 96-well plate with 2–5 μl of a saturated seed culture followed by incubation for 48 h at 30 °C. Where indicated, MnCl2 or BAPTA1 was added to the medium prior to inoculation, and the pH was adjusted to 6.0 with NaOH. Cultures were thoroughly mixed by gentle vortexing, and growth was monitored by measuring absorbance at 600 nm in a SPECTRAmax 340 microplate reader (Molecular Devices). Relative growth was expressed as the fraction of the absorbance of the control culture (no additions). YEpHR1, a yeast 2μ plasmid carrying the PMR1 coding sequence under control of a tandem repeat of a yeast heat shock element has been described previously (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). YCpHR1 is an identical construct made in a single copy/CEN backbone derived from plasmid YCplac33 (18Gietz R.D. Sugino A. Gene (Amst.). 1988; 74: PubMed Scopus Google Scholar). acid were introduced into or of into the the chain 1997; PubMed Scopus Google and a of in The PMR1 was to of the into plasmids and YCpHR1 was by of by and of Golgi from yeast were as described (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). was determined by a A.L. J. Biol. Chem. Full Text PDF PubMed Google of containing with acid and as were for electrophoresis by with acid as described (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). and the of Pmr1 were performed as described previously (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). 45Ca transport assays of and Golgi were by as described previously (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). A and were added to of In assays of Mn2+ 45Ca was at and MnCl2 was added as of were with at a to protein of in containing pH and were for with added to a of at 30 the were by of acid to were on and by and the resulting were in gel for yeast strains a copy of Pmr1, a P-type found in the distinct Ca2+ and defects (12Antebi A. Fink G.R. Mol. Biol. Cell. 1992; 3: 633-654Crossref PubMed Scopus (378) Google Scholar, 13Lapinskas P.J. Cunningham K.W. Liu X.F. Fink G.R. Culotta V.C. Mol. Cell. Biol. 1995; 15: 1382-1388Crossref PubMed Google Scholar, 14Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar). of pmr1 mutants to of Ca2+ or by such as BAPTA or to be to a of high affinity transport of these ions into the secretory where they functions in protein sorting, and has been that BAPTA toxicity may be by the of of Ca2+ or to growth media G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (349) Google Scholar), that the two ions can play roles in yeast yeast media have a of Ca2+ to and the is at low the growth by BAPTA in pmr1 mutants a of the of to Mn2+ toxicity is a of loss of Mn2+ transport. for Mn2+ is and be by into the secretory pathway by Pmr1 and from the Thus, we that sensitivity to BAPTA and Mn2+ toxicity be a of the Ca2+ and Mn2+ of Pmr1 mutations were made at cation-binding and in the predicted transmembrane segments M4, M5, M6, M7, and of were with and in the of also as and In one an was introduced to in M5 so as to the sequence of the SERCA pump. Pmr1 mutants were introduced into the yeast strain K616 which we have previously to be devoid of endogenous Ca2+ pump (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). low of the pump for mutants were expressed under control of the heat shock from single copy/CEN and grown at 30 °C. We found that in the of heat of Pmr1 was similar to of from the endogenous mutant was screened for sensitivity to BAPTA and Mn2+ in growth as described under containing wild and the were also into strain K616 as Mutants into one of three as exemplified in Class 1 such as mutant were indistinguishable from wild type in both BAPTA and Mn2+ assays and were to have normal transport Class 2 such as mutant a null for both BAPTA and suggesting a loss of Interestingly, mutants differential to BAPTA and were as Class 3 is mutant Q783A 1), in which to BAPTA is at wild type to Mn2+ is similar to the null suggesting that Ca2+ transport is is Two other mutants and showed of BAPTA loss of Mn2+ These are ion selectivity A of all mutants in this study is in I. of the residues for mutagenesis in membrane segments M4 and were found to be for the and were Class 2 with or completely of residues (M5), Asn774 and and in Additional of Asp778 with and also to the of this in cation of pmr1 mutant and protein by BAPTA and Mn2+ was from low copy of mutants in the pmr1 null strain as described in the Mutants into three wild type for both BAPTA and 1), null for both BAPTA and Mn2+ (Class 2), and differential to BAPTA and Mn2+ (Class were with in the presence or of and the presence of an as described in the is from grown at 30 were on gradients as described under and of were to and with of Pmr1 to Golgi or endoplasmic reticulum was determined on the basis of with Growth by BAPTA and Mn2+ was from low copy of mutants in the pmr1 null strain as described in the Mutants into three wild type for both BAPTA and 1), null for both BAPTA and Mn2+ (Class 2), and differential to BAPTA and Mn2+ (Class were with in the presence or of and the presence of an as described in the is Yeast from grown at 30 were on gradients as described under and of were to and with of Pmr1 to Golgi or endoplasmic reticulum was determined on the basis of with (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). in a the first step in a analysis of we in membrane of yeast heat from plasmids (10Sorin A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). evidence for the and membrane of all mutant Pmr1 proteins, with in as in the in Class 2 mutants and on 2 and plasmids were from the yeast strains and by and to the of the mutant The to be related to altered or of the protein M. and R. and was to be a first of as a of Pmr1 Yeast were with and to as described under Where indicated, were with The presence of an in mutants and with Golgi and normal of Pmr1 on as in 3 and I. the of this in mutants and with and of mutant Pmr1 was on well defined of yeast analysis was to ion transport to Pmr1 in the Golgi and can be considered a of structural ATPases are and in the yeast endoplasmic reticulum S. Allen K.E. A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). the of a subset of Pmr1 a of is found in I. We have previously a clear of the Golgi in from endoplasmic the in the of the of the Class 1 mutants showed normal trafficking to the with their normal the other two classes of normal as in 3 for were in the endoplasmic or completely the was and was a striking in targeting the of the with and to and loss of with a Golgi from this study was that with of the mutant Pmr1 polypeptide gel possibly an protein that was to in be that mutants showing to show trafficking under normal culture of 30 which the possibly of heat on protein Individual of the were transport described the strain was devoid of endogenous Ca2+ pump activity, Pmr1 transport was to Golgi membrane containing Class 1 mutants transport from to of wild type as for mutant in In contrast, Class 2 mutants detectable transport the control and in 3). the ion selectivity mutants (Class Q783A wild type of Ca2+ transport mutants and were to and of wild respectively. important step in mutant analysis is the of the structural of mutant We have previously described the of as a of protein Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). Golgi were with in the presence or of to and mutants were screened for as in the in was in all mutants with normal Golgi similar to that in the wild type protein and Interestingly, was a loss of with defects in in mutants showing was mutants showing and the of for all mutants in this We have into Golgi from strains wild type Pmr1 from the null mutant and R. with the role of Pmr1 in Mn2+ transport. approach is to Mn2+ of 45Ca transport to Mn2+ transport activity, as Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). we the of in the ion selectivity (Class mutant that Mn2+ is a of 45Ca transport in Golgi containing wild type Pmr1, with at under where the is in the for A. Rosas G. Rao R. J. Biol. Chem. 1997; 272: 9895-9901Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, and 11Wei Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). These that ion selectivity of wild type Pmr1 is Mn2+ In contrast, for the Q783A mutant at a to wild These results are with a in the affinity for and hypersensitivity to Mn2+ toxicity 1), in this In this we an to screen for acid residues critical for ion transport and selectivity in yeast mutants of the secretory pathway/Golgi ion pump, a of this we used site-directed mutagenesis of and residues within the transmembrane segments most likely to the Mutants were in one of three growth in or Class 1 mutants hypersensitivity to BAPTA or a first of normal analysis that these mutants showed normal trafficking to the transport and to be the of to of the residues for mutagenesis in this study into this and were for pump as was in site-directed mutagenesis studies of this on the Na+/K+-ATPase, and SERCA (reviewed in 4MacLennan D.H. Rice W.J. Odermatt A. Ann. N. Y. Acad. Sci. 1997; 834: 175-185Crossref PubMed Scopus (21) Google Scholar, 7Lingrel J.B. Croyle M.L. Woo A.L. Arguello J.M. Acta Physiol. Scand. 1998; 163: 69-77Google Scholar, and A. DeWitt N.D. Gupta S. Slayman C.W. Acta Physiol. Scand. 1998; Google Scholar). Class 2 mutants were to both BAPTA and indicative of a of pump which was by the of 45Ca transport in Golgi between structural and we protein trafficking and sensitivity to Mutants that showed normal Golgi trafficking also that showed to The also gel for that are These are with polypeptide and that are to structural and structural with the the by of the side chain may be as a it is clear of a side in of was more that of is that within the of the differences in for the of and may be the results of our of the residues in transmembrane segments M4 or were for loss of was at and and trafficking defects were also with of Asn774 (M6), and resulting in loss of However, and mutants of Asp778 (M6) showed normal trafficking and polypeptide yet lacked transport activity, this a for the cation-binding similar have been for mutations at residues equivalent to Asn774 and Asp778 of the plasma membrane mutant was in endoplasmic mutant showed normal trafficking to the plasma membrane D. D. F. E. Biochemistry. 1996; PubMed Scopus Google Scholar). showed a loss of Ca2+ transport D. D. F. E. Biochemistry. 1996; PubMed Scopus Google Scholar, A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). of the equivalent residues in the Na+/K+-ATPase, Asp804 and prevent trafficking to the plasma as by binding in J.B. Croyle M.L. Woo A.L. Arguello J.M. Acta Physiol. Scand. 1998; 163: 69-77Google Scholar), are also with loss of transport Thus, these two appear to be common components of the cation-binding site in most P-type is the fungal where to Asp778 of a salt bridge with in of both residues with (6Sen Gupta S. DeWitt N.D. Allen K.E. Slayman C.W. J. Biol. Chem. 1998; 273: 34328-34334Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The growth on BAPTA and Mn2+ toxicity in pmr1 mutants are of distinguishing between and residues and can be used to screen large numbers of generated mutants in future studies. We also show that the can identify of cation selectivity. Three such were identified in this in the mutants showed sensitivity to Mn2+ toxicity to that of the Ca2+ BAPTA in growth suggesting that the a more on Mn2+ transport. However, in a we have on a with the within the in Pmr1, was indistinguishable from wild type in the Mn2+ toxicity Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar), BAPTA toxicity in this mutant was between wild type and the pmr1 null We showed that Mn2+ affinity was to wild was a in affinity in this mutant Y. Marchi V. Wang R. Rao R. Biochemistry. 1999; 38: 14534-14541Crossref PubMed Scopus (56) Google Scholar). we show that Q783A has normal Ca2+ transport a in the affinity for one such with a similar on ion selectivity has been in studies on other ion of Ser775 in M5 with in the Na+/K+-ATPase a in K+ Na+ affinity R. A. Arguello J.M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google J.M. Biochemistry. 1998; PubMed Scopus Google Scholar). is that two of five residues required for Ca2+ binding in SERCA were in A likely is that SERCA, which has a of two Ca2+ Pmr1 one cation In the sequence of Pmr1 and and occupy positions equivalent to Glu771 and (M6) of SERCA, with a number of cation-binding These differences the of the secretory pathway Ca2+-ATPases as a distinct from related to the well known endoplasmic reticulum pumps.