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J. Biol. Chem., Vol. 275, Issue 31, 23927-23932, August 4, 2000

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

Ying Wei, Jun Chen, Gisele Rosas, D. Andrew Tompkins, P. Andrew Holt, and Rajini Rao

From the Department of Physiology, The Johns Hopkins University School of Medicine, Baltimore Maryland 21205

Received for publication, March 28, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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. 1). 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 (2, 3). 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. 4), and cysteine residues engineered into M4 and M6 can be disulfide-linked (5). 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 (6).

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. 4). 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. 7). 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 (8, 9). 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 residues cannot 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 (10, 11), as well as others (12-14), 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 (15, 16).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Media, Strains, and Growth Assays-- 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 (17), 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 (10). 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).

Plasmids and Mutagenesis-- 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 (10). YCpHR1 is an identical construct made in a single copy/CEN backbone derived from plasmid YCplac33 (18). Amino acid substitutions were introduced into an EcoRI-PstI fragment or a HindIII-EcoRI fragment of PMR1 subcloned into the bacterial vector pBlueScript (Stratagene) using the inverse polymerase chain reaction reaction (19) and a pair of oligonucleotide primers in each case. The PMR1 fragment was entirely sequenced to rule out unwanted mutations. Reconstruction of the mutagenized fragments into plasmids YEpHR1 and YCpHR1 was by standard cloning techniques.

Cell Fractionation and Biochemical Methods-- Preparations of total membranes, subcellular fractionation by sucrose gradient centrifugation, and isolation of Golgi vesicles from yeast were exactly as described earlier (10). Protein concentration was determined by a modified Lowry assay (20) following precipitation of samples containing sucrose with 10% trichloroacetic acid and using bovine serum albumin as standard. Samples were prepared for electrophoresis by precipitating with trichloroacetic acid as described (10). SDS-PAGE and Western blotting using polyclonal antibody raised against the C-terminal one-third of Pmr1 were performed as described previously (10).

⁴⁵Ca²⁺ Transport Assays-- ATP-dependent ⁴⁵Ca transport assays of sucrose gradient fractions and pooled Golgi membranes were done by rapid filtration as described previously (10). Concanamycin A (Sigma; 10 nM) and carbonyl cyanide p-chlorophenylhydrazone (25 µM) were added to eliminate activity of vacuolar H⁺/Ca²⁺ exchange. In assays of Mn²⁺ competition, ⁴⁵Ca was at 0.4 µCi/ml (nominally, 0.8 µM), and MnCl₂ was added as indicated.

Trypsinolysis-- Preparations of total membranes (1 mg/ml) were treated with trypsin (TosPheCH₂Cl-treated, Sigma) at a trypsin to protein ratio of 1:10 in buffer containing 20 mM Tris-HCl, pH 7.0, 0.1 M KCl, and 5 mM MgCl₂. When specified, samples were preincubated for 5 min with Na₂ATP (pH 6.7) added to a final concentration of 5 mM. After 5 min at 30 °C, the digests were terminated by addition of trichloroacetic acid to 10%. Samples were chilled on ice (30 min) and collected by microcentrifugation (30 min), and the resulting pellets were resuspended in gel loading buffer for electrophoresis.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Three Classes of pmr1 Mutants Are Revealed by Phenotypic Screening of BAPTA and Mn²⁺ Toxicity-- Mutant yeast strains lacking a functional copy of Pmr1, a P-type ATPase found in the Golgi, exhibit distinct Ca²⁺ and Mn²⁺-related cellular defects (12-14). Hypersensitivity of pmr1 mutants to depletion of extracellular Ca²⁺ or Mn²⁺, by chelating agents such as BAPTA or EGTA, appears to be due to a lack of high affinity transport of these ions into the secretory pathway, where they serve essential functions in protein sorting, glycosylation, and secretion. It has been shown that BAPTA toxicity may be overcome by the introduction of minimal free concentrations of either Ca²⁺ (~0.84 nM) or Mn²⁺ (~0.16 pM) to growth media (14), indicating that the two ions can play largely surrogate roles in supporting yeast growth. Because standard yeast media have nearly a 100-fold excess of Ca²⁺ relative to Mn²⁺, and the latter is efficiently removed at low chelator concentrations, the observed growth inhibition by BAPTA in pmr1 mutants represents a titration of the remaining Ca²⁺. Hypersensitivity of pmr1 mutants to Mn²⁺ toxicity is a specific consequence of loss of Mn²⁺ transport. Although essential for growth, excess Mn²⁺ is toxic and must be removed by delivery into the secretory pathway by Pmr1 and subsequent exit from the cell. Thus, we reasoned that sensitivity to BAPTA and Mn²⁺ toxicity would be a preliminary indication of the Ca²⁺ and Mn²⁺ transporting activity of Pmr1 mutants.

35 mutations were made at potential cation-binding sites (Asp, Glu, Asn, Gln, Ser, Thr, and Tyr) in the predicted transmembrane segments M4, M5, M6, M7, and M8 of Pmr1. Residues were substituted with Ala, and in the case of acidic residues, also as follows: Asp  Asn, Glu and Glu  Asp, Gln. In one case, an Asp was introduced to replace Ala⁷⁴⁹ in M5 so as to mimic the sequence of the SERCA pump. Pmr1 mutants were introduced into the yeast strain K616 (pmr1pmc1cnb1), which we have previously shown to be devoid of endogenous Ca²⁺ pump activity (10). To ensure low level expression of the pump for phenotype analysis, mutants were expressed under control of the heat shock element, from single copy/CEN plasmids, and grown at 30 °C. We found that in the absence of heat shock, leak-through expression of Pmr1 was similar to native levels of expression from the endogenous promoter (not shown). Each mutant was screened for sensitivity to BAPTA and Mn²⁺ in quantitative liquid growth assays, as described under "Experimental Procedures." Plasmids containing wild type PMR1 (YCpHR1) and the empty vector (YCpH2) were also transformed into strain K616 as controls.

Mutants fell into one of three classes, as exemplified in Fig. 1. Class 1 mutants, such as mutant T826A, were indistinguishable from wild type in both BAPTA and Mn²⁺ tolerance assays and were assumed to have largely normal transport function. Class 2 mutants, such as mutant D778A, displayed a null phenotype for both BAPTA and Mn²⁺ tolerance, suggesting a complete loss of function. Interestingly, some mutants displayed differential tolerance to BAPTA and Mn²⁺ and were grouped as Class 3 mutants. An extreme example is mutant Q783A (Fig. 1), in which tolerance to BAPTA is at wild type levels, whereas tolerance to Mn²⁺ is similar to the null mutant, suggesting that Ca²⁺ transport is retained but Mn²⁺ transport is lost. Two other mutants (E329A and A749D) showed intermediate profiles of BAPTA tolerance but complete loss of Mn²⁺ tolerance. These are putative ion selectivity mutants.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Representative examples of three classes of pmr1 mutants. Host strain K616 (pmr1pmc1cnb1) was transformed with a low copy vector (YCpH1) carrying either wild type PMR1 (WT) or mutants T826A, D778A, and Q783A. Cells were grown in YNB medium supplemented with BAPTA (A) or Mn2+ (B) as indicated for 48 h at 30 °C. Growth (A600) was normalized to control (no additions). T826A was representative of Class 1 mutants and resembled the wild type in both growth assays. D778A, a Class 2 mutant, displayed a hypersensitivity to growth inhibition by BAPTA and Mn2+, similar to the null strain (vector). The Class 3 mutant Q783A had differential sensitivity to BAPTA and Mn2+, suggestive of a change in ion selectivity. , WT; , vector; , T826A-Class 1; , D778A-Class 2; , Q783A-Class 3.

A complete classification of all 35 mutants reported in this study is listed in Table I. None of the residues targeted for mutagenesis in membrane segments M4 and M8 were found to be essential for function; although the apparently conservative substitutions E329D (M4) and D856E (M8) were Class 2 (loss-of-function) mutants, substitution with Ala restored function either partially (E329A) or completely (D856A). Alanine substitutions of residues Gln⁷⁴² (M5), Asn⁷⁷⁴ and Asp⁷⁷⁸ (M6), and Thr⁸¹⁷ (M7) resulted in loss-of-function phenotypes. Additional substitution of Asp⁷⁷⁸ with Glu and Asn also failed to restore function, confirming the importance of this residue in cation transport.

Biogenesis, Targeting, and Ion Transport Characteristics of pmr1 Mutants-- As the first step in a systematic biochemical analysis of mutants, we checked expression levels in rapid, small scale, total membrane preparations of yeast transformants, following heat shock-induced expression from multicopy (2µ) plasmids (10). Western blots using anti-Pmr1 polyclonal antibody provided evidence for the biogenesis and membrane insertion of all mutant Pmr1 proteins, albeit with some variation in expression levels, as shown in the samples in Fig. 2. Curiously, Class 2 mutants N774A, D778E, T817A, and D856E had distinctly faster mobility on SDS-PAGE (Figs. 2 and 4). Mutant plasmids were rescued from the transformed yeast strains and analyzed by restriction enzyme mapping and DNA sequencing to confirm the integrity of the mutant gene (not shown). The faster mobility appeared not to be related to altered glycosylation or phosphorylation of the protein² and was tentatively assumed to be a first indication of abnormal conformation.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Western blot of yeast total membranes showing expression levels of mutant Pmr1 ATPases. Wild type (WT), host strain K616 (pmr1), and a representative set of mutants are indicated. 20 µg of protein was loaded in each lane, and the blot was probed with anti-Pmr1 polyclonal antibody. Note the faster mobility of mutants N774A, D778E, and D856E also apparent in Fig. 4.

Trafficking of mutant Pmr1 proteins was examined on well defined sucrose density gradient fractionations of yeast lysates. This analysis was essential to normalize ion transport activity to Pmr1 expression levels in the Golgi fractions and can be considered a second indication of structural problems, because incorrectly folded ATPases are retained and subsequently degraded in the yeast endoplasmic reticulum (21). Fig. 3 shows the subcellular localization of a subset of Pmr1 mutants; a complete listing of organellar localization is found in Table I. We have previously demonstrated a clear separation of the lighter Golgi fractions (fractions 4-6 in Fig. 3) from endoplasmic reticulum, the latter being distributed in the denser half of the sucrose gradient (fractions 8-11). All of the Class 1 mutants showed normal trafficking to the Golgi, consistent with their normal phenotype. Among the other two classes of mutants, some displayed normal trafficking, as depicted in Fig. 3 for D778N. Others were retained in the endoplasmic reticulum, either partially (A749D) or completely (D778E). When the target residue was acidic (Asp⁷⁷⁸ and Asp⁸⁵⁶), there was a striking difference in targeting depending upon the nature of the substitution. Surprisingly, substitution with another acidic residue (Asp  Glu and Glu  Asp) led to ER retention and concomitant loss of function (Table I), whereas substitution with a neutral residue allowed correct Golgi targeting. Another interesting finding from this study was that complete ER retention consistently correlated with faster mobility of the mutant Pmr1 polypeptide upon SDS gel electrophoresis, possibly indicating an abnormal protein conformation that was resistant to unfolding in SDS. It should be noted that mutants showing ER retention continued to show trafficking problems under normal culture temperatures of 30 °C, which obviate the possibly deleterious effects of heat shock-induced expression (at 37 °C) on protein targeting.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Trafficking and Ca2+ transport activity of Pmr1 ATPases in subcellular fractions. Fractions from sucrose density gradients of yeast lysates were analyzed for 45Ca transport activity (A) and Pmr1 expression on Western blots (B) as described in the the legend to Fig. 2. Mutants with high (N859A), partial (E329A), and no detectable activity (E329D, D778N, and D778E) are shown. Note the difference in localization between Golgi (wild type, E329A, E329D, D778N, and N859A) and endoplasmic reticulum (D778E).

Individual fractions of the sucrose gradient were assayed for ⁴⁵Ca transport activity. As described earlier, the host strain was essentially devoid of endogenous Ca²⁺ pump activity, whereas plasmid-encoded Pmr1 transport activity was largely restricted to Golgi membrane containing fractions. All Class 1 mutants had significant transport activity ranging from ~10 to 150% of wild type levels as depicted for mutant N859A in Fig. 3. In contrast, Class 2 mutants had no detectable transport activity above the vector-transformed control (mutants E329D, D778N, and D778E in Fig. 3). Of the putative ion selectivity mutants (Class 3), Q783A had wild type levels of Ca²⁺ transport activity (not shown), whereas mutants A749D and E329A (Fig. 3) were reduced to 10 and 30% of wild type, respectively.

Limited Proteolysis as a Probe of Polypeptide Folding-- An important step in mutant analysis is the assessment of the structural integrity of mutant polypeptides. We have previously described the use of limited proteolysis as a probe of overall protein conformation (11). Golgi membranes were treated with trypsin in the presence or absence of ATP to detect ligand-induced conformational changes, and mutants were screened for specific proteolytic intermediates, as illustrated in the examples shown in Fig. 4. An ATP-protectable 52-kDa tryptic fragment was detected in all mutants with normal Golgi trafficking, similar to that seen in the wild type protein (Fig. 4; E329D, N859A, and D778N). Interestingly, there was a progressive loss of ATP-protection with increasing defects in localization; thus, in mutants showing partial ER retention, ATP protection against trypsinolysis was poor, whereas mutants showing complete ER retention had no ATP-protectable fragments (Fig. 4; N774A, D778E, and D856E). Table I lists the efficiency of ATP protection against proteolysis for all mutants in this study.

View larger version (49K): [in this window] [in a new window]   Fig. 4.   Limited trypsinolysis as a conformational probe of Pmr1 ATPases. Yeast total membranes were treated with trypsin and subjected to Western analysis, as described under "Experimental Procedures." Where indicated, membranes were preincubated with 5 mM ATP. The presence of an ATP-protectable 52-kDa tryptic fragment (asterisk) in mutants E329D, N859A, and D778N correlates with Golgi localization and normal mobility of Pmr1 polypeptides on SDS-PAGE, as shown in Figs. 2 and 3 and Table I. Conversely, the absence of this fragment in mutants N774A, D778E, and D856E correlates with abnormal mobility and localization.

Manganese Inhibition of Calcium Transport Is a Measure of Mn²⁺ Selectivity-- We have observed saturable ⁵⁴Mn uptake into Golgi vesicles isolated from strains expressing wild type Pmr1 but not from the null mutant K616,³ consistent with the proposed role of Pmr1 in Mn²⁺ transport. An alternative approach is to use Mn²⁺ inhibition of ⁴⁵Ca transport to assess Mn²⁺ transport activity, as reported earlier (11). Here, we examine the effectiveness of Mn²⁺ inhibition in the putative ion selectivity (Class 3) mutant Q783A. Fig. 5 shows that Mn²⁺ is a potent inhibitor of ⁴⁵Ca transport in Golgi vesicles containing wild type Pmr1, with half-maximal inhibition occurring at 0.2 µM, under conditions where the Ca²⁺ concentration is nominally 0.8 µM (approximately 10-fold in excess over the K_m for Ca²⁺; Refs. 10, and 11). These data suggest that ion selectivity of wild type Pmr1 is Mn²⁺ > Ca²⁺. In contrast, half-maximal inhibition for the Q783A mutant ocurred at 12 µM, a 60-fold shift relative to wild type. These results are consistent with a dramatic reduction in the affinity for Mn²⁺, and consequently hypersensitivity to Mn²⁺ toxicity (Fig. 1), in this mutant.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Manganese inhibition of 45Ca transport. 45Ca uptake into purified Golgi vesicles was assayed in the presence of added Mn2+, as indicated. 45Ca was fixed at 0.8 µM in the absence of chelators. The data points are averages of duplicate determinations, and the line is a best fit of the equation v = Vmax·I/(K0.5 + I), where I is the Mn2+ concentration and v is the measured rate of 45Ca transport. Derived values of K0.5 for wild type Pmr1 (WT) and mutant Q783A are in the text.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study, we describe an effective strategy to screen for amino acid residues critical for ion transport and selectivity in yeast mutants of the secretory pathway/Golgi ion pump, Pmr1. As a test of this approach, we used alanine-scanning site-directed mutagenesis of acidic and polar residues within the transmembrane segments most likely to constitute the translocation pore. Mutants were readily classified in one of three groups based upon cell growth in BAPTA- or Mn²⁺-supplemented medium. Class 1 mutants did not display hypersensitivity to either BAPTA or Mn²⁺, a first indication of normal function. Further analysis confirmed that these mutants showed normal trafficking to the Golgi, had significant ⁴⁵Ca transport activity and appeared to be correctly folded based upon the ability of ATP to protect against trypsinolysis. Most of the residues targeted for mutagenesis in this study fell into this class (Fig. 6) and were not essential for pump function, as was observed in previous site-directed mutagenesis studies of this scale on the Na⁺/K⁺-ATPase, H⁺-ATPase, and SERCA (reviewed in Refs. 4, 7, and 22).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Summary of mutants. Transmembrane segments M4-M8 are depicted as -helices, and residues targeted for mutagenesis in this study are color-coded based on the resulting phenotype (see text). When multiple substitutions were made, the effect of the least deleterious substitution is represented. The Class 2 mutants depicted in bold type showed structural and trafficking defects.

Class 2 mutants were hypersensitive to both BAPTA and Mn²⁺ toxicity, indicative of a complete lack of pump function, which was confirmed by the lack of ⁴⁵Ca transport activity in isolated Golgi fractions. To distinguish between structural and functional derangements, we analyzed protein trafficking and sensitivity to trypsinolysis. Mutants that showed normal Golgi trafficking also displayed ATP-protectable trypsin fragments, whereas those that showed ER retention failed to generate ATP-protectable tryptic peptides. The latter also displayed increased mobility upon gel electrophoresis, for reasons that are currently unresolved. These observations are consistent with abnormal polypeptide folding and indicate sites that are sensitive to structural perturbations. Unexpectedly, Asp  Glu and Glu  Asp substitutions caused structural problems, whereas replacement with the neutral alanine did not. Although the perturbation caused by introduction of the longer glutamate side chain may be rationalized as a steric effect, it is not clear why substitution of aspartate, a shorter carboxylate side chain, in place of glutamate was more deleterious than that of alanine. One possibility is that within the confines of the membrane, differences in dihedral angle preferences for the carboxyl group of aspartate and glutamate may be important.

Fig. 6 summarizes the results of our study. None of the target residues in transmembrane segments M4 or M8 were essential for function, although substitution-specific loss of function was observed at Glu³²⁹ and Asp⁸⁵⁶. Structural and trafficking defects were also seen with alanine substitution of Gln⁷⁴² (M5), Asn⁷⁷⁴ (M6), and Thr⁸¹⁷ (M7), resulting in complete loss of function. However, alanine and asparagine substitution mutants of Asp⁷⁷⁸ (M6) showed normal trafficking and polypeptide conformation yet lacked transport activity, making this residue a good candidate for the cation-binding pocket. Strikingly similar findings have been reported for mutations at residues equivalent to Asn⁷⁷⁴ and Asp⁷⁷⁸ of the plasma membrane Ca²⁺-ATPase: thus, mutant N879A was retained in endoplasmic reticulum, whereas mutant D883A showed normal trafficking to the plasma membrane (23). Both showed a complete loss of Ca²⁺ transport activity (23, 24). Alanine substitutions of the equivalent residues in the Na⁺/K⁺-ATPase, Asp⁸⁰⁴ and Asp⁸⁰⁸, however, do not prevent trafficking to the plasma membrane, as judged by ouabain binding ability in intact cells (7), but are also associated with loss of transport activity. Thus, these two sites appear to be common components of the cation-binding site in most P-type ATPases. One exception is the fungal H⁺-ATPase, Pma1, where Asp⁷³⁰ (equivalent to Asp⁷⁷⁸ of Pmr1) forms a salt bridge with Arg⁶⁹⁵ in M5; simultaneous substitution of both residues with alanine does not alter function (6).

The simple growth screens based on BAPTA and Mn²⁺ toxicity in pmr1 mutants are capable of distinguishing between essential and nonessential residues and can be used to screen large numbers of randomly generated mutants in future studies. We also show that the screens can identify potential sites of cation selectivity. Three such sites were identified in this study; in each case, the mutants showed greater sensitivity to Mn²⁺ toxicity than to that of the Ca²⁺ chelator BAPTA in growth assays, suggesting that the mutation had a more deleterious effect on Mn²⁺ transport. However, in a previous study, we have reported on a mutation with the converse effect; D53A, within the N-terminal modulatory EF hand-like domain in Pmr1, was indistinguishable from wild type in the Mn²⁺ toxicity assay (11), but BAPTA toxicity in this mutant was intermediate between wild type and the pmr1 null mutant.³ We showed that apparent Mn²⁺ affinity was slightly increased relative to wild type, whereas there was a 11-fold reduction in Ca²⁺ transport affinity in this mutant (11). Here, we show that Q783A has normal Ca²⁺ transport but a 60-fold reduction in the apparent affinity for Mn²⁺. Only one such mutation with a similar dramatic effect on ion selectivity has been reported in studies on other ion pumps; substitution of Ser⁷⁷⁵ in M5 with alanine in the Na⁺/K⁺-ATPase causes a 30-fold decrease in only K⁺ but not Na⁺ affinity (25, 26).

It is worth noting that only two of five residues required for Ca²⁺ binding in SERCA were essential in Pmr1. A likely interpretation is that unlike SERCA, which has a stoichiometry of two Ca²⁺ transported per ATP, Pmr1 transports only one cation transported per ATP. In the sequence of Pmr1 and its homologues, Ala and Met occupy positions equivalent to Glu⁷⁷¹ (M5) and Thr⁷⁹⁹ (M6) of SERCA, consistent with a fewer number of cation-binding sites. These differences support the classification of the newly emergent secretory pathway Ca²⁺-ATPases as a group distinct from but related to the well known endoplasmic reticulum pumps.

	FOOTNOTES

^* This work was supported by American Cancer Society Grants IRG11-33 and JFRA 538, American Heart Association Grant-in-aid 95012290, and National Institutes of Health Grant GM52414 (to R. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Physiology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore MD 21205. Tel.: 410-955-4732; Fax: 410-955-0461; E-mail: rrao@jhmi.edu.

Published, JBC Papers in Press, May 5, 2000, DOI 10.1074/jbc.M002618200

² D. M. Mandal and R. Rao, unpublished results.

³ Y. Wei and R. Rao, unpublished results.

	ABBREVIATIONS

The abbreviations used are: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PAGE, polyacrylamide gel electrophoresis; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; ER, endoplasmic reticulum.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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