INTRODUCTION

The Ca²⁺ pump from plasma membranes (PMCA)¹ is a calmodulin regulated, P₂-type ATPase (1) responsible for maintaining the intracellular Ca²⁺ homeostasis in eukaryotic cells by actively extruding Ca²⁺ to the extracellular space. Four mammalian genes coding for different PMCA isoforms have been identified, each of them producing different mRNAs by alternative splicing processes (2). The PMCA polypeptide would transverse the lipid bilayer about 10 times, and most of the pump, including two major loops and both terminal ends, would be exposed to the cytosol (3). The PMCA contains regions highly conserved in all P-type ATPases and other regions that are distinctive of the PMCA because they are found only in the PMCA isoforms. The regions of the PMCA encompassing the aspartate residue that forms the phosphorylated intermediate (4) and the putative ATP binding site (5) belong to the type of highly conserved regions. In contrast the C-terminal region after the transmembrane segment 10, which is involved in the regulation of the activity of the enzyme by calmodulin (6), and the N-terminal region upstream the first transmembrane segment belong to the regions that are not homologous to that of the other P-type ATPases.

Recently a PMCA mutant called hPMCA4b(ct120) with a deletion of the C-terminal 120 residues including the calmodulin-binding site was expressed in COS-1 cells and found to be fully active, and as expected its activity no longer regulated by calmodulin (7).

We have now investigated the functional relevance of the N-terminal region of the PMCA, which extends from the initial methionine to about the beginning of the first transmembrane segment. With this aim, we constructed a mutant called hPMCA4b(d18-75)(ct120) by removing the nucleotide sequence coding for amino acids 18-75 from the cDNA of hPMCA4b(ct120). The measurements of the activity of the hPMCA4b(d18-75)(ct120) expressed in COS-1 cells indicate that the residues 18-75 are essential for a functional Ca²⁺ pump.

MATERIALS AND METHODS

The construction of the cDNA of mutants hPMCA4b(ct120) and hPMCA4b(d18-75) was described previously, and the latter was called Hinm1 (3, 7). In addition to the deletion of residues 18-75, the presence of a new unique restriction site for MluI produced the replacement of serine 17 by threonine in the hPMCA4b(d18-75).

To obtain the hPMCA4b(d18-75)(ct120) mutant, the SalI-DraIII fragment was removed from hPMCA4b(d18-75) and cloned into h4PMCA(ct120). The wild-type and mutant cDNAs were cloned into the pMM₂ vector (7). For protein expression, COS-1 cells (9) were transfected by the DEAE-dextran-chloroquine method (8) and harvested after 48 h. The microsomal fraction was isolated as described previously (7). Protein concentration was estimated by means of the Bio-Rad protein assay, with bovine serum albumin as a standard.

SDS-electrophoresis and immunoblotting were carried out as described previously (10). Proteins were electrophoresed on a 7.5% acrylamide gel according to Laemmli (11) and subsequently transferred to Millipore Immobilon membranes. Nonspecific binding was blocked by incubating the membranes overnight at 4 °C in a solution of 160 mM NaCl, 0.05% Tween 20, and 1% nonfat dry milk. The membranes were incubated at 37 °C for 1 h with 5F10 monoclonal antibody from ascites fluid (dilution, 1:1000). For staining, biotinylated anti-mouse immunoglobulin G and avidin-horseradish peroxidase conjugate were used.

Ca²⁺ uptake assays were performed as described previously (10). The reaction mixture contained 100 mM KCl, 50 mM Tris-HCl (pH 7.3 at 37 °C), 5 mM NaN₃, 400 nM thapsigargin, 20 mM sodium phosphate, 6 mM ATP, 95 µM EGTA, and enough MgCl₂ and CaCl₂ to give a concentration of free Mg²⁺ of 800 µM and of free Ca²⁺ of 1 µM. The free concentrations of Mg²⁺ and Ca²⁺ were calculated using the program of Fabiato and Fabiato (12). Vesicles (5-10 µg of protein) were preincubated at 37 °C for 5 min, and the reaction was initiated by the addition of ATP. The reaction was terminated after 5 min by filtering the samples through a 0.45-µm filter. The ⁴⁵Ca taken up by the vesicles was then determined by counting in a liquid scintillation counter. Uptake activities were expressed per mg of COS-1 cell membrane protein. The activity of the expressed Ca²⁺ pump was estimated after subtracting the activity of the endogenous enzyme from COS-1 cells transfected with the empty plasmid pMM₂.

Ca²⁺ ATPase was measured by monitoring the [³²P]P_i liberated from [-³²P]ATP (13). The reaction was carried out at 37 °C for 30 min. The reaction mixture contained 50 mM Tris-HCl (pH 7.4 at 37 °C), 100 mM KCl, 0.1 mM MgCl₂, 0.1 mM EGTA, 0.1 mM ATP, 0.1 mM CaCl₂, 5 mM NaN₃, 0.5 mM ouabain, 4 µg/ml oligomycin, 400 nM thapsigargin, and 10 µg of membranes. The Ca²⁺ ATPase was taken as the difference of ATPase activities in the presence and the absence of Ca²⁺.

The phosphorylation reaction was carried out at 4 °C in medium containing 10 µg of microsomal protein, 100 mM KCl, 25 mM Tris-HCl (pH 7.4 at 4 °C), 400 nM thapsigargin, 5 mM MgCl₂, 0.02 µM CaCl₂, and 0.02 µM LaCl₃ in a reaction volume of 0.25 ml. La³⁺ is known to stabilize the phosphoenzyme of the plasma membrane Ca²⁺ pump (14). The reaction was initiated by the addition of 1 µM [-³²P]ATP and terminated after 30 s with 1 ml of stopping solution containing 10% trichloroacetic acid, 10 mM P_i, and 1 mM cold ATP at 4 °C. After adding 50 µl of 1 mg/ml of bovine serum albumin, the denatured proteins were collected by centrifugation at 20,000 × g for 10 min, washed once more with stopping solution, and washed once with distilled water. The precipitated samples were dissolved in sample buffer and separated by SDS-electrophoresis in a 7% acrylamide gel according to Sarkadi et al. (15). After drying the gel, autoradiographs were produced.

Digestion of COS-1 microsomes with trypsin was done on ice in a medium containing 10 µg of microsomes, 0.1 mM EGTA, 50 mM Tris-HCl (pH 7.7 at 4 °C), 0.25 mM sucrose, 0.15 mM KCl, 2 mM dithiothreitol. The reaction was initiated by the addition of trypsin to give a final concentration of 20 µg/ml and terminated after 15 or 30 s with 10-fold excess of soybean trypsin inhibitor. Controls of undigested microsomes (0 s) were done by adding the inhibitor before the trypsin.

RESULTS

COS-1 cells were transfected with the vector without insert or with either the hPMCA4b(ct120) or the hPMCA4b(d18-75)(ct120) DNAs. Different amounts of membranes from the transfected cells were submitted to SDS gel electrophoresis followed by immunoblotting using antibody 5F10, which recognizes an epitope located between amino acids 724-783 in the central part of the Ca²⁺ pump protein (3). The immunoblot is shown in Fig. 1. One band of about 140 kDa corresponding to the endogenous Ca²⁺ pump from COS-1 cells was detected in membranes from cells transfected with the empty vector pMM₂. An additional band of about 120 kDa was observed in membranes from hPMCA4b(ct120) transfected cells. Membranes from COS-1 cells transfected with hPMCA4b(d18-75)(ct120) DNA showed a band at about 114 kDa, the expected molecular mass of the hPMCA4b(d18-75)(ct120) protein. The intensity of the bands of the hPMCA4b(ct120) and hPMCA4b(d18-75)(ct120) were close, indicating that both proteins were expressed at a similar level.

As a test for the topology of the hPMCA4b(d18-75)(ct120) Ca²⁺ pump, microsomes containing the expressed protein were exposed briefly to the action of trypsin. Fig. 2 shows an immunoblot of the proteolyzed membranes using antibody 5F10. In agreement with previous reports (7), the treatment of COS-1 membranes expressing hPMCA4b(ct120) with 20 µg/ml of trypsin for 30 s resulted in the appearance of a major band of approximately 76 kDa.

Because the N-terminal part of the molecule containing amino acid residues 1-315 is removed early during proteolysis and is not recognized by 5F10 (3) providing the insertion and folding of the enzyme was the same, differences are not expected between the proteolytic pattern of the hPMCA4b(ct120) and hPMCA(d18-75)(ct120). As shown in Fig. 2 this was indeed the case, suggesting that both the hPMCA4b(ct120) and the hPMCA(d18-75)(ct120) proteins exposed the same sites to trypsin and hence produced similar proteolytic fragments containing the epitope for antibody 5F10.

Results in Table I show that the Ca²⁺ uptake of microsomes from cells transfected with the hPMCA4b(ct120) DNA was about 12 times higher than that from control microsomes from cells transfected with the empty vector. The Ca²⁺ transport activity of microsomes expressing the hPMCA4b(d18-75)(ct120) protein was not significantly different from that of the control, indicating that the mutant enzyme was not able to transport Ca²⁺. As also shown in Table I, the Ca²⁺ ATPase activity of microsomes from COS-1 cells transfected with the hPMCA4b(d18-75)(ct120) DNA was similar to that of the control, indicating that the hPMCA4b(d18-75)(ct120) was also not capable of hydrolyzing ATP in a Ca²⁺-dependent manner.

Table I. Ca2+ transport and Ca2+ ATPase activities of microsomes isolated from cells transfected with PMM2, hPMCA4b(ct120) or hPMCA4b(d18-75)(ct120) Ca2+ uptake and Ca2+ ATPase from COS-1 microsomes were estimated as described under ``Materials and Methods.'' Averages (± standard deviation) of duplicate measurements from three to five experiments done with different membrane preparations are shown. DNA transfected Ca2+ transport Ca2+ ATPase nmol Ca2+/mg membrane protein/min nmol Pi/mg membrane protein/min pMM2 0.14  ± 0.12 0.45  ± 0.25 hPMCA4b(ct120) 1.80  ± 0.34 6.08  ± 1.08 hPMCA4b(d18-75)(ct120) 0.25  ± 0.12 0.33  ± 0.43

In control experiments (not shown), the effects of the deletion of amino acid residues 18-75 on the activity of the full-length hPMCA4b was investigated. The hPMCA4b(d18-75) enzyme was also inactive, indicating that the C-terminal regulatory end of the PMCA was not involved in the mechanism leading to inactivation.

The first step of the normal Ca²⁺ pump cycle involves the reaction of the enzyme with Ca²⁺ and ATP to form a phosphoenzyme. The ability of the hPMCA4b(d18-75)(ct120) mutant to form a phosphorylated intermediate was investigated. Fig. 3 shows that two bands corresponding to the endogenous PMCA and sarco/endoplasmic reticulum Ca²⁺ pumps were observed when membranes from COS-1 cells transfected with the empty vector were phosphorylated with ATP in the presence of Ca²⁺ plus La³⁺. As expected an additional band in the molecular mass region of 120 kDa was observed in membranes containing the hPMCA4b(ct120) enzyme. The phosphorylation pattern of membranes containing the hPMCA4b(d18-75)(ct120) protein was similar to that of the control, indicating that no phosphorylation attributable to the hPMCA4b(d18-75)(ct120) mutant had occurred.

DISCUSSION

In the present work we have investigated the functional consequences of a deletion in the N-terminal region of the hPMCA4b between the initial methionine and the first transmembrane domain. Mutant hPMCA4b(d18-75)(ct120) was expressed in amounts comparable with those of the hPMCA4b(ct120), indicating that the N-terminal deletion did not significantly affect the level of expression of the PMCA. The measurements of Ca²⁺ transport and ATP hydrolysis indicated that the mutant protein was unable to perform any of these functions. In addition, no detectable amounts of phosphoenzyme were formed by the hPMCA4b(d18-75)(ct120) enzyme. Thus the lack of Ca²⁺ transport and ATPase activities of mutant hPMCA4b(d18-75)(ct120) could be ascribed to its inability to react with Ca²⁺ and ATP to form the phosphoenzyme.

Deletion of several amino acid residues could result in alterations of the folding or insertion in the membrane of the mutant protein. Although this possibility cannot be discarded, several observations suggest that this may not be the case of the hPMCA4b(d18-75)(ct120) mutant: (i) the expressed hPMCA4b(d18-75)(ct120) protein was always found associated with membranes, and after cell lysis it was recovered in the microsomal fraction, (ii) immunoblots of membranes from COS-1 cells transfected with the hPMCA4b(d18-75)(ct120) DNA showed only one major product of about 114 kDa in amounts similar to those of the hPMCA4b(ct120), indicating that both proteins were equally stable to degradation by intracellular proteases, and (iii) digestion with trypsin and immunoblotting of the hPMCA4b(ct120) and the hPMCA4b(d18-75)(ct120) revealed similar proteolytic fragments, suggesting that both proteins exposed the same limited number of sites to the protease.

Early analysis by SDS-polyacrylamide gel electrophoresis of the proteolytic fragments produced by treatment of red cell membranes with trypsin has shown that the protease acts on several specific sites of the PMCA resulting in major peptides of 90, 81, and 76 kDa (15, 16, 17). The appearance of the 76-kDa fragment in the SDS-polyacrylamide gel electrophoresis is concomitant with a highly active PMCA. Based on these results (15) concluded that the 76-kDa polypeptide was a fully competent Ca²⁺ transporter and therefore that the N- and C-terminal amino acid residues missing in the 76-kDa fragment are not essential for activity. However, as mentioned in Ref. 16, the effective separation of the N-terminal fragment containing the transmembrane domains 1 and 2 from the 76-kDa product in the absence of SDS was not proved.

Recently, the expression of N-terminally and C-terminally truncated PMCA mutants allowed to directly assess the activity of the fragments produced by trypsinolysis. Although truncation of the hPMCA4b generating a C terminus similar to that of the 76 kDa fragment produces a highly active enzyme (7), a mutant starting with the N terminus of the 90-kDa polypeptide (PMCA105) is inactive (18). As was suggested previously (18), the lack of activity of the PMCA105 mutant is probably related to the absence of a large portion of the pump containing the first two transmembrane domains and the region highly conserved in all P-ATPases, which has been called the transducing domain. Results in this paper show that a smaller deletion between the N terminus and the first transmembrane domain, a region previously assumed irrelevant for the function of the enzyme, suffices for inactivation. Studies of mutants with smaller deletions and substitutions of amino acids in the N-terminal region will be needed to obtain a more precise knowledge of the residues involved and their role during Ca²⁺ transport by the PMCA.