From the
Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Dr., Oxford OX3 7BN, United Kingdom, the ¶Laboratorium voor Fysiologie, Katholieke Universiteit Leuven, Campus Gasthuisberg O/N, Herestraat 49, B-3000 Leuven, Belgium, and the ||Department of Physiology, University of Aarhus, Ole Worms Allé 160, DK-8000 Aarhus C, Denmark
Received for publication, January 16, 2003 , and in revised form, April 9, 2003.
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ABSTRACT |
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INTRODUCTION |
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The first functional study on SPCA in animals was conducted on Caenorhabditis elegans. C. elegans SPCA was shown to transport Ca2+ and Mn2+ with high affinity into the Golgi following heterologous expression in COS-1 cells (11). Overexpression of human SPCA1 in yeast and Chinese hamster ovary cells was restricted to the Golgi compartment and, moreover, was able to complement the PMR1 null mutation, as demonstrated by the ability to transport Ca2+ and Mn2+ in yeast (12).
Key insights into structure/function relationships have been provided by functional analysis of over 250 point mutations in SERCA1a. This has led to identification of specific domains important in Ca2+ and ATP binding and phosphorylation, together with residues important in the conformational changes associated with the various stages of the catalytic cycle (13, 14) (Scheme 1). In contrast, mutagenic studies of the SPCA class have been limited and have only involved the S. cerevisiae protein. Mutations in the active phosphorylation site, Asp-372, had the expected effect of abolishing Ca2+ transport activity (15). Residues in the putative N-terminal EF hand-like motif were reported to have a modulatory effect on ion transport, through their importance in ion binding and participation in long range interactions involved in ATP binding and phosphorylation (16). Other reports have targeted the oxygen-containing side chains of the predicted transmembrane domains M4M8, likely to be involved in the coordination of Ca2+ and Mn2+ ions (17), and have defined Gln-783 as crucial in Mn2+ selectivity of PMR1 (18).
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We have previously described a spectrum of mutations scattered throughout the ATP2C1 gene in HHD patients (2, 19). Although nonsense, frameshift, and splice-site mutations are predicted to cause nonsense-mediated mRNA decay (20) or aberrant splicing, the underlying cause of disease in patients carrying missense mutations cannot be predicted a priori. In this study we investigated the molecular and physiological basis of HHD in patients carrying these mutations, providing new insights into SPCA structure/function relationships. Site-directed mutagenesis was used to introduce these disease-causing point mutations into the cDNA sequence of a novel splice variant of hSPCA1, which was identified and functionally characterized in this study. More than 50% of the mutants studied here showed low levels of protein expression, despite normal levels of mRNA and correct localization to the Golgi compartment. Other mutants are characterized by lack of ion transport, caused by specific alterations to the partial reactions of the catalytic cycle, such as defects in Ca2+ and Mn2+ binding and inability of the phosphoenzyme intermediate to undergo the energy-transducing E1P
E2-P conformational transition.
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EXPERIMENTAL PROCEDURES |
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Reverse Transcriptase-PCR AnalysesFollowing extraction of total RNA from human keratinocytes with TRIzol (Invitrogen), both reverse transcription of ATP2C1 and 3' rapid amplification of cDNA ends (RACE)-PCR, were performed using the MarathonTM cDNA amplification kit (Clontech, Palo Alto, CA). The gene-specific primer, ATP2C13'GSP (5'-CCAAGTCTGTGTTTGAGATTGGACTCTGC-3'), corresponded to nucleotides (nt) 26182646 in the ATP2C1d sequence. Products were subcloned into the PCR-cloning vector pGEM-T (Promega, Madison, WI) and sequenced in both directions. Splice variants identified by sequencing were subsequently amplified by PCR using ATP2C13'GSP and antisense primers Ex27R (5'-TTGCCCTTCTAAATGATCCTC-3') or Ex28R (5'-GGAAGAGCTGCAGGAAGATG-3') in standard reaction conditions. Ex27R and Ex28R, respectively, represent the inverse complement of nt 30393059 and 30483067 in the ATP2C1a and ATP2C1d sequences. PCR conditions were as follows: 4 min of denaturation at 95 °C and 30 cycles of 30 s at 95 °C,30sat58 °C, and 30 s at 72 °C.
First strand cDNA was prepared for LightCyclerTM-based real-time PCR analysis (Roche Applied Science) following total RNA extraction from transfected COS-1 cells using the Absolutely RNATM RT-PCR Miniprep kit (Stratagene). A 276-bp PCR product was generated using ATP2C1 primers C7F (5'-TTGGTTGGCTGGTTACTGGG-3') and C7R (5'-AGCATGCAGACCATCTGAAGT-3'), corresponding to nt 973992 and the inverse complement of nt 12281248, respectively, in the ATP2C1d sequence. GAPDH amplification was performed, to control for equal RNA loading between samples, using primers GAPDHF (5'-ATCATCCCTGCCTCTACTGG-3') and GAPDHR (5'-TGCTGTAGCCAAATTCGTTG-3') to generate a 349-bp product. Sequences correspond to nt 120139 and the inverse complement of nt 449468 in the GAPDH sequence. Real-time PCR amplification was performed with 1 µl of cDNA product in a 20-µl reaction containing 0.5 µM of each primer and QuantiTectTM SYBR® Green PCR Master Mix (Qiagen, W. Sussex, UK). The PCR cycle consisted of 15 min of denaturation at 95 °C, followed by 45 cycles of 15 s at 95 °C, 20 s at 60 °C, and 20 s at 72 °C. Fluorescence data was acquired at the end of each extension cycle. To confirm amplification specificity, PCR products were subjected to a melting curve analysis, as described previously (21). Analysis of real-time PCR data was performed using the LightCyclerTM software (Roche Applied Science). Crossing points, defined as the cycle number at which all samples are exponentially amplifying, were recalculated into relative log concentrations of RNA according to PCR efficiency calculated from standard curves generated using serially diluted cDNA for GAPDH and ATP2C1 primer sets. ATP2C1 amplification was then corrected for equal GAPDH expression between samples. PCR products were isolated from the LightCyclerTM capillaries after cycling and visualized following electrophoresis on 1.5% agarose gels.
cDNA Construction, Site-specific Mutagenesis, COS-1 Cell Culture, and TransfectionA full-length cDNA clone encoding ATP2C1d was constructed using cDNA fragments amplified by 5' and 3' RACE-PCR and was subcloned into vector pSPORT1 (Invitrogen). The full-length ATP2C1d cDNA was then transferred into the mammalian expression vector pMT2, with the insert corresponding to the entire coding sequence (nt 125-end) of the deposited ATP2C1d nucleotide sequence. Missense mutations originally reported in HHD patients by Sudbrak et al. (2) and Dobson-Stone et al. (19) were introduced into the wild type ATP2C1 expression clone using the QuikChangeTM XL site-directed mutagenesis kit (Stratagene). The full-length cDNA clones were sequenced in both directions to ensure no additional sequence changes had been introduced during mutagenesis. Details of mutations introduced into the hSPCA1 protein sequence are given in Table I. COS-1 cells for immunocytochemistry, microsome preparation, and isotope flux were cultured and transiently transfected with FuGENETM 6 transfection reagent (Roche Applied Science) as previously described (11). For total RNA or protein extraction 1 x 106 COS-1 cells were seeded in 60-mm culture dishes 24 h before transfection.
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Preparation of Antiserum to hSPCA1 ProteinAntibodies were raised essentially as described before (11). ATP2C1 cDNA was amplified using primers ATP2C1CYTF (5'-GCATGCTGTGAAAAAGCTGCCTATTG-3'; ATP2C1d nt 11221141) and ATP2C1CYTR (5'-GTCGACGCAACCTTTGGTACTATTTG-3'; ATP2C1d nt 19541973) containing, respectively, SphI and SalI sites at their 5'-end. PCR cycling conditions were as follows: 94 °C for 2 min, followed by 30 cycles of 10 s at 94 °C, 30 s at 57 °C, and 60 s at 68 °C. After 10 cycles the extension time was increased to 90 s. The PCR amplification product encoded a fragment of hSPCA1 corresponding to the large cytoplasmic loop between transmembrane segments 4 and 5. The SphI/SalI-cut PCR fragment was ligated into the corresponding sites in the pQE-31 bacterial expression vector (Qiagen). Following expression in Escherichia coli using the QIAexpress Type IV System (Qiagen), the recombinant protein migrated at the expected size of 33 kDa on a 12% SDS-polyacrylamide gel.
Protein Preparation and Immunostaining AnalysesFor total protein extractions COS-1 cells were washed twice in phosphate-buffered saline before lysis in freshly prepared buffer (150 mM NaCl, 50 mM Tris HCl (pH 8), 5 mM EDTA, 2% SDS, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml each of leupeptin, pepstatin, chymostatin, and antipain). After 30-min incubation on ice, insoluble proteins were pelleted at 6000 x g. Soluble protein was retained in the supernatant. Microsomes were isolated from COS-1 cells as described by Verboomen et al. (22). Protein concentrations were determined by using the bicinchoninic acid method (Pierce, Rockford, IL). For Western blotting, proteins were separated and transferred onto Immobilon-P membranes (Millipore, Edinburgh, UK) using the NuPAGE® system with 412% Bis-Tris pre-cast gels (Invitrogen). For immunofluorescence, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. Cells and membranes were quenched with 3% bovine serum albumin and 1% goat serum, followed by incubation with primary and secondary antiserum as described in the figure legends. Membranes were then incubated with ECF substrate (Amersham Biosciences) and fluorescent imaging was performed using a Storm 840 PhosphorImager in combination with ImageQuaNTTM software (Amersham Biosciences).
45Ca2+/54Mn2+ FluxesThese radioactive isotope fluxes were essentially performed as described earlier (11). Cells were loaded with 120 mM KCl, 30 mM imidazole-HCl (pH 6.8), 5 mM ATP, 0.44 mM potassium EGTA (pH 6.8), 10 mM NaN3, and 2 µM thapsigargin to inhibit SERCA activity. Unless otherwise stated, cells were loaded for 90 min and 5 mM potassium oxalate was included in the loading medium. MgCl2, CaCl2, and MnCl2 were added to obtain a free Mg2+ concentration of 0.5 mM (unless otherwise stated) and the indicated concentrations of free Ca2+ and Mn2+. Calcium ionophore A23187 [GenBank] (10 µM) was included in the efflux medium as indicated.
Analysis of Phospho-intermediatesPhosphorylation from [-32P]ATP, processing of the acid-precipitated microsomal proteins, and acidic SDS-PAGE electrophoresis were essentially performed as described before (11). The rate of dephosphorylation of the phosphoenzyme intermediate formed from ATP was analyzed after treatment with EGTA and ADP for serial time intervals prior to acid quenching (23, 24). Phosphorylation was performed in 100 µl of a solution containing 160 mM KCl, 17 mM potassium Hepes (pH 7), 5 mM NaN3, 1 mM dithiothreitol, and 2 µM thapsigargin. Phosphorylation from 0.5 mM 32Pi was performed at 25 °C as before (23). Quantification of the separated phosphoenzyme band was performed by imaging using the Packard CycloneTM storage phosphor system (Packard Bioscience, Berkshire, UK). Appropriate background phosphorylation levels were subtracted before data analysis as described (24).
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RESULTS |
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Characterization of the Transport Capacity for Ca2+ and Mn2+ of Novel Splice Variant hSPCA1dFor functional characterization, hSPCA1d was transiently expressed in COS-1 cells. First, its ability to transport Ca2+ and Mn2+ was investigated. Because the fraction of Golgi-derived membranes accounts for only a small proportion of total ER in microsomal fractions, conventional techniques used to measure SERCA-mediated Ca2+ transport activity into isolated microsomes (22) could not be employed here. Alternatively, all Ca2+ uptake measurements were performed using detergent-permeabilized whole cells (25). COS-1 cells transfected with empty vector (control) or wild type hSPCA1d cDNA were first permeabilized with saponin in a medium mimicking cytosolic composition and then loaded with 45Ca2+ in the presence of sodium azide (NaN3), to inhibit mitochondrial pump activity, and in the presence of thapsigargin, to inhibit SERCA pump activity. Efflux of the Ca2+ taken up by active pumps was then followed for 16 min in Ca2+-free medium, with the ionophore A23187 [GenBank] being added after 8 min. Fig. 2A shows that hSPCA1d-transfected cells exhibit a significantly higher thapsigargin-insensitive Ca2+-uptake compared with control cells. The addition of A23187 [GenBank] enhanced the release of stored Ca2+, suggesting that the Ca2+ had been transported into a membrane-delineated compartment. These results support the theory that hSPCA1d represents a thapsigargin-insensitive Golgi pump. Furthermore, the addition of 5 mM potassium oxalate to the loading medium stimulated an increase in the Ca2+-uptake capacity of hSPCA1 by more than 50%, when compared with uptake in the absence of oxalate. Oxalate acts to increase the ability of hSPCA1d to transport Ca2+ into the Golgi by precipitating Ca2+ and thus reducing the free Ca2+ concentration within the Golgi lumen. Fig. 2B compares the time-dependent loading of 45Ca2+ in hSPCA1d-expressing COS-1 cells versus control cells. A higher Ca2+ content was observed in transfected cells, compared with control cells, with increasing loading time. However, because more cells are prone to detach from the culture plate with prolonged loading times, subsequent experiments were all maintained at 90-min loading. Fig. 2C shows that half-maximal activation of hSPCA1d occurred at 0.20 µM free Ca2+, which is equivalent to that of 0.25 µM Ca2+, previously reported for the orthologue from C. elegans (11) and 0.26 µM reported for hSPCA1a, following heterologous expression in yeast (12).
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In the next series of experiments, the ability of hSPCA1d to transport Mn2+ was assessed. First, the ability of Mn2+ to inhibit 45Ca2+ uptake was evaluated, as an indirect indicator of Mn2+-uptake capacity. Fig. 3A clearly shows that, at higher Ca2+ concentrations, more Mn2+ was needed to inhibit Ca2+ uptake. Half-maximal inhibition was observed at 0.86 and 0.36 µM Mn2+ when loaded with 1 or 0.1 µM Ca2+, respectively. The respective values reported for C. elegans SPCA were 1 and 0.2 µM Mn2+. Fig. 3 (B and C) provide direct evidence that hSPCA1d can transport Mn2+. COS-1 cells transfected with hSPCA1d showed an increased level of 54Mn2+ uptake relative to control cells (Fig. 3B). Following the addition of ionophore A23187 [GenBank] after 4 min of efflux, the rate of Mn2+ release was increased. Fig. 3C shows efflux curves from cells loaded with 1 µM Mn2+ at various free Ca2+ concentrations in the loading medium. The Mn2+ uptake became increasingly inhibited by the increased Ca2+ concentration, thus demonstrating the competition between Ca2+ and Mn2+.
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Expression and Cellular Localization of Wild Type and HHD Mutant hSPCA1dAs a first step in the analysis of the HHD mutants, expression and cellular localization were compared with that of the wild type, because even minor mutation-induced structural abnormalities could, in principle, lead to protein degradation and/or cellular mistargeting.
The expression of wild type hSPCA1d in COS-1 cells transfected with the corresponding cDNA was clearly demonstrated by both immunocytochemistry and Western blot analysis (Fig. 4, A and B) using a newly generated rabbit polyclonal antibody raised against the large cytoplasmic loop of the hSPCA1 protein. The low level of hSPCA1 reactivity seen in control COS-1 cells indicated that our antiserum was able to detect the low level of endogenous COS-1 SPCA1 homologue. Immunocytochemical analysis of wild type hSPCA1-expressing COS-1 cells illustrates that the protein was correctly targeted to the Golgi. This result was further confirmed by co-localization of hSPCA1d with the key Golgi markers TGN46 (Fig. 4A) and mannosidase II (data not shown). Additionally, all nine HHD mutants studied were also correctly targeted to the Golgi, as exemplified by the cellular localization of D742Y. Wild type and mutant protein expression levels were compared by Western blot analysis of total protein extracted from transfected COS-1 cells (Fig. 4B, top row). The hSPCA1-specific immunoreactive bands migrated slightly below the predicted molecular mass of 104 kDa. P201L, G309C, I580V, and D742Y expression was comparable to wild type, whereas L341P, C344Y, C411R, T570I, and G789R were only marginally expressed above background. Equal protein loading was confirmed by re-staining of the stripped membrane with an antibody against -tubulin (Fig. 4B, bottom row). The low levels of hSPCA1d protein observed for L341P, C344Y, C411R, T570I, and G789R were not accompanied by correspondingly low mRNA levels, as documented by real-time PCR, performed in the presence of SYBR Green 1. ATP2C1 mRNA copy numbers were calculated to be 250-fold higher in COS-1 cells transfected with the wild type cDNA, compared with endogenous levels. The mRNA levels for all nine HHD mutants did not differ significantly from the wild type ATP2C1 mRNA level (data not shown). PCR products isolated from the LightCyclerTM capillaries after cycling are shown in Fig. 4C.
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Formation of hSPCA1 Phosphoenzyme Intermediate from ATP and PiA characteristic of the reaction mechanism of phosphorylation-type ion transport ATPases, cycling under normal (forward) conditions, is the transient transfer of the -phosphate of ATP to the protein. This phosphorylation reaction requires that one or more of the transported ions are bound to the enzyme. Conversely, in the SERCA enzymes, phosphorylation from inorganic phosphate in the reverse reaction is inhibited by the presence of Ca2+ (24, 26), presumably because Ca2+ binding transforms the Pi-reactive E2 conformation into the E1 conformation (see Scheme 1). It has not previously been determined whether the hSPCA1 enzyme responds in a similar way and whether Mn2+ exerts similar effects as Ca2+ on this enzyme. An interesting difference between the SERCA and SPCA is that, although the transport stoichiometry in the former is two per cycle, the latter is likely to bind and transport only one Ca2+ or Mn2+ per cycle, because only the residues binding one of the two Ca2+ ions in SERCA (corresponding to the so-called "site II") are conserved in SPCA. In the next series of experiments, we investigated the ability of the overexpressed wild type and HHD mutant hSPCA1d to phosphorylate from [
-32P]ATP or 32Pi in a Ca2+- and Mn2+-dependent way. Phosphorylation from ATP was performed using conditions known to favor accumulation of the E1
P form of SERCA in the presence of Ca2+ (0 °C, neutral pH, presence of alkaline metal ions) (23). Fig. 5B clearly shows that as little as 5 µM Ca2+ or Mn2+ is required to activate the phosphoryl transfer from ATP in wild type hSPCA1d. Phosphorylation from Pi was performed in conditions known to favor SERCA E2-P accumulation in the absence of Ca2+ (acidic pH, absence of alkali metal ions, and presence of the organic solvent dimethyl sulfoxide (26)). Fig. 5C shows that these conditions also allow formation of phosphoenzyme from Pi in the SPCA class. Furthermore, 2.5 mM Ca2+ or Mn2+ completely prevented phosphorylation from Pi in wild type hSPCA1d, indicating that Ca2+ and Mn2+ have a similar effect on the E1-E2 conformational equilibrium of the SPCA class to that shown for Ca2+ with SERCA. These phosphorylation assays furthermore permitted assessment of the intactness of the ion binding sites in the SPCA mutants, following the same principles as those previously applied to SERCA1a mutants (13, 14). Phosphorylation reactions were performed with equal amounts of hSPCA1 protein, taking into account any small differences in the level of hSPCA1 expression found among the different microsome preparations by Western blot analysis (Fig. 5A). This enabled the direct comparison between wild type and mutant phosphoenzyme levels. Our results indicate that Ca2+ or Mn2+ bind with normal affinity to P201L and I580V, causing activation of phosphorylation from ATP (Fig. 5B) and inhibition of phosphorylation from Pi (Fig. 5C). As seen in Fig. 5B, P201L phosphorylation from ATP was stimulated to a similar level as the wild type by 5 µM Ca2+ and Mn2+, whereas a higher level of phosphorylation was observed for I580V. The latter increase can be explained by the reduced rate of E1
P
E2-P conversion observed for this mutant, as described below, because the amount of phosphoenzyme accumulated at a given Ca2+ concentration depends on the rate of phosphoenzyme turnover as well as the Ca2+-dependent rate of phosphoenzyme formation.
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In contrast, G309C and D742Y were unable to form significant amounts of phosphoenzyme from ATP in the presence of 5 µM Ca2+ or Mn2+ (Fig. 5B), although a high level of phosphoenzyme was produced from Pi (Fig. 5C). Because these mutations involve residues within the transmembrane sector, located far from the nucleotide binding and phosphorylation domains, the lack of phosphoenzyme formation from ATP is not likely to be caused by a direct effect on nucleotide binding or catalysis but is, rather, due to defective ion binding and/or signal transduction from the ion binding site to the phosphorylation domain. The inability of D742Y to bind either of the transported ions is further verified by the observation that phosphorylation of D742Y from Pi remained high in the presence of 2.5 mM Ca2+ or Mn2+; a concentration that completely prevented phosphorylation from Pi in the wild type (Fig. 5C). Interestingly, phosphorylation of G309C from Pi remained high in the presence of 2.5 mM Mn2+, but was inhibited by 2.5 mM Ca2+ (Fig. 5C). This shows that, although this mutation entirely abolishes the ability to bind Mn2+, the enzyme can still bind Ca2+, albeit with a reduced affinity, because the phosphorylation from ATP was not activated by 5 µM Ca2+.
Dephosphorylation from E1PThe decay of the E1
P phosphoenzyme intermediate formed from ATP can occur either in the forward direction, via the conformational change to E2-P and subsequent E2-P hydrolysis, or in the reverse direction, through transfer of the phosphoryl group to ADP, yielding ATP (see Scheme 1). The rate of phosphoenzyme decay in the forward direction can be monitored by determination of residual phosphoenzyme levels upon treatment with EGTA (1 mM, to remove Ca2+ and, thereby, prevent further phosphorylation) for serial time intervals, prior to acid quenching. This assay can therefore be used to identify mutants in which the high energy, phosphorylated E1
P conformation can only move forward slowly, if at all, into the E2-P conformation. By addition of ADP (1 mM) to dephosphorylate E1
P in the reverse direction, the amount of residual phosphoenzyme in the E2-P (ADP-insensitive) form can also be measured. Therefore, mutants defective in either the E1
P
E2-P conversion or E2-P hydrolysis can be identified. The slow decay of I580V phosphoenzyme relative to wild type shown in Fig. 6 demonstrates the limited ability of this mutant to go through the E1
P
E2-P conformational change. Approximately 80% of I580V phosphoenzyme remained after 120 s treatment with EGTA, compared with only 10% of the wild type. The amount of phosphoenzyme in the ADP-insensitive (E2-P) conformation was similar to wild type levels, demonstrating that this mutant was not defective in E2-P hydrolysis. Conversely, the rate of dephosphorylation and the ratio of E1
P to E2-P for P201L were similar to wild type levels.
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DISCUSSION |
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The ubiquitous expression of ATP2C1 mRNA has previously been demonstrated in several tissues (3). We have further studied the mRNA tissue distribution pattern in 76 fetal and adult human tissues by dot blot hybridization analysis and confirmed that hSPCA1 is ubiquitously expressed.2 However, the physiological significance of the presence of four alternatively spliced transcripts in keratinocytes remains to be investigated. Studies of SERCA and PMCA have revealed that divergence in the extreme C-terminal can be responsible for some functional differences between the splice variants (22, 3336). Unfortunately, due to the high degree of sequence overlap at both the DNA and protein levels, it is impossible to generate hSPCA1 variant-specific DNA probes or antibodies that would be required to study any subtle differences in function or expression resulting from divergence in C-terminal sequence. Interestingly, some indications that functional differences do exist between the splice variants can be drawn from a previous study, where we identified a splice acceptor mutation in intron 26 of an HHD patient (19). With the likely effect of impaired splicing to exon 27, it can be inferred that hSPCA1a, hSPCA1b, and hSPCA1d cannot be produced from the mutant allele, because these splice variants contain amino acid stretches encoded by different parts of exon 27 (see Fig. 1). Although the reported mutation should not affect the formation of the ATP2C1c splice variant, the manifestation of classic HHD symptoms in the patient carrying this mutation does not suggest any functional compensation brought about by hSPCA1c. This lack of compensation is most likely due to the disruption of transmembrane segment 10, resulting from the exclusion of exon 27, and suggests that hSPCA1c has very limited functional capability, if any. For this reason, together with the low level of ATP2C1c mRNA expressed in keratinocytes, we have not attempted further characterization of this variant. Instead we have focused on the longest variant expressed in keratinocytes, ATP2C1d (hSPCA1d), which encodes optional 3' exons 27 and 28 in their entirety and, therefore, was thought most appropriate for the consecutive study of disease-causing point mutations.
Recently, the first functional evidence that hSPCA1 is a bona fide member of the SPCA class was reported following heterologous expression of hSPCA1a in yeast (12). Using an isotope flux-based procedure, we show here that hSPCA1d can transport both Ca2+ and Mn2+ into the Golgi apparatus of COS-1 cells with high affinity and in a thapsigargin-insensitive manner. A novelty of the experimental system used here relative to that previously applied in studies of C. elegans SPCA1 (11), is the addition of 5 mM potassium oxalate to the loading medium. We have shown that the addition of oxalate greatly increases the uptake capacity of the hSPCA1-containing store. The ability of oxalate to penetrate the Golgi membrane has been a point of some debate; however, Ca2+ oxalate precipitations have previously been reported in the Golgi of smooth muscle (37). The Ca2+ buffering capacity of oxalate enabled the loading time during isotope flux to be extended to 90 min without saturation of internal stores. This increased the observed uptake of hSPCA1d relative to background levels, thus strengthening the credibility of the data.
By overexpression of hSPCA1d in COS-1 cells, we have demonstrated that the enzyme has a high affinity for Ca2+ equal to that previously demonstrated for hSPCA1a (12) and C. elegans SPCA1 (11). By comparison, S. cerevisiae PMR1 was shown to have an even higher affinity for Ca2+ (K0.5 = 70 nM) (15). We have shown for the first time the direct transport of Mn2+ by hSPCA1 and provide evidence for the competitive uptake of Ca2+ and Mn2+, through inhibition of Ca2+ uptake by increasing concentrations of Mn2+ and vice versa. Half-maximal inhibition of Ca2+ uptake occurred at a Mn2+ concentration approximately equivalent to the Ca2+ concentration required for half-maximal stimulation of the uptake, indicating that the affinity of the hSPCA1d transport ATPase for Ca2+ and Mn2+ is approximately the same. In contrast, similar studies performed on S. cerevisiae PMR1 showed this latter protein to have a somewhat lower affinity for Ca2+ than Mn2+ (17). Conclusions drawn from the isotope fluxes were further verified by the results of phosphoenzyme studies, which indicated that maximal phosphoprotein formation from ATP was achieved at micromolar concentrations of Ca2+ and Mn2+, demonstrating the high affinity nature of the binding sites. Furthermore, phosphorylation was activated to an equal extent by Ca2+ and Mn2+, supporting the notion that these ions are bound with equal affinities.
The large number of nonsense, frameshift, and splice site mutations identified in the ATP2C1 gene of HHD patients support the theory that haploinsufficiency of hSPCA1, via nonsense-mediated mRNA decay of the mutant allele, is a prevalent mechanism for the dominant inheritance of HHD. However, the precise functional consequence of the missense mutations found in patient ATP2C1 cannot be predicted a priori. All nine of the HHD missense mutations studied here occur at residues that are invariant across a range of hSPCA1 orthologues. This high degree of conservation suggests that the residues involved are essential for correct protein function. In this study we have provided evidence that these missense mutations are responsible for low expression levels, or defects in Ca2+ and Mn2+ binding, or the energy-transducing E1P
E2-P conformational change. By looking at the precise effect of the mutation on protein function, these results outline the underlying cause of HHD in these patients. Furthermore, this work provides an insight into structure/function relationships in the SPCA class.
We have shown that the low expression level of mutant proteins L341P, C344Y, C411R, T570I, and G789R is not the result of impaired mRNA levels. Instead, we suggest that these non-conservative mutations introduce structural perturbations into the hSPCA1 protein, resulting in either abnormal protein folding or destabilization of the correctly folded protein, thus making it sensitive to endoplasmic reticulum-mediated quality control. Indeed, such an effect was previously observed when deletions or specific substitutions of a few residues were made in the N-terminal region of SERCA1a (38). In this study of SERCA1a, the mutations were shown to have no effect on transcription, translation, or integration of the protein into the membrane but, rather, induced protein degradation at a rate substantially faster than the wild type. The detection of some mutant L341P, C344Y, C411R, T570I, and G789R proteins, which had correctly localized to the Golgi in COS-1 cells, supports the hypothesis that these amino acid substitutions might induce an abnormally rapid rate of degradation of the mutant proteins, because it indicates that at least some of the mutant protein does escape endoplasmic reticulum-mediated quality control. Moreover, these results further support the theory of haploinsufficiency as a mechanism for the dominant inheritance of HHD, by suggesting that epidermal cells are sensitive to levels of hSPCA1.
Only one of the hSPCA1 mutations (T570I) resulting in a low level of protein expression occurred at an amino acid residue that has been studied at its conserved site in SERCA1a (Thr-625). It has been proposed that Thr-625 in SERCA1a, which is an integral part of the highly conserved TGD motif, might participate in the formation of the nucleotide-binding site (39). Conservative mutations in Thr-625 were shown to result in a Ca2+ transport deficiency and impaired abilities to form a phosphorylated intermediate with ATP or Pi (40). Interestingly, as shown here for T570I, the change T625V in SERCA1a also resulted in a marked decrease in expression compared with the level observed for the wild type. Analogous to conclusions drawn from this study, the authors suggested that such a large change in amino acid polarity might have led to a structural alteration.
The evidence presented in this study suggests that Asp-742 is equally important for the binding of Ca2+ and Mn2+, whereas Gly-309 seems to influence Mn2+ binding more profoundly than Ca2+ binding. These conclusions are based on the observations that D742Y and G309C phosphorylation from ATP was not activated by 5 µM Ca2+ or Mn2; ion concentrations known to almost saturate the wild type enzyme, as judged by the results of our isotope fluxes. Phosphorylation of D742Y from Pi was not inhibited at 2.5 mM of either ion, whereas phosphorylation of G309C from Pi was inhibited at 2.5 mM Ca2+, but not at 2.5 mM Mn2+. The latter finding shows that G309C is unable to bind Mn2+ even at 2.5 mM, whereas Ca2+ is bound with a moderately reduced affinity, intermediate between 5 µM and 2.5 mM. An equivalent assay was originally used to demonstrate the inability of SERCA1a mutants with respect to Ca2+ binding (41, 42). The importance of the hSPCA1 residue Asp-742 in ion binding is not surprising, in view of the results obtained in intensive mutagenic and structural studies of Asp-800, the equivalent amino acid in SERCA1a. All evidence points toward this residue forming an integral part of the Ca2+-binding site, by acting as a Ca2+-binding ligand through the donation of side-chain oxygen atoms (4145). More recently, Ca2+ and Mn2+ uptake has been shown to be inhibited by mutations at the equivalent residue, Asp-778, in S. cerevisiae PMR1 (17), supporting our evidence that ion binding is conserved at this site within the SPCA class. It is interesting to note that, although Asp-800 donates a side-chain oxygen atom to each of the two Ca2+-binding sites in SERCA1a (41, 45), this residue still plays a crucial role in SPCA ion binding, where only one ion-binding site, corresponding to site II in SERCA1a (45), appears to be conserved (based on comparison with the SERCA1a sequence). Furthermore, our studies show that, in the wild type hSPCA1, Ca2+ or Mn2+ binding inhibits phosphorylation from Pi, similar to the effect of Ca2+ binding in SERCA1a (41, 42), despite the fact that in SERCA1a this effect is caused by Ca2+ binding at the site (site I) that apparently does not exist in SPCA. Hence, it can be concluded that the inhibition of Pi phosphorylation occurs irrespective of which Ca2+ site is being occupied, supporting the notion that inhibition is caused by displacement of the E1-E2 conformational equilibrium away from the Pi-reactive E2 form (see Scheme 1).
The difference between the effects of HHD mutant G309C on Ca2+ and Mn2+ binding observed here suggest that Gly-309 is important for Mn2+ transport selectivity. Gly-309 is located immediately adjacent to the putative ion-binding residue, Glu-308, in transmembrane segment M4. Studies of the equivalent residue, Gly-310, in SERCA1a infer that mutations could sterically hinder ion binding, with the Ca2+ affinity of the enzyme decreasing as the size of the substituted side chain in this position was increased (41). Recently, a mutation in the putative transmembrane segment M4 Ca2+-binding site of S. cerevisiae was shown to have a more deleterious effect on the transportation of Mn2+ in comparison to Ca2+ (17). This observation supports our conclusion that this region of the protein plays an important role in hSPCA1 Mn2+ transport selectivity. Furthermore, structural information has highlighted the close interaction between transmembrane segments M4 and M6 in SERCA1a (45). With the recent identification of Gln-783 in transmembrane segment M6 as an important residue in Mn2+ selectivity of S. cerevisiae PMR1 (18), it seems conceivable that specific residues in M4 and M6 could interact to control uptake of this ion in the SPCA class.
The relatively conservative mutation, I580V, has been identified as a conformational change mutant in this study. No information regarding this or adjacent residues in the phosphorylation domain has been reported for the equivalent site in SERCA1a. However, it now seems that the I580V mutation disrupts the crucial conformational change associated with the transformation of the enzyme from the "high energy" state (E1P), to the "low energy" state (E2-P). Interestingly, in the crystal structure of SERCA1a (45) the equivalent residue Ile-635 has been shown to be located close to the hinge between the phosphorylation and nucleotide-binding domains, which could explain the involvement in the conformational change if a hinge-bending movement occurs in relation to the E1
P
E2-P transition.
The fact that the P201L mutation was not detected in 100 control chromosomes suggests that it does not represent a benign polymorphism. However, we found little evidence that the hSPCA1 enzymatic cycle was directly affected in this mutant protein, which correctly localized to the Golgi and was expressed to a similar level as the wild type. These results are concurrent with previous observations made following investigation of the equivalent mutation, P195L, in SERCA1a (46). Interestingly, a 15% reduction in the Vmax for Ca2+ transport was observed for P201L in isotope fluxes, whereas the apparent affinity for Ca2+ was close to wild type levels (data not shown). However, the validity of comparing the ion transport capacity of different proteins by isotope flux was questioned in view of the differential levels of toxicity exerted on cells by the transfection of individual constructs. The potentially variable loss of cells from culture plates transfected with different constructs would have a direct effect on the ion uptake figures obtained during each experiment. Nevertheless, in view of this result, it is tempting to speculate that such a reduction could be the cause of the HHD phenotype in patients carrying this mutation. Alternatively, the reduction of the Vmax for Ca2+ transport could be explained by the existence of slower steps in the catalytic cycle compared with the wild type, such as those associated with the ion-binding transition (E2 E1(Ca2+/Mn2+)). However, it is also possible that the mutation might affect an important interaction with a regulatory protein present in keratinocytes.
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* This work was supported in part by the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Wellcome Trust Prize Studentship.
** Holder of a Wellcome Trust Senior Clinical Fellowship. To whom correspondence should be addressed: INSERM U563, Pavillon Lefebvre, Purpan Hospital, Place du Dr Baylac, 31059 Toulouse cedex 03, France. Tel.: 33-561-158-432; Fax: 33-561-499-036; E-mail: alain.hovnanian{at}toulouse.inserm.fr.
1 The abbreviations used are: PMCA, plasma membrane calcium adenosine triphosphatase; (h)SPCA1, (human) secretory pathway Ca2+/Mn2+-adenosine triphosphatase; HHD, Hailey-Hailey disease; SERCA, sarco(endo)plasmic reticulum calcium adenosine triphosphatase; PMR, plasma membrane ATPase-related; cDNA, complementary DNA; RACE, rapid amplification of cDNA ends; GSP, gene-specific primers; nt, nucleotide(s); D, donor; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid.
2 R. J. Fairclough, L. Dode, J. Vanoevelen, J. P. Andersen, L. Missiaen, L. Raeymaekers, F. Wuytack, and A. Hovnanian, unpublished data.
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