Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport

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 Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) 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 Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca 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, Asn(774) and Asp(778) 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 Ca(2+) and Mn(2+) 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.     

Golgi manganese transport is required for rapamycin signaling in Saccharomyces cerevisiae

The Pmr1 Golgi Ca2+/Mn2+ ATPase negatively regulates target of rapamycin complex (TORC1) signaling, the rapamycin-sensitive TOR complex in Saccharomyces cerevisiae. Since pmr1 causes resistance to rapamycin and tor1 causes hypersensitivity, we looked for genetic interactions of pmr1 with tor1. Deletion of TOR1 restored two wild-type phenotypes. Loss of TOR1 restored the ability of the pmr1 strain to grow on media containing 2 mm MnCl2 and conferred wild type as well as the wild-type sensitivity to rapamycin. Mn2+ additions to media partially suppressed rapamycin resistance of wild type and pmr1 tor1, suggesting that Tor1 and Tor2 are regulated by manganese. We parsed the roles of Ca2+ and Mn2+ transport and the compartments in rapamycin response using separation-of-function mutants available for Pmr1. A strain containing the D53A mutant (Mn2+ transporting) of Pmr1 is rapamycin sensitive, but the Q783A mutant (Ca2+ transporting) strain is rapamycin resistant. Mn2+ transport into the Golgi lumen appears to be required for rapamycin sensitivity. Overexpression of Ca2+ pump SERCA1, Ca2+/H+ antiporter Vcx1, or a Mn2+ transporting mutant of Vcx1 (Vcx1-M1) failed to restore rapamycin sensitivity, and loss of Pmr1 but not other transporters of Ca2+ or Mn2+ results in rapamycin resistance. Overexpression of Ccc1, a Fe2+ and Mn2+ transporter that has been localized to Golgi and the vacuole, does restore rapamycin sensitivity to pmr1Delta. We conclude that Mn2+ in the Golgi inhibits TORC1 signaling.     

Functional consequences of alterations to Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Glu309, Glu771, and Asp800 in the Ca(2+)-ATPase of rabbit fast twitch muscle sarcoplasmic reticulum with their corresponding amides. These residues are predicted to lie in the transmembrane domain and have been suggested as oxygen ligands for Ca2+ binding at high affinity sites (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478). The Glu309----Gln and Asp800----Asn mutants were unable to form a phosphoenzyme from ATP at the Ca2+ concentrations examined (up to 12.5 mM), whereas the Glu771----Gln mutant phosphorylated from ATP at 2.5 mM Ca2+. In all three mutants, Ca2+ at concentrations well below 12.5 mM prevented or inhibited phosphorylation with Pi, suggesting that at least one calcium-binding site was functioning in each mutant. In the mutants Glu309----Gln and Glu771----Gln, the ADP-insensitive phosphoenzyme intermediate was unusually stable, as indicated by a very low rate of dephosphorylation observed in kinetic experiments and by an increased apparent affinity for Pi determined in equilibrium phosphorylation experiments. These data indicate a central role of Glu309 and Glu771 in the energy-transducing conformational changes and/or in the activation of phosphoenzyme hydrolysis.     

An N-terminal EF hand-like motif modulates ion transport by Pmr1, the yeast Golgi Ca(2+)/Mn(2+)-ATPase.

Pmr1, a novel member of the family of P-type ATPases, localizes to the Golgi compartment in yeast where it provides Ca(2+) and Mn(2+) for a variety of normal secretory processes. We have previously characterized Ca(2+) transport in isolated Golgi vesicles, and described an expression system for the analysis of Pmr1 mutants in a yeast strain devoid of background Ca(2+) pump activity [Sorin, A., Rosas, G., and Rao, R. (1997) J. Biol. Chem. 272, 9895-9901]. Here we show, using recombinant bacterial fusions, that an N-terminal EF hand-like motif in Pmr1 binds Ca(2+). Increasing disruptions of this motif led to progressive loss of pump function; thus, the single point mutations D51A and D53A retained pump activity but with drastic reductions in the affinity for Ca(2+) transport, while the double mutant was largely unable to exit the endoplasmic reticulum. In-frame deletions of the Ca(2+)-binding motif resulted in complete loss of function. Interestingly, the single point mutations conferred differential affinities for transport of Ca(2+) and Mn(2+) ions. Further, the proteolytic stability of the catalytic ATP-binding domain is altered by the N-terminal mutations, suggesting an interaction between these two regions of polypeptide. These studies implicate the N-terminal domain of Pmr1 in the modulation of ion transport, and may help elucidate the role of N-terminal metal-binding sites of Cu(2+)-ATPases, defective in Wilson and Menkes disease.     

Packing interactions between transmembrane helices alter ion selectivity of the yeast Golgi Ca2+/Mn2+-ATPase PMR1

PMR1 is the yeast secretory pathway pump responsible for high affinity transport of Mn2+ and Ca2+ into the Golgi, where these ions are sequestered and effectively removed from the cytoplasm. Phenotypic growth assays allow for convenient screening of side chains important for Ca2+ and Mn2+ transport. Earlier we demonstrated that mutant Q783A at the cytoplasmic interface of M6 could transport Ca2+, but not Mn2+. Scanning mutagenesis of side chains proximal to residue Gln-783 in membrane helices M2, M4, M5, and M6 revealed additional residues near the cytoplasmic interface, notably Leu-341 (M5), Phe-738 (M5), and Leu-785 (M6) that are sensitive to substitution. Importantly, we obtained evidence for a packing interaction between Val-335 in M4 and Gln-783 in M6 that is critical for Mn2+ transport. Thus, mutant V335G mimics the Mn2+ transport defect of Q783A and mutant V335I can effectively suppress the Mn2+-defective phenotype of Q783A. These changes in ion selectivity were confirmed by cation-dependent ATP hydrolysis using purified enzyme. Other substitutions at these sites are tolerated individually, but not in combination. Exchange of side chains at 335 and 783 also results in ion selectivity defects, suggesting that the packing interaction may be conformation-sensitive. Homology models of M4, M5, and M6 of PMR1 have been generated, based on the structures of the sarcoplasmic reticulum Ca2+-ATPase. The models are supported by data from mutagenesis and reveal that Gln-783 and Val-335 show conformation-sensitive packing at the cytoplasmic interface. We suggest that this region may constitute a gate for access of Mn2+ ions.     

Mutational analysis of the role of Glu309 in the sarcoplasmic reticulum Ca(2+)-ATPase of frog skeletal muscle.

Site-specific mutagenesis was used to analyse the role of the residue, Glu309, in the function of the Ca(2+)-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 microM Ca2+ was unable to prevent phosphorylation from Pi, consistent with previous observations on the Ca(2+)-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476-478]. At neutral pH, however, micromolar concentrations of Ca2+ were sufficient to inhibit phosphorylation of the Glu309----Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca2+ site was relatively intact in this mutant. The Glu309----Lys mutant was unable to form a phosphoenzyme from ATP at all Ca2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu309----Ala mutant occurred at 12.5 mM Ca2+, but not at Ca2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E2P intermediate in the Glu309----Lys mutant. A less pronounced stabilization of E2P was observed with the Glu309----Ala mutant, suggesting a possible role of the charge at the position of Glu309 in phosphoenzyme hydrolysis.     

Inhibition of the intracellular Ca2+ transporter SERCA (Sarco-Endoplasmic Reticulum Ca2+-ATPase) by the natural polyphenol epigallocatechin-3-gallate

The use of a microsomal preparation from skeletal muscle revealed that both Ca(2+) transport and Ca(2+)-dependent ATP hydrolysis linked to Sarco-Endoplasmic Reticulum Ca(2+)-ATPase are inhibited by epigallocatechin-3-gallate (EGCG). A half-maximal effect was achieved at approx. 12?μM. The presence of the galloyl group was essential for the inhibitory effect of the catechin. The relative inhibition of the Ca(2+)-ATPase activity decreased when the Ca(2+) concentration was raised but not when the ATP concentration was elevated. Data on the catalytic cycle indicated inhibition of maximal Ca(2+) binding and a decrease in Ca(2+) binding affinity when measured in the absence of ATP. Moreover, the addition of ATP to samples in the presence of EGCG and Ca(2+) led to an early increase in phosphoenzyme followed by a time-dependent decay that was faster when the drug concentration was raised. However, phosphorylation following the addition of ATP plus Ca(2+) led to a slow rate of phosphoenzyme accumulation that was also dependent on EGCG concentration. The results are consistent with retention of the transporter conformation in the Ca(2+)-free state, thus impeding Ca(2+) binding and therefore the subsequent steps when ATP is added to trigger the Ca(2+) transport process. Furthermore, phosphorylation by inorganic phosphate in the absence of Ca(2+) was partially inhibited by EGCG, suggesting alteration of the native Ca(2+)-free conformation at the catalytic site.     

Stimulation of adenosine triphosphatase activity of sarcoplasmic reticulum by adenylyl methylene diphosphate.

The effects of adenylyl methylene diphosphate (AMD), a non-hydrolyzable ATP analogue, were examined in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. The Ca2+-dependent APTase activity measured at 5 degrees C and pH 7.0 in 5.2 micrometer [gamma-32P]ATP and in the absence of added alkali metal salts was stimulated by added AMD. The steady state level of phosphoenzyme, however, was not decreased greatly by added AMP under these conditions. The hydrolysis of the phosphoenzyme formed at the steady state in the absence of added alkali metal salts was accelerated by added AMD to an extent that can account for the stimulation of the ATPase activity. At 5 degrees C and pH 7.0 the maximum stimulation of phosphoenzyme hydrolysis by AMD and the Km value for this ATP analogue were 4.3-fold and 40 micrometer, respectively. These results provide further support for our previous conclusion (Shigekawa, M., Dougherty, J.P. and Katz, A.M. (1978) J.Biol. Chem. 253, 1442--1450) that 2 classes of ATP site exist in the calcium pump ATPase in the absence of added alkali metal salts, one being the catalytic site and the other being the regulation site which activates the activity of the catalytic site.     

Functional consequences of alterations to Gly310, Gly770, and Gly801 located in the transmembrane domain of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Site-specific mutagenesis was used to replace Gly310, Gly770, and Gly801, located in the transmembrane domain of the sarcoplasmic reticulum Ca(2+)-ATPase, with either alanine or valine. In addition, Gly310 was substituted with proline. In the Gly310----Ala mutant, the Vmax for Ca2+ transport and ATPase activity was reduced to about 40% of the wild type activity, but the apparent Ca2+ affinity was close to normal. The Gly310----Val and Gly310----Pro mutants were devoid of Ca2+ transport or ATPase activity and displayed more than a 20-fold reduction in the apparent Ca2+ affinities measured in the phosphorylation assays with either ATP or Pi. In these mutants, the rate of phosphoenzyme hydrolysis was reduced, and the ADP-insensitive phosphoenzyme intermediate accumulated. The apparent affinity for Pi was increased in the absence, but not in the presence, of dimethyl sulfoxide. The properties of this new class of Ca(2+)-ATPase mutants ("E2/E2P" type) are consistent with a conformational state in which the protein-phosphate interaction is stabilized and the Ca(2+)-protein interaction is destabilized. The Gly770----Ala mutant transported Ca2+ with a Vmax close to that of the wild type, but displayed more than a 20-fold reduction of apparent Ca2+ affinity. The Gly770----Val mutant was not phosphorylated from either ATP or Pi. The Gly801----Ala mutant transported Ca2+ with a Vmax of 126% that of the wild type, hydrolyzed ATP at the same Vmax as the wild type in the presence of calcium ionophore, and displayed a 3-fold reduction in apparent Ca2+ affinity. The Gly801----Val mutant was unable to transport Ca2+ and to be phosphorylated from ATP, even at a Ca2+ concentration of 1 mM, but Ca2+ in the micromolar range inhibited phosphorylation from Pi. The ability to bind ATP with normal affinity was retained. The properties of this mutant are consistent with a disruption of one of the two Ca2+ binding sites required for phosphorylation with ATP.     

Critical role of Val-304 in conformational transitions that allow Ca2+ occlusion and phosphoenzyme turnover in the Ca2+ transport ATPase

Site-directed mutations were produced in the distal segments of the Ca(2+)-ATPase (SERCA) transmembrane region. Mutations of Arg-290 (M3-M4 loop), Lys-958, and Thr-960 (M9 - M10 loop) had minor effects on ATPase activity and Ca(2+) transport. On the other hand, Val-304 (M4) mutations to Ile, Thr, Lys, Ala, or Glu inhibited transport by 90-95% while reducing ATP hydrolysis by 83% (Ile, Thr, and Lys), 56% (Ala), or 45% (Glu). Val-304 participates in Ca(2+) coordination with its main-chain carbonyl oxygen, and this function is not expected to be altered by mutations of its side chain. In fact, despite turnover inhibition, the Ca(2+) concentration dependence of residual ATPase activity remained unchanged in Val-304 mutants. However, the rates (but not the final levels) of phosphoenzyme formation, as well the rates of its hydrolytic cleavage, were reduced in proportion to the ATPase activity. Furthermore, with the Val-304 --> Glu mutant, which retained the highest residual ATPase activity, it was possible to show that occlusion of bound Ca(2+) was also impaired, thereby explaining the stronger inhibition of Ca(2+) transport relative to ATPase activity. The effects of Val-304 mutations on phosphoenzyme turnover are attributed to interference with mechanical links that couple movements of transmembrane segments and headpiece domains. The effects of thermal activation energy on reaction rates are thereby reduced. Furthermore, inadequate occlusion of bound Ca(2+) following utilization of ATP in Val-304 side-chain mutations is attributed to inadequate stabilization of the Glu-309 side chain and consequent defect of its gating function.     

CrATP-induced Ca2+ occlusion in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum.

Use of the nonphosphorylating beta,gamma-bidentate chromium(III) complex of ATP to induce a stable Ca(2+)-occluded form of the sarcoplasmic reticulum Ca(2+)-ATPase was combined with molecular sieve high performance liquid chromatography of detergent-solubilized protein to examine the ability of the Ca(2+)-ATPase mutants Gly-233-->Glu, Gly-233-->Val, Glu-309-->Gln, Gly-310-->Pro, Pro-312-->Ala, Ile-315-->Arg, Leu-319-->Arg, Asp-703-->Ala, Gly-770-->Ala, Glu-771-->Gln, Asp-800-->Asn, and Gly-801-->Val to occlude Ca2+. This provided a new approach to identification of amino acid residues involved in Ca2+ binding and in the closure of the gates to the Ca2+ binding pocket of the Ca(2+)-ATPase. The "phosphorylation-negative" mutant Asp-703-->Ala and mutants of ADP-sensitive phosphoenzyme intermediate type were fully capable of occluding Ca2+, as was the mutant Gly-770-->Ala. Mutants in which carboxylic acid-containing residues in the putative transmembrane segments had been substituted ("Ca(2+)-site mutants") and mutant Gly-801-->Val were unable to occlude either of the two calcium ions. In addition, the mutant Gly-310-->Pro, previously classified as ADP-insensitive phosphoenzyme intermediate type (Andersen, J.P., Vilsen, B., and MacLennan, D.H. (1992). J. Biol. Chem. 267, 2767-2774), was unable to occlude Ca2+, even though Ca(2+)-activated phosphorylation from MgATP took place in this mutant.     

Ca(2+)-binding site in Rhodobacter sphaeroides cytochrome C oxidase

Cytochrome c oxidase (COX) from R. sphaeroides contains one Ca(2+) ion per enzyme that is not removed by dialysis versus EGTA. This is similar to COX from Paracoccus denitrificans [Pfitzner, U., Kirichenko, A., Konstantinov, A. A., Mertens, M., Wittershagen, A., Kolbesen, B. O., Steffens, G. C. M., Harrenga, A., Michel, H., and Ludwig, B. (1999) FEBS Lett. 456, 365-369] and is in contrast to the bovine oxidase, which binds Ca(2+) reversibly. A series of R. sphaeroides mutants with replacements of the E54, Q61, and D485 residues, which form the Ca(2+) coordination sphere in subunit I, has been generated. The substitutions for the E54 residue do not assemble normally. Mutants with the Q61 replacements are active and retain the tightly bound Ca(2+); their spectra are not perturbed by added Ca(2+) or EGTA. The D485A mutant is active, binds to Ca(2+) reversibly, like the mitochondrial oxidase, and exhibits the red shift in the heme a absorption spectrum upon Ca(2+) binding for both reduced and oxidized states of heme a. The K(d) value of 6 nM determined by equilibrium titrations is much lower than that reported for the homologous D477A mutant of Paracoccus denitrificans or for bovine COX (K(d) = 1-3 microM). The rate of Ca(2+) binding with the D485A oxidase (k(on) = 5 x 10(3) M(-1) s(-1)) is comparable to that observed earlier for bovine COX, but the off-rate is extremely slow (approximately 10(-3) s(-1)) and highly temperature-dependent. The k(off) /k(on) ratio (190 nM) is about 30-fold higher than the equilibrium K(d) of 6 nM, indicating that formation of the Ca(2+)-adduct may involve more than one step. Sodium ions reverse the Ca(2+)-induced red shift of heme a and dramatically decrease the rate of Ca(2+) binding to the D485A mutant COX. With the D485A mutant, 1 Ca(2+) competes with 1 Na(+) for the binding site, whereas 2 Na(+) compete with 1 Ca(2+) for binding to the bovine oxidase. This finding indicates that the aspartic residue D442 (a homologue of R. sphaeroides D485) may be the second Na(+) binding site in bovine COX. No effect of Ca(2+) binding to the D485A mutant is evident on either the steady-state enzymatic activity or several time-resolved partial steps of the catalytic cycle. It is proposed that the tightly bound Ca(2+) plays a structural role in the bacterial oxidases while the reversible binding with the mammalian enzyme may be involved in the regulation of mitochondrial function.     

Inhibition of human SERCA3 by PL/IM430. Molecular analysis of the interaction

The monoclonal antibody PL/IM430 has previously been reported to uncouple Ca(2+) transport from ATP hydrolysis in platelet membranes (Hack, N., Wilkinson, J. M., and Crawford, N. (1988) Biochem. J. 250, 355-361). More recently, we have demonstrated that this antibody is specific for human SERCA3 (Poch, E., Leach, S., Snape, S., Cacic, T., MacLennan, D. H., and Lytton, J. (1998) Am. J. Physiol. 275, C1449-C1458). In this paper, we have extended the analysis of the PL/IM430-SERCA3 interaction. Using HEK293 cells to express human SERCA3a, we were able to measure both ATP-mediated, oxalate-dependent (45)Ca(2+) uptake and Ca(2+)-dependent ATP hydrolysis activities due exclusively to SERCA3. Treatment with PL/IM430 inhibited both activities almost identically, with a maximal inhibition of 81 and 73% and a half-maximal concentration of 8.3 and 5.9 microg/ml, for Ca(2+) uptake and ATP hydrolysis, respectively. We conclude that PL/IM430 does inhibit SERCA3 activity but does not uncouple Ca(2+) transport from ATP hydrolysis. Using a combination of partial proteolysis, GST fusion protein expression, and mutation of residues that differ between rat and human SERCA3, we have identified human SERCA3 amino acids Pro(8) and Glu(192) as essential to forming the PL/IM430 epitope. PL/IM430 thus recognizes a linearly noncontiguous set of amino acids within the actuator domain of human SERCA3. We propose that PL/IM430 inhibits SERCA3 activity by sterically preventing movement of the actuator domain into a catalytically critical position in the E2 conformation of the enzyme.     

Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP

Ca(2+) transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle was studied. A passive Ca(2+) load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca(2+) but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca(2+)/P(i) coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca(2+) exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca(2+) bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca(2+) affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model.     

Candida albicans Pmr1p, a secretory pathway P-type Ca2+/Mn2+-ATPase, is required for glycosylation and virulence

The cell surface of Candida albicans is the immediate point of contact with the host. The outer layer of the cell wall is enriched in highly glycosylated mannoproteins that are implicated in many aspects of the host-fungus interaction. Glycosylation of cell wall proteins is initiated in the endoplasmic reticulum and then elaborated in the Golgi as the protein passes through the secretory pathway. Golgi-bound mannosyltransferases require Mn(2+) as an essential cofactor. In Saccharomyces cerevisiae, the P-type ATPase Pmr1p transports Ca(2+) and Mn(2+) ions into the Golgi. To determine the effect of a gross defect in glycosylation on host-fungus interactions of C. albicans, we disrupted the PMR1 homolog, CaPMR1. This mutation would simultaneously inhibit many Golgi-located, Mn(2+)-dependent mannosyltransferases. The Capmr1Delta null mutant was viable in vitro and had no growth defect even on media containing low Ca(2+)/Mn(2+) ion concentrations. However, cells grown in these media progressively lost viability upon entering stationary phase. Phosphomannan was almost completely absent, and O-mannan was severely truncated in the null mutant. A defect in N-linked outer chain glycosylation was also apparent, demonstrated by the underglycosylation of surface acid phosphatase. Consistent with the glycosylation defect, the null mutant had a weakened cell wall, exemplified by hypersensitivity to Calcofluor white, Congo red, and hygromycin B and constitutive activation of the cell integrity pathway. In a murine model of systemic infection, the null mutant was severely attenuated in virulence. These results demonstrate the importance of glycosylation for cell wall structure and virulence of C. albicans.     

Interference with phosphoenzyme isomerization and inhibition of the sarco-endoplasmic reticulum Ca2+ ATPase by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene

ATP hydrolysis and Ca(2+) transport by the sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) are inhibited by 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br(2)-TITU) in the micromolar range (Berman, M. C., and Karlish, S. J. (2003) Biochemistry 42, 3556-3566). In a study of the mechanism of inhibition, we found that Br(2)-TITU allows the enzyme to bind Ca(2+) and undergo phosphorylation by ATP. The level of ADP-sensitive phosphoenzyme (i.e. E1P-2Ca(2+)) observed in the transient state following addition of ATP is much higher in the presence than in the absence of the inhibitor. Br(2)-TITU does not interfere with enzyme phosphorylation by P(i) in the reverse direction of the cycle (i.e. E2P) and produces only a slight inhibition of its hydrolytic cleavage. The inhibitory effect of Br(2)-TITU on steady state ATPase velocity is attributed to interference with the E1P-2Ca(2+) to E2P-2Ca(2+) transition. In fact, experiments on conformation-dependent protection from proteolytic digestion suggest that, in the presence of Br(2)-TITU, the loops connecting the "A" domain to the ATPase transmembrane region undergo greater fluctuation than expected in the E2 and E2P states. Optimal stability of the gathered headpiece domains is thereby prevented. These effects are opposite to those of thapsigargin, in which the mechanism of inhibition is related to stabilization of a highly compact ATPase conformation and interference with Ca(2+) binding and phosphoenzyme formation. Our experiments with Br(2)-TITU provide the first demonstration of a kinetic limit posed by an inhibitor on the E1P-2Ca(2+) to E2P-2Ca(2+) transition in the wild-type enzyme.     

Catalytic and regulatory ATP-binding sites of the red cell Ca2+ pump studied by irreversible modification with fluorescein isothiocyanate

Catalytic and regulatory binding sites for ATP on the red cell Ca2+ pump have been investigated using fluorescein isothiocyanate (FITC). Both (Ca2+ + Mg2+)-ATPase activity and ATP-dependent Ca2+ flux are selectively and irreversibly inactivated by FITC and the pump is protected from FITC by the presence of ATP. The time course of inactivation by FITC is characteristically biphasic. Analysis of the kinetics of inactivation by FITC and protection by ATP reveals the participation of both high and low affinity binding sites for ATP and FITC. The sites binding ATP or reacting with FITC do not, however, appear to co-exist on the same enzyme molecules. Thus, "flip-flop" mechanisms for (Ca2+ + Mg2+)-ATPase, involving negative interactions between high and low affinity ATP sites, are considered unlikely. The two affinities for ATP are most simply explained by assuming that the Ca2+ pump protein exists in alternative conformational forms, E1 having a high affinity for ATP and E2 having a low affinity for ATP. Ca2+ pumping and (Ca2+ + Mg2+)-ATPase involve interconversion between these forms. It is suggested that regulation of Ca2+ pump activity by Mg-ATP reflects acceleration of the conformational transition between the E1 and E2 forms, as well as a previously described acceleration of phosphoenzyme hydrolysis (Muallem, S., and Karlish, S. J. D. (1981) Biochim. Biophys. Acta 647, 73-86; Garrahan, P. J., and Rega, A. F. (1978) Biochim. Biophys. Acta 513, 59-65).     

Ca(2+) signals in Pmr1-GFP-expressing COS-1 cells with functional endoplasmic reticulum

We studied the role of the Pmr1-containing Ca(2+) store in COS-1 cells endowed with a functional endoplasmic reticulum. Transfected cells could be recognized by using a green-fluorescent-protein (GFP)-tagged form of Pmr1. Pmr1-GFP fluorescence showed a typical juxtanuclear Golgi-like distribution. Pmr1-GFP-containing cells with functional endoplasmic reticulum responded to 100 microM ATP with baseline Ca(2+) spiking, while non-transfected cells produced an initial Ca(2+) peak followed by a long-lasting plateau. The Ca(2+) signal often appeared after a long latency in Pmr1-GFP-expressing cells. ATP-stimulated Pmr1-GFP-expressing cells with functional endoplasmic reticulum responded after a latency period to extracellular Ca(2+) with a regenerative Ca(2+) signal, while non-transfected control cells responded with an immediate slow rise in free cytosolic Ca(2+) concentration. These results demonstrate the importance of the Pmr1-containing Ca(2+) store in generating or modifying cellular Ca(2+) signals.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme

The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.