Hugo P. Adamo

Intramolecular Fluorescence Resonance Energy Transfer between Fused Autofluorescent Proteins Reveals Rearrangements of the N- and C-terminal Segments of the Plasma Membrane Ca2+ Pump Involved in the Activation

The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca2+ pump (PMCA) isoform 4xb (hPMCA4xb). The fusion protein was successfully expressed in yeast and purified by calmodulin affinity chromatography. Despite the presence of the fused autofluorescent proteins BFP-PMCA-GFP performed similarly to the wild-type enzyme with respect to Ca2+-ATPase activity and sensitivity to calmodulin activation. In the autoinhibited state BFP-PMCA-GFP exhibited a significant intramolecular fluorescence resonance energy transfer (FRET) consistent with the location of the fluorophores at an average distance of 45Å. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca2+-calmodulin, partial proteolysis, or acidic lipids. Moreover, FRET decreased and became insensitive to calmodulin when hPMCA4xb was activated by mutation D170N in BFP-PMCA(D170N)-GFP. The results suggest that the ends of the PMCA are in close proximity in the autoinhibited conformation, and they separate or reorient when the PMCA achieves its final activated conformation.

Loss of Autoinhibition of the Plasma Membrane Ca2+ Pump by Substitution of Aspartic 170 by Asparagine ACTIVATION OF PLASMA MEMBRANE CALCIUM ATPase 4 WITHOUT DISRUPTION OF THE INTERACTION BETWEEN THE CATALYTIC CORE AND THE C-TERMINAL REGULATORY DOMAIN

The plasma membrane calcium ATPase (PMCA) actively transports Ca2+ from the cytosol to the extra cellular space. The C-terminal segment of the PMCA functions as an inhibitory domain by interacting with the catalytic core. Ca2+-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp170, in the putative “A” domain of human PMCA isoform 4xb, plays a critical role in autoinhibition. In the absence of calmodulin a PMCA containing a site-specific mutation of D170N had 80% of the maximum activity of the calmodulin-activated PMCA and a similar high affinity for Ca2+. The mutation did not change the activation of the PMCA by ATP. Deletion of the C-terminal segment further downstream of the calmodulin-binding site led to an additional increase in the maximal activity of the mutant, which suggests that the mutation did not affect the inhibition because of this portion of the C-terminal segment. The calmodulin-activated PMCA was more sensitive to vanadate inhibition than the autoinhibited enzyme. In contrast, inhibition of the D170N mutant required higher concentrations of vanadate and was not affected by calmodulin. Despite its higher basal activity, the mutant had an apparent affinity for calmodulin similar to that of the wild type enzyme, and its rate of proteolysis at the C-terminal segment was still calmodulin-dependent. Altogether these results suggest that activation by mutation D170N does not involve the displacement of the calmodulin-binding autoinhibitory domain from the catalytic core and may arise directly from changes in the accessibility to the calcium-binding residues of the pump.

Deletions in the acidic lipid-binding region of the plasma membrane Ca2+ pump. A mutant with high affinity for Ca2+ resembling the acidic lipid-activated enzyme.

The C-terminal segment of the loop between transmembrane helices 2 and 3 (AL region) of the plasma membrane Ca2+ pump (PMCA) is not conserved in other P-ATPases. Part of this region, just upstream from the third transmembrane domain, has been associated with activation of the PMCA by acidic lipids. cDNAs coding for mutants of the Ca2+pump isoform h4xb with deletions in the AL region were constructed, and the proteins were successfully expressed in either COS or Chinese hamster ovary cells. Mutants with deletions in the segment 296–349 had full Ca2+ transport activity, but deletions involving the segment of amino acids 350–356 were inactive suggesting that these residues are required for a functional PMCA. In the absence of calmodulin the V max of mutant d296–349 was similar to that of the recombinant wild type pump, but its K 0.5 for Ca2+ was about 5-fold lower. The addition of calmodulin increased theV max and the apparent Ca2+ affinity of both the wild type and d296–349 enzymes indicating that the activating effects of calmodulin were not affected by the deletion. At low concentrations of Ca2+ and in the presence of saturating amounts of calmodulin, the addition of phosphatidic acid increased about 2-fold the activity of the recombinant wild type pump. In contrast, under these conditions phosphatidic acid did not significantly change the activity of mutant d296–349. Taken together these results suggest that (a) deletion of residues 296–349 recreates a form of PMCA similar to that resulting from the binding of acidic lipids at the AL region; (b) the AL region acts as an acidic lipid-binding inhibitory domain capable of adjusting the Ca2+ affinity of the PMCA to the lipid composition of the membrane; and (c) the function of the AL region is independent of the autoinhibition by the C-terminal calmodulin-binding region.

Magnesium-ions accelerate the formation of the phosphoenzyme of the (Ca2+ + Mg2+)-activated ATPase from plasma membranes by acting on the phosphorylation reaction

Magnesium ions in the reaction medium at 37 degrees C increased up to 222 s-1 the kapp for phosphorylation by ATP of the Ca2(+)-ATPase of pig red cell membranes. This effect was observed after partial proteolysis with trypsin which makes the enzyme behave like the E1 conformer during phosphorylation. These findings lead to the conclusion that Mg2+ increased the rate of phosphorylation of the Ca2(+)-ATPase by acting directly on this reaction. The apparent dissociation constant of Mg2+ for this effect was 44 microM whereas the apparent dissociation constant for Mg2+ to accelerate the shift E2----E1 between conformers measured on the intact enzyme was 50 microM. This suggests that Mg2+ accelerated both reactions from a single class of site.

Amino acid residues 18-75 are essential for expression of an active plasma membrane Ca2+ pump.

The NH2and COOH-terminal segments of the P-type ATPases are the most variable regions of these enzymes. Recently a mutant of the plasma membrane Ca2+ pump (PMCA) called hPMCA4b (ct120) with a deletion of the COOH-terminal 120 amino acid residues was expressed in COS-I cells and found to be fully active.] We have now investigated the functional relevance of the NH2-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) containing a deletion of the NH2-terminal 18-75 and the COOH-terminal 120 amino acid residues. Immunoblots of COS1 cell membranes transfected with hPMCA4b(d18-75) (ct120) or hPMCA4b (ct120) DNA2,3 showed that both proteins were expressed in similar amounts, indicating that the NHz-terminal deletion did not significantly affect the level of expression of the PMCA. The expressed hPMCA4b(d18-75) (ct120) was found in one major band of the expected molecular mass ( I 14 kD), indicating that it was as stable as the wild-type enzyme to degradation by intracellular proteases. Because the removal of several amino acid residues may lead to proteins that are not correctly folded or inserted in the membrane, we tested the topology of the mutant by partial proteolysis. Tryptic digestion of the hPMCA4b(d18-75) (ct120) mutant resulted in the appearance of the same fragments obtained by proteolysis of the hPMCA4b (ct120) enzyme, indicating that both proteins exposed the same limited number of sites to the protease and suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein. Results in TABLE 1 show that Caz+ transport activity of microsomes from cells expressing the hPMCA4b(d18-75) (ct120) protein was not significantly different

Replacement of Val674 by Pro increases the sensitivity of the plasma membrane Ca2+ pump to inhibition by Mg2+.

A cDNA encoding a plasma membrane Ca2+ pump mutant V674P(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the V674P(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val674 to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the V674P(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val674 by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the V674P(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E2-E1. The study of the Mg2+ dependency of the Ca2+ transport showed that the V674P(ct120) mutant reached maximum activation at 100 microM Mg2+ in contrast with 500 microM in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 microM Mg2+. These results indicate that the decrease in the activity observed upon substitution of Val674 by Pro was due to a higher sensitivity to Mg2+ as inhibitor.

A Mutant of the Plasma Membrane Ca2+ Pump Highly Sensitive to Inhibition by Mg2+a

Val674 in the “nucleotide binding domain” of the plasma membrane Ca2+ pump (PMCA) is replaced by Pro in most of the other P-type ATPases. We previously found that restitution of the consensus Pro at position 674 of the PMCA causes a substantial reduction in the enzyme’s activity, and this effect seemed unrelated to ATP binding.’ In the present study we constructed and expressed in COS-I cells the mutant V674P (ct120) after knowing that removal of the COOH-tenninal 120 amino acid residues of hPMCA4be results in a mutant enzyme called hPMCA4b (ct120) which has higher activity independently of ~almodulin.~ The V674P and V674P (ct120) Ca2+ pumps could readily be detected in membranes from transfected COS-1 cell membranes by immunoblotting with monoclonal antibody 5F10: as single bands with the expected migration according to their molecular mass. The intensity of the bands was comparable to that of the hPMCA4b and hPMCA4b (ct120), respectively, indicating that the substitution V674P did not affect the level of expression of the Ca2+ pump. The capacity of the V674P and V674P (ct 120) enzymes to pump Ca2+ was compared to that of the hPMCA4b and hPMCA4b (ct120), respectively, by measuring the Ca2+ uptake rate by microsomal vesicles derived from transfected COS-1 cells. The results in TABLE 1 show that substitution of by Pro reduced the activity of the hPMCA4b and that of the hPMCA4b (ct120) to a similar extent. Despite its lower activity, the apparent affinity for Ca2+ of the V674P (ct120) enzyme was at least as high as that of hPMCA4b (ct120), indicating that the lower activity of the Vh74P (ct120) enzyme could not be accounted for by a reduction in the affinity for Ca2+ at the transport site. Study of the Mg2+ dependency of the Ca2+ transport of the V674P (ct 120) mutant