Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

The transport of calcium to the extracellular space carried out by plasma membrane Ca2+ pumps (PMCAs) is essential for maintaining low Ca2+ concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca2+-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca2+. Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the Vmax. Mutation of residue Glu99 to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the Vmax of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca2+. Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.Fil: Mazzitelli, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentin

Deletions in the N-terminal segment of the plasma membrane Ca2+ pump impair the expression of a correctly folded functional enzyme

Mutant cDNAs encoding h4 plasma membrane Ca2+ pumps with deletions in the N-terminal segment have been constructed and expressed in COS cells. As judged by immunoblotting, each construct was expressed at a high level similar to that of the wild-type enzyme. The removal of the first six amino acids had no effect on the Ca2+ transport activity, but deletions in the segment 15-75 reduced the activity to undetectable levels. The d(43-56)h4 mutant, lacking amino acids 43-56, was also efficiently expressed in stable form in CHO cells. The Ca2+ transport activity of d(43-56)h4 in this system was about 40% of that of the wild type. The d(43-56)h4 enzyme exhibited a similar affinity for Ca2+, a slightly increased apparent affinity for ATP, and a slightly lower sensitivity to inhibition by vanadate than the wild-type enzyme. Analysis of the phosphoenzyme intermediate formed in the presence of lanthanum showed that the phosphorylation reaction was not affected, but the maximum amount of phosphoenzyme was reduced to the same extent as the Ca2+ transport activity. These results suggest that the expressed d(43-56)h4 was a mixture of fully active and inactive enzyme. The d(43-56)h4 enzyme was more easily degraded by proteases and had a higher sensitivity to heat inactivation than the wild type suggesting that the loss of function was due to the improper folding and instability, of the mutant protein. On the basis of these findings, it appears that the N-terminal segment of the plasma membrane Ca2+ pump is neither essential for synthesis nor for catalytic activity but is critical for the expression of a correctly folded functional enzyme.Fil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Grimaldi, Mirta E.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Garcia Arguinzonis, Maisa I.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentin

Highly exposed segment of the Spf1p P5AATPase near transmembrane M5 detected by limited proteolysis

The yeast Spf1p protein is a primary transporter that belongs to group 5 of the large family of P-ATPases. Loss of Spf1p function produces ER stress with alterations of metal ion and sterol homeostasis and protein folding, glycosylation and membrane insertion. The amino acid sequence of Spf1p shows the characteristic P-ATPase domains A, N, and P and the transmembrane segments M1-M10. In addition, Spf1p exhibits unique structures at its N-terminus (N-T region), including two putative additional transmembrane domains, and a large insertion connecting the P domain with transmembrane segment M5 (D region). Here we used limited proteolysis to examine the structure of Spf1p. A short exposure of Spf1p to trypsin or proteinase K resulted in the cleavage at the N and C terminal regions of the protein and abrogated the formation of the catalytic phosphoenzyme and the ATPase activity. In contrast, limited proteolysis of Spf1p with chymotrypsin generated a large N-terminal fragment containing most of the M4-M5 cytosolic loop, and a minor fragment containing the C-terminal region. If lipids were present during chymotryptic proteolysis, phosphoenzyme formation and ATPase activity were preserved. ATP slowed Spf1p proteolysis without detectable changes of the generated fragments. The analysis of the proteolytic peptides by mass spectrometry and Edman degradation indicated that the preferential chymotryptic site was localized near the cytosolic end of M5. The susceptibility to proteolysis suggests an unexpected exposure of this region of Spf1p that may be an intrinsic feature of P5A-ATPases.Fil: Petrovich, Guido Daniel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Pavan, Carlos Humberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; ArgentinaFil: Noli Truant, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Estudios de la Inmunidad Humoral Prof. Ricardo A. Margni; ArgentinaFil: Adamo, Hugo Pedro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.Fil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Mazzitelli, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Petrovich, Guido Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: de Tezanos Pinto, Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rochi, Lucía. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

The role of the Ca2+ binding ligand Asn879 in the function of the plasma membrane Ca2+ pump

Asn879 in the transmembrane segment M6 of the plasma membrane Ca2+ pump (PMCA human isoform 4xb) has been proposed to coordinate Ca2+ at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca2+-ATPases but is replaced by Asp, Glu, and other residues in closely related 2P-type ATPases of different ionic specificity. Previous mutagenesis studies have shown that the substitution of Ala for Asn abolish the activity of the enzyme (Adebayo et al. 1995, Guerini et al. 1996). We have constructed a mutant PMCA in which the Asn879 was substituted by Asp. The mutant protein was expressed in S. cerevisiae,solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30 % of the wild type a Ca2+-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca2+. The decrease in the Ca2+-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca2+ plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca2+ revealing a major effect of the mutation on the Ca2+-independent reactions of the transport cycle. At a pH low enough to protonate the Asp carboxylate the pNPPase activity of Asn879Asp increased, suggesting that the binding of protons to Asn879 is essential for the activities catalyzed by E2¬-like forms of the enzyme.Fil: Rinaldi, Debora Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

The strategic function of the P5-ATPase ATP13A2 in toxic waste disposal

The P-type ATPase ATP13A2 protein was originally associated with a form of Parkinson´s Disease (PD)known as Kufor Rakeb Syndrome (KRS). However, in the last years it has been found to underlay variantsof neuronal ceroid-lipofuscinoses and hereditary spastic paraplegia. These findings expand the clinicaland genetic spectrum of ATP13A2-associated disorders, which are commonly characterized by lysosomaldysfunction. Nowadays it is well known that lysosomes are not merely related to the degradation andrecycling of cellular waste, but are also involved in fundamental processes such as secretion, plasmamembrane repair, signaling, energy metabolism and autophagy. The essential role of lysosomes in thesecellular processes has significant implications for health and disease. ATP13A2 is localized in lysosomesand late endosomes and its mutation leads to lysosome dysfunction, diminishes the exosome secretionand impairs autophagic flux. In this review, we first describe ATP13A2-associated disorders and theirrelation with the endolysosomal pathway. We then describe the ATP13A2-involvement in iron homeostasisand its potential linkage with new pathologies like cancer, and finally, we consider the putativerole of ATP13A2 in lipid processing and degradation, opening the interesting possibility of a broader roleof this protein providing protection against a variety of disease-associated changes affecting cellularhomeostasis.Fil: de Tezanos Pinto, Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas ; Argentin

The Human P5B-ATPase ATP13A2 is not a Ca2+ Transporting Pump

The human gene ATP13A2 has been proposed to code for an ATP powered ion transporter of the P5B subfamily. Mutations of the human gene ATP13A2 were found to underlie an autosomal recessive form of early-onset parkinsonism (PD) with pyramidal degeneration and dementia. The ion transported by the ATP13A2 pump is not known, but several studies have shown that the P5-ATPases influence the homeostasis of intracellular Ca2+, and thus it has been suggested that they transport Ca2+. In order to evaluate this possibility Chinese hamster ovary (CHO) cells stably expressing the human ATP13A2 protein have been obtained and the Ca2+ transport activity of ATP13A2 was assessed by measuring the ATP-dependent uptake of Ca2+ into microsomal vesicles. As a positive control vesicles containing the human plasma membrane Ca2+ pump (PMCA) were used. No significant differences were found between vesicles containing the ATP13A2 protein and the control. Moreover, Ca2+ was unable to induce the formation of the P-ATPase acylphosphate intermediate in vesicles containing the expressed ATP13A2. These results favor the idea that the ATP13A2 does not transport Ca2+.Fil: de Tezanos Pinto, Felicitas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

The plasma membrane Ca2+ pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca2+

The plasma membrane Ca2+ ATPase catalyzed the hydrolysis of ATP in the presence of  millimolar concentrations of EGTA and no added Ca2+ at a rate near 1.5% of that attained at saturating concentrations of Ca2+. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca2+-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca2+-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca2+ and is catalyzed by E2-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.Fil: Mazzitelli, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Rinaldi, Debora Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin

The Spf1p P5A-ATPase “arm-like” domain is not essential for ATP hydrolysis but its deletion impairs autophosphorylation

The yeast Spf1p P5A-ATPase actively translocates membrane spanning peptides of mislocalized proteins from the endoplasmic reticulum. Loss of Spf1p function causes a pleiotropic ER stress-phenotype associated with alterations of homeostasis of metal ions, lipids, protein folding, glycosylation, and membrane insertion. A unique characteristic of P5A-ATPases is the presence of an extended insertion which was called the “arm-like” domain connecting the phosphorylation domain (P) with transmembrane segment M5 near the peptidyl-substrate binding pocket. Here we have constructed and characterized a Δarm mutant of Spf1p lacking a segment of 117 amino acids of the “arm-like” domain. The Δarm mutant was capable of hydrolyzing ATP at maximal rates of 50% of that of the wild type enzyme. With the non-nucleotide substrate analog pNPP, the hydrolytic activity of the mutant dropped to 10%. The mutant showed an apparent affinity for ATP similar to the wild type. When incubated with ATP the Δarm mutant produced a lower level of the catalytic phosphoenzyme in amounts proportionate to the ATPase activity. These results indicate that the “arm-like” domain is not essential for hydrolytic activity and suggest that it is needed for the stabilization of Spf1p in a phosphorylation-ready conformation.Fil: Grenon, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Corradi, Gerardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Petrovich, Guido Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Mazzitelli, Luciana Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Adamo, Hugo Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentin