RESUMO
The Spf1p protein from Saccharomyces cerevisiae belongs to the family of P5A-ATPases that have recently been shown to protect the endoplasmic reticulum by dislocating misinserted membrane proteins. The loss of function of P5A-ATPases leads to endoplasmic reticulum stress with a pleiotropic phenotype including protein, sterol and metal ion dyshomeostasis. Like other P-ATPases, Spf1p requires Mg2+. We found that free Mg2+ stimulated the Spf1p ATPase activity along a double hyperbolic curve with two components of K1/2 = 14 and 800 µM Ca2+, Mn2+ and Co2+ lowered about 50% of the Spf1p ATPase with relatively low affinity (Ki â¼75 µM) and the activity was fully recovered after metal ion chelation with EGTA. In contrast, low concentrations of Zn2+ and Cd2+decreased the activity to less than 20% and lead to slow irreversible inactivation of the enzyme. After the treatment with Zn2+, Spf1p exhibited a reduced apparent affinity for ATP and formed lower levels of the catalytic phosphoenzyme. The inactivation by Zn2+ occurred preferentially at a pH > 6 and could be prevented by adding either ATP or ADP to the inactivation media. These results suggest that Zn2+ inactivated Spf1p by binding to amino acid residues from the nucleotide binding-phosphorylation domains that are protonated at lower pH. Alternatively the binding of nucleotides may indirectly compete with a conformational change leading to the Zn2+-inactive form of the enzyme. Exposure of yeast cells to high concentrations of Zn2+ led to changes similar to the phenotype characteristic of the Spf1Δ cells. Altogether, our data, point out a possible mechanism by which the inhibition of P5A-ATPases could potentiate metal ion-induced ER stress and proteotoxicity.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Adenosina Trifosfatases , Proteínas de Saccharomyces cerevisiae , Zinco , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Íons , Fosforilação , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Zinco/farmacologia , Metais/farmacologiaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Hidrólise , FosforilaçãoRESUMO
P5-ATPases are important for processes associated with the endosomal-lysosomal system of eukaryotic cells. In humans, the loss of function of P5-ATPases causes neurodegeneration. In the yeastSaccharomyces cerevisiae, deletion of P5-ATPase Spf1p gives rise to endoplasmic reticulum stress. The reaction cycle of P5-ATPases is poorly characterized. Here, we showed that the formation of the Spf1p catalytic phosphoenzyme was fast in a reaction medium containing ATP, Mg(2+), and EGTA. Low concentrations of Ca(2+)in the phosphorylation medium decreased the rate of phosphorylation and the maximal level of phosphoenzyme. Neither Mn(2+)nor Mg(2+)had an inhibitory effect on the formation of the phosphoenzyme similar to that of Ca(2+) TheKmfor ATP in the phosphorylation reaction was â¼1 µmand did not significantly change in the presence of Ca(2+) Half-maximal phosphorylation was attained at 8 µmMg(2+), but higher concentrations partially protected from Ca(2+)inhibition. In conditions similar to those used for phosphorylation, Ca(2+)had a small effect accelerating dephosphorylation and minimally affected ATPase activity, suggesting that the formation of the phosphoenzyme was not the limiting step of the ATP hydrolytic cycle.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/genética , Fosforilação/fisiologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPase group are present in humans. Loss-of-function mutations in the ATP13A2 gene (PARK9, OMIM 610513) underlay a form of Parkinson's disease (PD) known as the Kufor-Rakeb syndrome (KRS), which belongs to the group of syndromes of neurodegeneration with brain iron accumulation (NBIA). Here we report that the cytotoxicity induced by iron exposure was two-fold reduced in CHO cells stably expressing the ATP13A2 recombinant protein (ATP13A2). Moreover, the iron content in ATP13A2 cells was lower than control cells stably expressing an inactive mutant of ATP13A2. ATP13A2 expression caused an enlargement of lysosomes and late endosomes. ATP13A2 cells exhibited a reduced iron-induced lysosome membrane permeabilization (LMP). These results suggest that ATP13A2 overexpression improves the lysosome membrane integrity and protects against the iron-induced cell damage.
Assuntos
Cloretos/toxicidade , Compostos Férricos/toxicidade , Lisossomos/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetulus , Relação Dose-Resposta a Droga , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/enzimologia , Lisossomos/patologia , Mutação , Tamanho das Organelas/efeitos dos fármacos , Permeabilidade , ATPases Translocadoras de Prótons/genética , TransfecçãoRESUMO
The transport of calcium to the extracellular space carried out by plasma membrane Ca(2+) pumps (PMCAs) is essential for maintaining low Ca(2+) concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca(2+)-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 Ca(2+). 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 Glu(99) 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 Ca(2+). 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.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Calmodulina/metabolismo , Catálise , Citosol/metabolismo , Deleção de Genes , Glutamina/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Lisina/metabolismo , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/metabolismoRESUMO
P-type ion pumps are membrane transporters that have been classified into five subfamilies termed P1-P5. The ion transported by the P5-ATPases is not known. Five genes, ATP13A (ATPase type 13A) 1-ATP13A5, that belong to the P5-ATPase group have been identified in humans. Mutations of the human gene ATP13A2 underlie a form of PD (Parkinson's disease). Previous studies have suggested a relation between polyamines and P5B-ATPases. We have recently shown that the cytotoxicity induced by the polyamine analogue paraquat (1,1'-dimethyl-4,4'-bipyridinium), which is an environmental agent related to PD development, was increased in ATP13A2-expressing CHO (Chinese-hamster ovary) cells. In the present study we showed that ATP13A2-expressing CHO cells exhibit a 2-fold higher accumulation of spermidine. Increasing concentrations of spermidine reduced the viability of CHO cells stably expressing ATP13A2. The higher levels of spermidine attained by the ATP13A2-expressing CHO cells were correlated with an increase in the ATP-dependent spermidine uptake in an isolated subcellular fraction containing lysosomes and late endosomes. The results of the present study support the idea that the human P5B-ATPase ATP13A2 is involved in polyamine uptake.
Assuntos
Doença de Parkinson/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Espermidina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/metabolismo , Humanos , Cinética , Lisossomos/metabolismo , Doença de Parkinson/genética , ATPases Translocadoras de Prótons/genética , TemperaturaRESUMO
The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of ~0.3-1 µmol of P(i)/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg(2+). No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg(2+) dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg(2+) acts as a cofactor. The addition of Mn(2+) to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Trifosfato de Adenosina/imunologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/química , Magnésio/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.
Assuntos
Ativação Enzimática , Fosfolipídeos/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Animais , Calmodulina/química , Cromatografia de Afinidade , Eritrócitos/enzimologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/isolamento & purificação , Ligação Proteica , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Coloração e Rotulagem , TitulometriaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Membrana Celular/enzimologia , Proteólise , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Domínios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
RESUMO
Asn879 in the transmembrane segment M6 of the plasma membrane Ca(2+) pump (PMCA human isoform 4xb) has been proposed to coordinate Ca(2+) at the transport site through its carboxylate. This idea agrees with the fact that this Asn is conserved in other Ca(2+)-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 abolishes 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 Saccharomyces cerevisiae, solubilized and purified by calmodulin affinity chromatography. The Asn879Asp PMCA mutant exhibited about 30% of the wild type Ca(2+)-dependent ATPase activity and only a minor reduction of the apparent affinity for Ca(2+). The decrease in the Ca(2+)-ATPase of the mutant enzyme was in parallel with the reduction in the amount of phosphoenzyme formed from Ca(2+) plus ATP. Noteworthy, the mutation nearly eliminated the ability of the enzyme to hydrolyze pNPP which is maximal in the absence of Ca(2+) revealing a major effect of the mutation on the Ca(2+)-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 E(2)-like forms of the enzyme.
Assuntos
Asparagina/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Asparagina/química , Asparagina/genética , Transporte Biológico , Western Blotting , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Fosforilação , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Vanadatos/metabolismoRESUMO
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 E(2)-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.
Assuntos
Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Animais , Membrana Celular/metabolismo , Ácido Egtázico/metabolismo , Eritrócitos/enzimologia , Hidrólise , SuínosRESUMO
P5 ATPases are eukaryotic pumps important for cellular metal ion, lipid and protein homeostasis; however, their transported substrate, if any, remains to be identified. Ca2+ was proposed to act as a ligand of P5 ATPases because it decreases the level of phosphoenzyme of the Spf1p P5A ATPase from Saccharomyces cerevisiae. Repeating previous purification protocols, we obtained a purified preparation of Spf1p that was close to homogeneity and exhibited ATP hydrolytic activity that was stimulated by the addition of CaCl2. Strikingly, a preparation of a catalytically dead mutant Spf1p (D487N) also exhibited Ca2+-dependent ATP hydrolytic activity. These results indicated that the Spf1p preparation contained a co-purifying protein capable of hydrolyzing ATP at a high rate. The activity was likely due to a phosphatase, since the protein i) was highly active when pNPP was used as substrate, ii) required Ca2+ or Zn2+ for activity, and iii) was strongly inhibited by molybdate, beryllium and other phosphatase substrates. Mass spectrometry identified the phosphatase Pho8p as a contaminant of the Spf1p preparation. Modification of the purification procedure led to a contaminant-free Spf1p preparation that was neither stimulated by Ca2+ nor inhibited by EGTA or molybdate. The phosphoenzyme levels of a contaminant-free Spf1p preparation were not affected by Ca2+. These results indicate that the reported effects of Ca2+ on Spf1p do not reflect the intrinsic properties of Spf1p but are mediated by the activity of the accompanying phosphatase.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Biocatálise , Cloreto de Cálcio/metabolismo , Ensaios Enzimáticos , Hidrólise , Mutação , Naftóis , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Fosforilação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , TriazinasRESUMO
The human plasma membrane Ca2+ pump (isoform 4xb) was expressed in Saccharomyces cerevisiae and purified by calmodulin-affinity chromatography. Under optimal conditions the recombinant enzyme (yPMCA) hydrolyzed ATP in a Ca2+ dependent manner at a rate of 15 micromol/mg/min. The properties of yPMCA were compared to those of the PMCA purified from human red cells (ePMCA). The mobility of yPMCA in SDS-PAGE was the expected for the hPMCA4xb protein but slightly lower than that of ePMCA. Both enzymes achieved maximal activity when supplemented with acidic phospholipids. However, while ePMCA in mixed micelles of phosphatidylcholine-detergent had 30% of its maximal activity, the yPMCA enzyme was nearly inactive. Increasing the phosphatidylcholine content of the micelles did not increase the activity of yPMCA but the activity in the presence of phosphatidylcholine improved by partially removing the detergent. The reactivation of the detergent solubilized yPMCA required specifically acidic lipids and, as judged by the increase in the level of phosphoenzyme, it involved the increase in the amount of active enzyme. These results indicate that the function of yPMCA is highly sensitive to delipidation and the restitution of acidic lipids is needed for a functional enzyme.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Metabolismo dos Lipídeos , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácidos/metabolismo , Trifosfato de Adenosina/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Calmodulina , Membrana Celular/metabolismo , Cromatografia de Afinidade , Detergentes/farmacologia , Ativação Enzimática/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Micelas , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tensoativos/farmacologiaRESUMO
The purified PMCA supplemented with phosphatidylcholine was able to hydrolyze pNPP in a reaction media containing only Mg(2+) and K(+). Micromolar concentrations of Ca(2+) inhibited about 75% of the pNPPase activity while the inhibition of the remainder 25% required higher Ca(2+) concentrations. Acidic lipids increased 5-10 fold the pNPPase activity either in the presence or in the absence of Ca(2+). The activation by acidic lipids took place without a significant change in the apparent affinities for pNPP or K(+) but the apparent affinity of the enzyme for Mg(2+) increased about 10 fold. Thus, the stimulation of the pNPPase activity of the PMCA by acidic lipids was maximal at low concentrations of Mg(2+). Although with differing apparent affinities vanadate, phosphate, ATP and ADP were all inhibitors of the pNPPase activity and their effects were not significantly affected by acidic lipids. These results indicate that (a) the phosphatase function of the PMCA is optimal when the enzyme is in its activated Ca(2+) free conformation (E2) and (b) the PMCA can be activated by acidic lipids in the absence of Ca(2+) and the activation improves the interaction of the enzyme with Mg(2+).
Assuntos
4-Nitrofenilfosfatase/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/fisiologia , Membrana Celular/enzimologia , Lipídeos/farmacologia , Magnésio/metabolismo , 4-Nitrofenilfosfatase/antagonistas & inibidores , 4-Nitrofenilfosfatase/sangue , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Química Encefálica , ATPases Transportadoras de Cálcio/sangue , Bovinos , Ativação Enzimática , Fosfatos/farmacologia , Fosfatidilcolinas/farmacologia , Potássio/farmacologia , Suínos , Vanadatos/farmacologiaRESUMO
Mutants of the plasma membrane Ca(2+) pump (human isoform 4xb) with deletions in the linker between domain A and transmembrane segment M3 (A(L) region) were constructed and expressed in Chinese hamster ovary cells. The total or partial removal of the amino acid segment 300-349 did not change the maximal Ca(2+) transport activity, but mutants with deletions involving residues 300-338 exhibited a higher apparent affinity for Ca(2+) than the wild type h4xb enzyme. Deletion of the putative acidic lipid interacting sequence (residues 339-349) had no observable functional consequences. The removal of either residues 300-314 or 313-338 resulted in a similar increase in the apparent Ca(2+) affinity of the pump although the increase was somewhat lower than that obtained by the deletion 300-349 suggesting that both deletions affected the same structural determinant. The results show that alterations in the region of the alternative splicing site A change the sensitivity to Ca(2+) of the human isoform 4 of the PMCA.
Assuntos
ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Sítios de Splice de RNA/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Células CHO , ATPases Transportadoras de Cálcio/química , Proteínas de Transporte de Cátions/química , Cricetinae , Cricetulus , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Dados de Sequência Molecular , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Angiotensin (Ang) (1-7) is the endogenous ligand for the G protein-coupled receptor Mas, a receptor associated with cardiac, renal, and cerebral protective responses. Physiological evidence suggests that Mas receptor (MasR) undergoes agonist-dependent desensitization, but the underlying molecular mechanism regulating receptor activity is unknown. We investigated the hypothesis that MasR desensitizes and internalizes on stimulation with Ang-(1-7). For this purpose, we generated a chimera between the MasR and the yellow fluorescent protein (YFP; MasR-YFP). MasR-YFP-transfected HEK 293T cells were incubated with Ang-(1-7), and the relative cellular distribution of MasR-YFP was observed by confocal microscopy. In resting cells, MasR-YFP was mostly localized to the cell membrane. Ang-(1-7) induced a redistribution of MasR-YFP to intracellular vesicles of various sizes after 5 minutes. Following the time course of [(125)I]Ang-(1-7) endocytosis, we observed that half of MasR-YFP underwent endocytosis after 10 minutes, and this was blocked by a MasR antagonist. MasR-YFP colocalized with Rab5, the early endosome antigen 1, and the adaptor protein complex 2, indicating that the R is internalized through a clathrin-mediated pathway and targeted to early endosomes after Ang-(1-7) stimulation. A fraction of MasR-YFP also colocalized with caveolin 1, suggesting that at some point MasR-YFP traverses caveolin 1-positive compartments. In conclusion, MasR undergoes endocytosis on stimulation with Ang-(1-7), and this event may explain the desensitization of MasR responsiveness. In this way, MasR activity and density may be tightly controlled by the cell.
Assuntos
Angiotensina I/farmacologia , Endocitose/fisiologia , Endossomos/metabolismo , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Angiotensina I/metabolismo , Células Cultivadas , Clatrina/metabolismo , Células HEK293 , Humanos , Fragmentos de Peptídeos/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidoresAssuntos
ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Fosfatidilserinas/farmacologia , Proteínas de Transporte de Cátions , Clonagem Molecular , Concentração de Íons de Hidrogênio , Cinética , ATPases Transportadoras de Cálcio da Membrana Plasmática , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
The blue and green fluorescent proteins (BFP and GFP) have been fused at the N- and C-terminal ends, respectively, of the plasma membrane Ca(2+) 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 Ca(2+)-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 45A. The FRET intensity in BFP-PMCA-GFP decreased when the enzyme was activated either by Ca(2+)-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.
Assuntos
Membrana Celular/enzimologia , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Substituição de Aminoácidos , Cálcio/metabolismo , Calmodulina/metabolismo , Ativação Enzimática/fisiologia , Proteínas de Fluorescência Verde/genética , Humanos , Mutação de Sentido Incorreto , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genéticaRESUMO
The plasma membrane calcium ATPase (PMCA) actively transports Ca(2+) 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. Ca(2+)-calmodulin binds to the C-terminal segment and stops inhibition. Here we showed that residue Asp(170), 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 Ca(2+). 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.