Autoinhibition mechanism of the plasma membrane calcium pump isoforms 2 and 4 studied through lipid-protein interaction.
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.
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, CONICET, Junín 956 (1113), Buenos Aires, Argentina.
Irene C
IC
Mangialavori
Gerardo
G
Corradi
Débora E
DE
Rinaldi
María Candelaria
MC
de la Fuente
Hugo P
HP
Adamo
Juan Pablo F C
JP
Rossi
D000818:Animals; D002147:Calmodulin / Q000737:chemistry; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D010743:Phospholipids / Q000737:chemistry*; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D013194:Staining and Labeling; D017186:Titrimetry
D002147:Calmodulin; D007527:Isoenzymes; D010743:Phospholipids; D011994:Recombinant Proteins; D053499:Plasma Membrane Calcium-Transporting ATPases