Involvement of the cytoplasmic loop L6-7 in the entry mechanism for transport of Ca2+ through the sarcoplasmic reticulum Ca2+-ATPase.
We previously found that mutants of conserved aspartate residues of sarcoplasmic reticulum Ca(2+)-ATPase in the cytosolic loop, connecting transmembrane segments M6 and M7 (L6-7 loop), exhibit a strongly reduced sensitivity toward Ca(2+) activation of the transport process. In this study, yeast membranes, expressing wild type and mutant Ca(2+)-ATPases, were reacted with Cr small middle dotATP and tested for their ability to occlude (45)Ca(2+) by HPLC analysis, after cation resin and C(12)E(8) treatment. We found that the D813A/D818A mutant that displays markedly low calcium affinity was capable of occluding Ca(2+) to the same extent as wild type ATPase. Using NMR and mass spectrometry we have analyzed the conformational properties of the synthetic L6-7 loop and demonstrated the formation of specific 1:1 cation complexes of the peptide with calcium and lanthanum. All three aspartate Asp(813)/Asp(815)/Asp(818) were required to coordinate the trivalent lanthanide ion. Overall these observations suggest a dual function of the loop: in addition to mediating contact between the intramembranous Ca(2+)-binding sites and the cytosolic phosphorylation site (Zhang, Z., Lewis, D., Sumbilla, C., Inesi G., and Toyoshima, C. (2001) J. Biol. Chem. 276, 15232-15239), the L6-7 loop, in a preceding step, participates in the formation of an entrance port, before subsequent high affinity binding of Ca(2+) inside the membrane.
Menguy
Thierry
T
Section de Biophysique des Fonctions Membranaires, DBJC, CEA et CNRS URA 2096 and LRA17V Université de Paris XI, CE Saclay, 91191 Gif sur Yvette Cedex, France.
Corre
Fabienne
F
Juul
Birte
B
Bouneau
Laurence
L
Lafitte
Daniel
D
Derrick
Peter J
PJ
Sharma
Parveen S
PS
Falson
Pierre
P
Levine
Barry A
BA
Møller
Jesper V
JV
le Maire
Marc
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D017136:Ion Transport; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D021241:Spectrometry, Mass, Electrospray Ionization
D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium