Calcium transport by sarcoplasmic reticulum Ca(2+)-ATPase. Role of the A domain and its C-terminal link with the transmembrane region.
After treatment of sarcoplasmic reticulum Ca(2+)-ATPase with proteinase K (PK) in the presence of Ca(2+) and a protecting non-phosphorylated ligand (e.g. adenosine 5'-(beta,gamma-methylenetriphosphate), we were able to prepare in high yield an ATPase species that only differs from intact ATPase because of excision of the MAATE(243) sequence from the loop linking the A domain with the third transmembrane segment. The PK-treated ATPase was unable to transport Ca(2+) and to catalyze ATP hydrolysis, but it could bind two calcium ions with high affinity and react with ATP to form a classical ADP-sensitive phosphoenzyme, Ca(2)E1P, with occluded Ca(2+). The ability of Ca(2)E1P to become converted to the Ca(2+)-free ADP-insensitive form, E2P, was strongly reduced, as was the ability of PK-treated ATPase to react with orthovanadate or to form an E2P intermediate from inorganic phosphate in the absence of Ca(2+). PK-treated ATPase also reacted with thapsigargin to form a complex with altered properties, and the tryptic cleavage "T2" site in the A domain was no longer protected in the absence of Ca(2+). It is probable that disrupting the C-terminal link of the A domain with the transmembrane region severely compromises reorientation of A and P domains and the functionally critical cross-talk of these domains with the membrane-bound Ca(2+) ions.
Department of Biophysics, University of Aarhus, Ole Worms Allé 185, 8000 Aarhus C, Denmark.
Jesper V
JV
Möller
Guillaume
G
Lenoir
Christophe
C
Marchand
Cédric
C
Montigny
Marc
M
le Maire
Chikashi
C
Toyoshima
Birte Staehr
BS
Juul
Philippe
P
Champeil
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D017136:Ion Transport; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
D008081:Liposomes; D010446:Peptide Fragments; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D019286:Endopeptidase K; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium