Proteolytic studies on the transduction mechanism of sarcoplasmic reticulum Ca2+-ATPase: common features with other P-type ATPases.
After proteinase K-induced excision of five amino acid residues in the semiconserved polypeptide chain linking the end of the A domain with the S3/M3 transmembrane segment we find that Ca(2+) transport is blocked while partial reactions like Ca(2+) binding, ATP phosphorylation, and Ca(2+)-occlusion are left intact. However, formation of the so-called E2P state (either from the phosphorylated species formed in the presence of ATP and Ca(2+) or from the Ca(2+)-depleted unphosphorylated species) is blocked. We conclude that the proteinase K-treated ATPase, while maintaining many of the partial reactions, is incapable of energy transduction because of the absence of an E2P state with Ca(2+) binding sites exposed to the intravesicular space. Sequence comparisons and mutagenesis data point to an important role in energy transduction of P-type ATPases of a conserved motif located at the end of the A domain.
Møller
Jesper V
JV
Department of Biophysics, University of Aarhus, Denmark. jvm@biophys.au.dk
Lenoir
Guillaume
G
Le Maire
Marc
M
Juul
Birte Staehr
BS
Champeil
Philippe
P
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D019286:Endopeptidase K / Q000378:metabolism*; D007700:Kinetics; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D019286:Endopeptidase K; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium