Mutations of either or both Cys876 and Cys888 residues of sarcoplasmic reticulum Ca2+-ATPase result in a complete loss of Ca2+ transport activity without a loss of Ca2+-dependent ATPase activity. Role of the CYS876-CYS888 disulfide bond.
Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca(2+)-ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys(876)-Cys(888) disulfide bond in the expressed wild type. All the three mutants possessed high Ca(2+)-ATPase activity. In contrast, no Ca(2+) transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from P(i). Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca(2+)-ATPase; activation of the catalytic site upon high affinity Ca(2+) binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADP-insensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca(2+) from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys(876)-Cys(888) disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca(2+) pathway.
Daiho
T
T
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. daiho@asahikawa-med.ac.jp
Yamasaki
K
K
Saino
T
T
Kamidochi
M
M
Satoh
K
K
Iizuka
H
H
Suzuki
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003545:Cysteine; D004220:Disulfides; D007700:Kinetics; D018482:Muscle, Skeletal / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014162:Transfection
D004220:Disulfides; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D002118:Calcium