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

Abstract

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 ADPinsensitive 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.The Ca 2ϩ -ATPase of adult fast-twitch skeletal muscle sarcoplasmic reticulum (SERCA1a) 1 is a 994-residue membranebound protein (1, 2) that catalyzes Ca 2ϩ transport coupled to ATP hydrolysis (3, 4). In the catalytic cycle, the enzyme is activated by binding of two Ca 2ϩ ions to the transport sites from the cytoplasmic side, and then ␥-phosphoryl group of ATP is transferred to Asp 351 (5-7) to form ADP-sensitive EP, which can react with ADP to form ATP (8 -10). Upon formation of this EP, the two Ca 2ϩ ions are occluded. A subsequent rate-limiting transition of ADP-sensitive EP to ADP-insensitive EP, which cannot react with ADP, results in release of the Ca 2ϩ ions into the lumen. Finally, ADP-insensitive EP is hydrolyzed to form P i and the dephosphoenzyme. This EP can also be formed from P i in the absence of Ca 2ϩ by reversal of its hydrolysis (11, 12). The Ca 2ϩ -ATPase contains ten transmembrane-helices (M1 to M10), and the bound two Ca 2ϩ ions are shown to be located side by side near the center of four helices, M4, M5, M6, and M8 in the crystal structure (13). The ATP binding site and phosphorylation site are located on the large cytoplasmic loop between M4 and M5 (13, 14). Luminal loops are short except for the one connecting M7 and M8, the loop 7-8 (approximate...

Characterization of Calumenin-SERCA2 Interaction in Mouse Cardiac Sarcoplasmic Reticulum

“…The SERCA2-L4 is the longest luminal loop in SERCA2 connecting transmembrane loops M7 and M8 (7). The L4 loop of SERCA2 contains two cysteine residues, which form a disulfide bond and regulate the luminal Ca 2ϩ balance by modulating SERCA2 activity (49). A previous study in Xenopus oocytes shows that ER chaperone ER protein 57 interacts with SERCA2b in a Ca 2ϩ -dependent manner and regulates SERCA Ca 2ϩ uptake activity (16).…”

Distinct Types of Abnormality in Kinetic Properties of Three Darier Disease-causing Sarco(endo)plasmic Reticulum Ca2+-ATPase Mutants That Exhibit Normal Expression and High Ca2+ Transport Activity

“…Actually, lumenal Loop 3-4 connected directly to M3 at the immediate C-terminal region of Ile 274 (Fig. 2) have been predicted to form the lumenal Ca 2ϩ gate (11) with essential contribution of 60). In the atomic structure E2(TG) (11) (and such interactions are less significant in E1Ca 2 structure (8)) (Fig.…”

Distinct Natures of Beryllium Fluoride-bound, Aluminum Fluoride-bound, and Magnesium Fluoride-bound Stable Analogues of an ADP-insensitive Phosphoenzyme Intermediate of Sarcoplasmic Reticulum Ca2+-ATPase

“…Our results further suggested that the stabilization energy provided by intimate contacts between the three domains in E2P will provide energy for moving transmembrane helices to open the Ca 2ϩ release pathway to lumen and thus release the bound Ca 2ϩ ions. During the subsequent E2P hydrolysis to E2, the compactly organized state of cytoplasmic domains becomes more relaxed (10,11), and the postulated Ca 2ϩ release pathway (the area surrounded by M3-M5 (8)) and lumenal gate (formed with contribution of lumenal loop connecting M7-M8 (L78) (18) and possibly by its interaction with L34 (9)) should be closed to prevent possible Ca 2ϩ leakage and prepare for the entry of new Ca 2ϩ from the cytoplasmic side to the transport sites (9). In the crystal structure of E2 fixed with TG, the postulated lumenal gate and pathway are, in fact, closed (9).…”