Structure and Function of the Region Near Phosphorylation Site of Sarcoplasmic Reticulum Calcium Pump

The author previously suggested that Arg - D1198 - D1 and His - D15 - D1 of The NH - D22 - D2-terminalregion in sarcoplasmic reticulum Ca D12+ D1-ATPase (SERCA 1a) are located near the phosphorylationsite.The functional role of Arg D1198 was investigated by site-directed mutagenesis. Theturnover rate of the mutant R198K was almost the same as that of the wild typewhereas the tumover rates of the mutants R198Q, R198Aand R198I were substantially lower than that of the wild type. the turnover rate was further reducedin the mutant R198E. the rate of dephosphorylation of the ADP-insensitive phosphoenzyme was reducedsubstantially in the mutant R198Q，more strongly in the mutants R198A and R198I and most strongly in the mutant R198Ebut to a much lesser extent in the R198K.这些results indicate that the positive charge and highArg hydrophilicity of Arg 1198 hydrophilicity D1 are critical for rapid hydrolysis of the ADP-insensitivephosphoenzyme.Amino acid residues in the NH D22 -terrninal r…More egion (Glu D12 D1- ala D114 D1) of SERCA1a were deleted or substituted，and the mutants were expressed in COS-1 cells. Deletion of any single residue in theD13 D1- ser D16 D1 region，deletion of two or more consecutive residues in the Ala - D13 - thr - D19 - D1 region，or substitution of A4K, A4D，H5K caused strongly reduced expression. Deletion of any single residue in theD13 D1- ser D16 D16 D1 region caused only a small decrease in the specific Ca D12+ D1.transport速率,whereas其他mutants showing low expression levels had greatly reduced specific Ca D12+ D1transport rates. Translation, transcription，and integration into the microsomal membranes were not impaired in the mutants which showed very low表达式水平在COS-1 cells. Degradation of这些mutants was substantially faster than thatwild type. Lactacystin, a specific inhibitor of proteasome，inhibited the degradation accelerated by single-residue deletion of Ala D13 . These resultssuggest that the NH D22 D2-terminal region (Ala D13 D1- thr D19 D1) of SERCA 1a is sensitive toendoplasmic reticulum-mediated quality control and is thus critical for either correct foldingof this protein or stabilization of the correctly folded SERCA 1a protein or both. Less

The author previously suggested that ArgィイD1198ィエD1 and HisィイD15ィエD1 of the NHィイD22ィエD2-terminal region in sarcoplasmic reticulum CaィイD12+ィエD1-ATPase (SERCA 1a) are located near the phosphorylation site.The functional role of ArgィイD1198ィエD1 was investigated by site-directed mutagenesis. The turnover rate of the mutant R198K was almost the same as that of the wild type, whereas the tumover rates of the mutants R198Q, R198A, and R198I were substantially lower than that of the wild type. The turnover rate was further reduced in the mutant R198E. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I and most strongly in the mutant R198E, but to a much lesser extent in the R198K. These results indicate that the positive charge and high hydrophilicity of ArgィイD1198ィエD1 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.Amino acid residues in the NHィイD22ィエD2-terrninal r … More egion (GluィイD12ィエD1-AlaィイD114ィエD1) of SERCA1a were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the AlaィイD13ィエD1-SerィイD16ィエD1 region, deletion of two or more consecutive residues in the AlaィイD13ィエD1-ThrィイD19ィエD1 region, or substitution of A4K, A4D, and H5K caused strongly reduced expression. Deletion of any single residue in the AlaィイD13ィエD1-SerィイD16ィエD1 region caused only a small decrease in the specific CaィイD12+ィエD1. Transport rate, whereas other mutants showing low expression levels had greatly reduced specific CaィイD12+ィエD1 transport rates. Translation, transcription, and integration into the microsomal membranes were not impaired in the mutants which showed very low expression levels in COS-1 cells. Degradation of these mutants was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of AlaィイD13ィエD1. These results suggest that the NHィイD22ィエD2-terminal region (AlaィイD13ィエD1-ThrィイD19ィエD1) of SERCA 1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of this protein or stabilization of the correctly folded SERCA 1a protein or both. Less