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

The author previously suggested that Arg I D1198文件D1 and His I D15文件D1 of the NH文件D22文件D2-terminal region in sarcoplasmic reticulum Ca文件D12+文件D1-ATPase(SERCA1a)are located near the phosphorylation site.The functional role of Arg I D1198文件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 I D1198 ii D1 are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme.Amino acid residues in the NH I D22 ii D2-terrninal r…More egion(Glu I D12 I D 1-Ala D 114 I D 1)of SERCA 1a were deleted or substituted，and the mutants were expressed in COS-1 cells.Deletion of any single residue in the Ala I D13文件D1-Ser D 16文件D 1 region，deletion of two or more consecutive residues in the Ala I D 13文件D1-Thr I D 19文件D 1 region，or substitution of A4K，A4D，and H5K caused strongly reduced expression.Deletion of any single residue in the Ala I D13 I D 1-Ser I D 16 I D 1 region caused only a small decrease in the specific Ca I D 12+I D 1.Transport rate，whereas other mutants showing low expression levels had greatly reduced specific Ca I D12+ii 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-1cells.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 I D13 I D 1.These results suggest that the NH I D22文件D2-terminal region(Ala I D13文件D1-ThrI D19文件D1)of SERCA1a 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 SERCA1a protein or both。Less：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