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

The Author previously suggested that Argy D1198 y D1 and His y D15 y D1 of the NH y D22 y D2-terminal region in sarcoplasmic reticulum Ca y D12+ y D1-ATPase（SERCA1a） are located near the phosphorylation site. Argy D1198 y D1 was investigated by site-directed mutagenesis.突变体R198 K的逆转率几乎与野生类型的逆转率相同，而突变体R198 Q、R198 A和R198 I实际上低于野生类型的逆转率。在突变R198 E中，转弯率进一步降低。ADP不敏感磷酸酶的脱磷速率在突变R198 Q中被降低，在突变R198 A和R198 I中更强，在突变R198 E中也更强，但在R198 K中更少。These Results indicate that the positive charge and high hydrophilicity of Argy D1198-D1 are critical for rapid hydroylysis of the ADP-不敏感磷酸酶酶.氨基酸residues in the NH-D22-terrninal r ... More egion （Glu D12 D1-Ala D114 D1） of SERCA 1a已被删除或被替换，并且突变体在COS-1细胞中表达。在Ala-D13-Ser-D16-D1区域中的任何单一保留的删除，在Ala-D13-Thr-D19-D1区域中的删除，或在Ala-D13-Thr-D19-D1区域中的删除，或在A4 K、A4 D和H5 K中导致的强烈减少表达式的替代。Deletion of any single residue in the Ala D13-Ser D16-D1 region caused only a small decrease in the specific Ca D12+ D1。Transport rate， whereas other mutants showing low expression levels had greatly reduced specific CaイD12+イエD1 transport rates。翻译、转录和集成在COS-1细胞中显示出非常低的表达水平的突变体中没有受到影响。这些突变体的退化比野生类型的退化要快。Lactacystin， a specific inhibitor of proteasome， inhibited the degradation accelerated by single-residue deletion of AlaイD13イエD1。These Results suggest that the NH-D22-terminal region （Ala-D13-Thr-D19-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 folding SERCA1a protein or both。Less（低）

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