Deletions or specific substitutions of a few residues in the NH(2)-terminal region (Ala(3) to Thr(9)) of sarcoplasmic reticulum Ca(2+)-ATPase cause inactivation and rapid degradation of the enzyme expressed in COS-1 cells.
Amino acid residues in the NH(2)-terminal region (Glu(2) - Ala(14)) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala(3)-Ser(6) region or deletion of two or more consecutive residues in the Ala(3)-Thr(9) region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala(3)-Ser(6) region caused only a small decrease in the specific Ca(2+) transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca(2+) transport rates. In vitro expression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [(35)S]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels 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(3). These results suggest that the NH(2)-terminal region (Ala(3) -Thr(9)) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.
Daiho
T
T
Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. daiho@asahikawa-med.ac.jp
Yamasaki
K
K
Suzuki
H
H
Saino
T
T
Kanazawa
T
T
D000818:Animals; D019556:COS Cells; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000252:Calcium-Transporting ATPases