Deletions or specific substitutions of a few residues in the NH2-terminal region (Ala(3) to Thr(9)) of sarcoplasmic reticulum Ca2+-ATPase cause inactivation and rapid degradation of the enzyme expressed in COS-1 cells

Amino acid residues in the NH2-terminal region (Glu(2) - Ala(14)) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) we re deleted or substituted, and the mutants were expressed in COS-1 cells. D eletion of any single residue in the Ala(3)-Ser(6) region or deletion of tw o 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 Ca2+ transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression leve ls had greatly reduced specific Ca2+ transport rates. In vitro expression e xperiments 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 [S-35]m ethionine/cysteine labeling of transfected COS-1 cells demonstrated that de gradation of the mutants showing low expression levels was substantially fa ster than that of the wild type. Lactacystin, a specific inhibitor of prote asome, inhibited the degradation accelerated by single-residue deletion of Ala(3). These results suggest that the NH2-terminal region (Ala(3)-Thr(9)) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality contr ol and is thus critical for either correct folding of the SERCA1a protein o r stabilization of the correctly folded SERCA1a protein or both.