Deletions or Specific Substitutions of a Few Residues in the NH2-terminal Region (Ala3 to Thr9) of Sarcoplasmic Reticulum Ca2+-ATPase Cause Inactivation and Rapid Degradation of the Enzyme Expressed in COS-1 Cells

Abstract Amino acid residues in the NH2-terminal region (Glu2 – Ala14) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala3–Ser6 region or deletion of two or more consecutive residues in the Ala3–Thr9 region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala3–Ser6 region caused only a small decrease in the specific Ca2+ transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca2+ transport rates. In vitroexpression 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 [35S]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 Ala3. These results suggest that the NH2-terminal region (Ala3 –Thr9) 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.