Muscle-specific calpain, p94, is degraded by autolysis immediately after translation, resulting in disappearance from muscle.

We previously identified a third type of the calpain large subunit named p94 as a cDNA whose mRNA is expressed exclusively in skeletal muscle at levels approximately 10-fold more abundant than those of the conventional calpain subunit. Rat skeletal muscle fractions were screened by two anti-peptide antibodies raised against two specific sequences in p94, but the p94 protein could not be found. To examine this apparent discrepancy between the amounts of mRNA and protein, wild-type p94 was expressed in COS cells. Although p94 mRNA was expressed normally in COS cells, only very small amounts of the protein and its presumed degradation products were detected by the antibodies described above. A series of COOH-terminal deletion mutants was constructed and expressed in COS cells and L8 cells, a rat myoblast cell line. When IS2, one of the specific regions of p94, was completely eliminated, the truncated p94 proteins were expressed normally, and the amount of the expressed proteins was at least 100-fold higher than with wild-type p94. Moreover, when site-directed mutagenesis was introduced to change the presumed active-site cysteine of p94 to serine or alanine, the mutated p94 proteins were highly expressed like the IS2-deleted mutants. These results indicate the following. 1) The mRNA for p94 is normally transcribed in COS, L8, and muscle cells; 2) the p94 protein becomes active in the cytosol immediately after translation; 3) the p94 protein virtually disappears from cells by autocatalytic degradation; and 4) the p94-specific IS2 region plays an important role in this degradation. In vitro translation experiments support this idea. Furthermore, p94 shows nuclear localization when expressed in COS cells. The physiological function of p94 in muscle is discussed on the basis of the analysis of these transfectants.