The importance of conserved amino acid residues in p94 protease sub-domain IIb and the IS2 region for constitutive autolysis

p94/calpain 3, a skeletal muscle-specific member of calpain protease family, is characterized by apparent Ca2+-independence during exhaustive autolysis and concomitant proteolysis of non-self substrates. The purpose of our study was to comprehensively profile the structural basis of p94 enabling activation in the cytosol without an extra Ca2+. Ca2+-dependent p94 mutants were screened using “p94-trapping”, which is an application of yeast genetic reporter system called “proteinase-trapping”. Several amino acids were revealed as critical for apparent Ca2+-independent p94 activity. These results highlight the importance of conserved amino acids in domain IIb as well as in the p94-specific IS2 region.