Identification of Endogenous Substrates for Drosophila Calpain from a Salt-Extracted Fraction of Drosophila Ovaries

Drosophila calpain (Dm-calpain) produced in Escherichia coli has a distinct Ca2+-depen-dent activity. By using a recombinant Dm-calpain, we searched for its substrates occurring in Drosophila ovaries, where Dm-calpain is expressed. Among a number of major proteins, several proteins in a salt-extracted fraction were selectively degraded by Dm-calpain in a Ca2+-dependent manner. The major substrates were identified by microsequencing the lysylendopeptidase-digested proteins. Three ribosomal proteins, the L5, L7, and L8 subunits of the 60S ribosome, were found to be potential Dm-calpain substrates. In addition, the α subunit of elongation factor-1 (EF-1α), a multi-functional protein involved in both protein synthesis and cytoskeletal regulation, was shown to be cleaved by Dm-calpain into several distinct fragments when expressed as a GST-fusion protein. Endogenous EF-1α in ovary extracts was also shown by western blot analysis to be similarly degraded. These observations suggest that Dm-calpain may regulate protein synthesis and cytoskeletal structure through its degradative or processing activity.