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+-dependent 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 alpha subunit of elongation factor-1 (EF-1alpha), 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-1alpha 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.
Amano
S
S
Department of Molecular Biology, Institute of Molecular and Cellular Biosciences, The University of Tokyo.
Kawasaki
H
H
Ishiura
S
S
Kawashima
S
S
Suzuki
K
K
Emori
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004331:Drosophila melanogaster / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006868:Hydrolysis; D010053:Ovary / Q000378:metabolism*; D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D020648:Peptide Elongation Factor 1; D010445:Peptide Elongation Factors; D011994:Recombinant Proteins; D002154:Calpain