© 1999 by The Society for Integrative and Comparative Biology
Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans1
Department of Biology, Cell and Molecular Biology Program, and Program in Molecular, Cellular and Integrative Neurosciences, Colorado State University Fort Collins, Colorado 8052
Correspondence: 2E-mail: don{at}lamar.ColoState.edu
The claw muscles of large-clawed decapod crustaceans undergo a programmed atrophy in preparation for molting, or ecdysis. This is mediated by five cytosolic proteinases organized into two proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent. The calcium-dependent system consists of four calcium-dependent cysteine proteinases (CDPs I, IIa, IIb, and III; native masses 310, 125, 195, and 59 kDa, respectively) that completely degrade myofibrillar proteins and are activated in atrophic muscles. Immunological analysis shows that the active-site sequence in CDP IIa (60-kDa subunit mass) is similar to that in mammalian CDPs (calpains), and that CDP IIb is homologous to a calpain-like gene isolated from Drosophila cDNA libraries. Increased intracellullar Ca2+ stimulates proteolysis in situ, indicating CDPs play an important role in muscle protein catabolism. The ubiquitin/proteasome-dependent system involves the ATP-dependent conjugation of multi-ubiquitin chains to protein by ubiquitin-conjugating enzymes. This acts as a signal for substrate degradation by the 26S proteasome, a multi-subunit complex consisting of a 20S proteasome catalytic "core" and two PA700 (19S) regulatory complexes. Polyubiquitin mRNA, ubiquitin-protein conjugates, and 20S proteasome are elevated about 5-, 8-, and 2-fold, respectively, during atrophy. A heat-induced form of the 20S proteasome hydrolyzes myosin, troponin, and tropomyosin to large fragments in vitro. Biochemical studies identified the branched-chain amino acid-preferring (BrAAP) activity, one of six distinct catalytic components in the complex, as the activity that carries out these initial cleavages. These results indicate that the ubiquitin/proteasome pathway is involved, but its precise role remains to be resolved.