TRANSGENE-CODED CHIMERIC PROTEINS AS REPORTERS OF INTRACELLULAR PROTEOLYSIS - STARVATION-INDUCED CATABOLISM OF A LACZ FUSION PROTEIN IN MUSCLE-CELLS OF CAENORHABDITIS-ELEGANS

The product of an integrated transgene provides a convenient and cell- specific reporter of intracellular protein catabolism in 103 muscle ce lls of the nematode Caenorhabditis elegans. The transgene is an in-fra me fusion of a 5'-region of the C. elegans unc-54 (muscle myosin heavy -chain) gene to the lacZ gene of Escherichia coli [Fire and Waterston (1989): EMBO J 8:3419-3428], encoding a 146-kDa fusion polypeptide tha t forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is in activated exponentially (t(1/2) = 17 h) following an initial lag of 8 h. The same rate constant (but no lag) is observed in animals starved in the presence of cycloheximide, implying that inactivation is cataly zed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t( 1/2) = 13 h) and a major 116-kDa intermediate (t(1/2) = 7 h) undergo e xponential physical degradation alter a lag oi 8 h. Degradation ic thu s paradoxically faster than inactivation, and a number oi characterist ic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (ac tive) protein. Some of these intermediates are conjugated to ubiquitin . We infer that the initial proteolytic cleavages occur in the cytosol , possibly by a ubiquitin-mediated proteolytic pathway and do not nece ssarily inactivate the fusion protein tetramer. (C) 1997 Wiley-Liss, I nc.