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 cells of the nematode Caenorhabditis elegans. The transgene is an in-frame 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 that forms active beta-galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t1/2 = 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 catalyzed by pre-existing proteases. Both the 146-kDa fusion polypeptide (t1/2 = 13 h) and a major 116-kDa intermediate (t1/2 = 7 h) undergo exponential physical degradation after a lag of 8 h. Degradation is thus paradoxically faster than inactivation, and a number of characteristic immunoreactive degradation intermediates, some less than one-third the size of the parent polypeptide, are found in affinity-purified (active) 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 necessarily inactivate the fusion protein tetramer.
Zdinak
L A
LA
Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.
Greenberg
I B
IB
Szewczyk
N J
NJ
Barmada
S J
SJ
Cardamone-Rayner
M
M
Hartman
J J
JJ
Jacobson
L A
LA
D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005508:Food Deprivation; D017930:Genes, Reporter; D007700:Kinetics; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019076:Transgenes; D014452:Ubiquitins / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D003513:Cycloheximide; D001616:beta-Galactosidase; D010450:Endopeptidases; D018995:Myosin Heavy Chains