Transgene-coded chimeric proteins as reporters of intracellular proteolysis: Starvation-induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans
✍ Scribed by Lisa A. Zdinak; Ian B. Greenberg; Nathaniel J. Szewczyk; Sami J. Barmada; Mark Cardamone-Rayner; James J. Hartman; Lewis A. Jacobson
- Publisher
- John Wiley and Sons
- Year
- 1997
- Tongue
- English
- Weight
- 240 KB
- Volume
- 67
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
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 5Ј-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 -galactosidase tetramers. The protein is stable in vivo in well-fed animals, but upon removal of the food source it is inactivated exponentially (t 1/2 ϭ 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 (t 1/2 ϭ 13 h) and a major 116-kDa intermediate (t 1/2 ϭ 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.