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Transgene‐coded chimeric proteins as reporters of intracellular proteolysis: Starvation‐induced catabolism of a lacZ fusion protein in muscle cells of Caenorhabditis elegans
Author(s) -
Zdinak Lisa A.,
Greenberg Ian B.,
Szewczyk Nathaniel J.,
Barmada Sami J.,
CardamoneRayner Mark,
Hartman James J.,
Jacobson Lewis A.
Publication year - 1997
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(19971001)67:1<143::aid-jcb15>3.0.co;2-i
Subject(s) - fusion protein , biology , cycloheximide , proteolysis , protein degradation , proteases , transgene , biochemistry , caenorhabditis elegans , ubiquitin , microbiology and biotechnology , ubiquitins , lac operon , protein biosynthesis , escherichia coli , ubiquitin ligase , gene , recombinant dna , enzyme
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 β‐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 = 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 = 13 h) and a major 116‐kDa intermediate (t 1/2 = 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. J. Cell. Biochem. 67:143–153, 1997. © 1997 Wiley‐Liss, Inc.