Transgene-coded chimeric proteins as reporters of intracellular proteolysis: Starvation-induced catabolism of alacZ fusion protein in muscle cells ofCaenorhabditis elegans

Abstract

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.

Methods to Assess Subcellular Compartments of Muscle in <em>C. elegans</em>

“…The use of transgenically encoded proteins allows assessment of muscle specific proteostasis. For example, use of animals containing β-galactosidase, fused to an N-terminal portion of myosin, that is synthesized under the control of a myosin promoter and enhancer allows assessment of cytosolic muscle protein content 19 . The presence of blue stain in wild-type adults demonstrates the presence of active β-galactosidase and the absence of pathologic cytosolic protein degradation.…”

“…The presence of blue stain in wild-type adults demonstrates the presence of active β-galactosidase and the absence of pathologic cytosolic protein degradation. A loss of staining in response to treatment suggests pathologic protein degradation is occurring (Figure 5), as β-galactosidase is stable for 72 -96 hr post adulthood in wild-type adults 13,14,19 …”

“…Thus, the worms are both stimulated and subjected to some degree of osmotic stress when picked into liquid. The degree of osmotic stress can be limited by using M9 or BU buffer 19 as opposed to water. Some of these limitations are alleviated by measuring habitual movement on plates using commercially available tracking systems 34 or free plug ins for ImageJ 35 .…”

“…The worm strain used here for assessing muscle protein degradation was PD55, which contains a muscle specific β-galactosidase that is continuously synthesized throughout development until adulthood is reached and which remains stable in the cytosol for the next 72-96 hr 19 . Thus, measurement of β-galactosidase levels during development predominantly indicates the impact of interventions on synthesis from the myosin promoter whereas the loss of β-galactosidase during adulthood indicates an intervention has triggered increased cytosolic protein degradation 19 .…”

“…Thus, measurement of β-galactosidase levels during development predominantly indicates the impact of interventions on synthesis from the myosin promoter whereas the loss of β-galactosidase during adulthood indicates an intervention has triggered increased cytosolic protein degradation 19 . The key step in assessing β-galactosidase activity is to ensure effective desiccation.…”