Temperature and induction effects on the degradation rate of an abnormal β-galactosidase in Escherichia coli

Abstract

Intracellular protein degradation was investigated using an unstable fragment of Escherichia coli β-galactosidase, the CSH11 mutant, as a model protein. This abnormal protein was expressed from a single copy gene in the chromosome and is converted to a detectable degradable intermediate. The in vivo degradation rates of both β-galactosidase fragments were measured using pulse-chase radioactive labeling techniques, and their intracellular concentrations were determined using α-complementation assays. In the physiological range of 30 to 37°C, the apparent degradation rate constant for the CSH11 fragment follows Arrhenius behavior; while the intermediate's apparent degradation rate constant is nearly unchanged. However, above 37°C the degradation rates of both fragments increase significantly. Analysis of the labeled intermediate's rate of change above 40°C reveals that the CSH11 fragment is being degraded by a second pathway which does not produce the intermediate. When the induction level of the abnormal β-galactosidase was varied the degradation rates of both fragments behaved similarly, but they unexpectedly decreased with increasing IPTG concentration. The two parallel degradation pathways for CSH11 apparently operated at only the lower IPTG levels. The measured degradation rates did not correlate directly with the intracellular concentration of abnormal proteins.