A novel proteolytic activity apparently initiating degradation of beta-galactosidase nonsense fragments in in vitro extracts of Escherichia coli.

Our previous in vivo studies demonstrated that large premature fragments of beta-galactosidase are degraded in Escherichia coli by a common pathway, and the initial event appears to be a site-specific cleavage (McKnight, J. L., and Fried, V. A. (1981) J. Biol. Chem. 256, 9652-9661). We now have developed a cell-free system that retains the specificity of this early cleavage event. Immunochemical techniques were used to isolate and quantitate the polypeptide substrate and products in pulse-chase experiments. The in vitro system has an activity that quantitatively converts the prematurely terminated A polypeptide of the lacZ non-sense mutant CSH-10 to the 90-kilodalton common B polypeptide intermediate observed in vivo. The activity is localized in the cytoplasm since the cleavage reaction is not affected by osmotic shock of whole cells or removal of the membrane fraction from cell-free extracts. The lon mutation capR9, which blocks this degradation pathway in vivo, does not affect the initial cleavage event in cell-free extracts of CSH-10 carrying this mutation. The in vitro cleavage event in extracts of lon+ CSH-10 or the isogenic lon- mutant is not stimulated by addition of ATP, not inhibited by depletion of ATP pools by hexokinase-2-deoxyglucose treatment, and not inhibited by EDTA or phenylmethylsulfonyl fluoride. These results suggest that the ATP-dependent proteolytic activity of the lon gene product does not directly catalyze this primary cleavage event.