Figure 4.

Timecourse of full-length CheA decay and the appearance of CheA proteolytic fragments. A) Triplicate, membrane-bound arrays reconstituted from serine receptor, CheA kinase and CheW coupling protein were incubated at 22°C for 504 hrs (21 days). Atthe indicated timepoints a sample was removed from each triplicate and pelleted to separate array-bound and supernatant CheA. Pelleted samples were resolved on SDS gels and western blotted with polyclonal anti-CheA antibody. The timecourses of full-length CheA decay were similar, generally within error, when monitored by western blotting (filled circles) and Coomassie staining (open circles). In addition to the full-length CheA band, two smaller bands were observed building up in the array western blots. These bands are observed at the molecular weights expected for CheA lacking the P1 domain (triangles, P2-P3-P4-P5) and for CheA lacking the P1-P2 domains (squares, P3-P4-P5). The sum of the western blot densities for full-length CheA, P2-P3-P4-P5, and P3-P4-P5 were also determined (diamonds). All data are relative to the initial level of full-length CheA. B) Sample western blot showing the full-length CheA band and the two degradation products initially and after 48 hours.