An in vitro study of the methylation of methyl-accepting chemotaxis protein of Escherichia coli. Construction of the system and effect of mutant proteins on the system.

An in vitro system for the methylation of methyl-accepting chemotaxis proteins (MCP's), which have been shown to be membrane integral proteins, was constructed. The system, consisting of the membrane, the cytoplasm, and labeled S-adenosyl methionine, showed the following characteristics. 1. The methylation of MCP in the membrane required the cytoplasm. The rate of incorporation of the labeled methyl group into MCP was dependent on the amount of the cytoplasm. 2. Incorporation of the labeled methyl moiety into MCP reached a steady state, and the level of the steady state incorporation was dependent on the concentration of the cytoplasm when the concentration of the membrane protein was constant. 3. The methyl moiety which had been incorporated into MCP before the steady state could be exchanged. It was suggested that the amount of methyl group introduced into MCP was equal to that of taken from MCP. 4. The methylated MCP was demethylated faster in the presence of a methyl donor than in its absence. 5. The membranes obtained from cheX-, cheB-, and cheZ mutants were inactive in the present in vitro system even when they were mixed with the wild type cytoplasm.