Precursor Forms of the Subunits of Nitrate Reductase inchlAandchlBMutants ofEscherichia coliK12

Abstract

The synthesis of nitrate reductase by a parental E. coli K12 strain and its isogenic chlA and chlB mutants was analyzed by protein double labeling with L-[4,5-3H]leucine and 35S and by immunoprecipitation using specific antiserum. The chlA and chlB mutants, although defective in nitrate reductase activity, retain the ability to synthesize the different polypeptides that are normally required for functional enzyme activity. These polypeptides are present in unequal quantities in the membrane and in the cytoplasm of the cells. The chlB mutant synthesizes 3 times more nitrate reductase than the chlA mutant. The subunit composition of the membrane-bound nitrate reductase present in the 2 mutants is different. Membrane preparations from the chlB mutant contain the 3 subunits .alpha., .beta., .gamma. in a ratio which is similar to the wild type. In the chlA mutant the 2 subunits .beta. and .gamma. are missing and the level of a subunit is very low. In the same membrane, a 48,000-MW subunit (polypeptide .beta.'') precipitable by nitrate reductase antiserum was found. The chlA and chlB mutants accumulate the 3 subunits .alpha., .beta. and .gamma. in different proportions and concentrations in the cytoplasm, unlike the parental strain. The cytoplasm from the chlA mutant also contains the .beta.'' polypeptide found in the membrane fraction of this mutant and in addition contain another polypeptide designated .alpha.'' of MW 105,000, which is precipitated by the nitrate reductase antiserum. The formation of particulate active nitrate reductase can be achieved by mixing the supernatant fractions of the chlA and chlB mutants (complementation) and procedes by 2 distinct but mutually dependent stages. Following reconstitution of activity the 2 peptides .alpha.'' and .beta.'' present in the supernatant fraction of the ChlA mutant, disappear. Analysis of the immunoprecipitate polypeptides present in both the soluble and particulate nitrate reductase protein after reconstitution suggests that the polypeptides are precursors of the .alpha. and .beta. subunits following a process that remains to be elucidated.