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Fatty Acid Acylation of Proteins in Bioluminescent Bacteria? Angel Rodriguez, Lee Wall, Denis Riendeau, and Edward Meighen* ABSTRACT: Acylation of proteins with [3H]tetradecanoic acid (+ATP) has zyxwvutsr been demonstrated in extracts of different strains of luminescent bacteria. The labeled polypeptides from Photobacterium phosphoreum (34K and 50K) have been identified as being involved in the acyl-protein synthetase activity that is part of a purified fatty acid reductase complex responsible for synthesis of long-chain aldehydes for the bioluminescent reaction. The two polypeptides (34K and 50K) have been separated from the acyl-CoA reductase enzyme (58K) of the complex and resolved from each other, and the 50K polypeptide was further purified to >95% homogeneity. Acylation of the 50K polypeptide, alone, occurred at a low rate; however, the rate and level of acylation were greatly stimulated by the addition of either the 34K or the 58K polypeptide. Cold S t u d i e s concerning the incorporation of fatty acids into proteins have increased over the last few years. The formation of acyl-protein intermediates during fatty acid metabolism is well documented particularly with respect to the covalent attachment of fatty acids to acyl-carrier protein (ACP) as part of the mechanism of fatty acid synthesis (Vagelos, 1973; Rock et al., 1981; Jaworski & Stumpf, 1974). Studies on fatty acyl intermediates of other proteins involved in fatty acid metab- olism are not extensive; however, evidence for this type of intermediate has been obtained in a few cases (Ayling et al., 1972; Bar-Tana et al., 1973). Interest in the acylation of proteins has also been stimulated by the recent discovery that fatty acids are covalently incorporated into specific membrane From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada. Received May 3, 1983. This research is supported by a Medical Research Council grant (MT-4314) and a Medical Research Council studentship (to L.W.). 0006-2960/83/0422-5604$01.50/0 chase experiments demonstrated that the acylated 5OK poly- peptide turned over in the presence of the 58K polypeptide but not in a mixture containing only the 34K and 50K poly- peptides. Furthermore, the acylated 50K polypeptide could function as the immediate substrate for the fatty acyl-CoA reductase enzyme (58K), being reduced with NADPH to aldehyde. The 34K polypeptide was acylated only when all three polypeptides (34K, 50K, and 58K) were present. Fatty acid reductase activity could be restored by mixing of only the 58K (acyl-CoA reductase) and 50K polypeptides, showing that the 50K polypeptide is responsible for fatty acid activation in the fatty acid reductase complex and raising the question of what role the 34K polypeptide plays in fatty acid utilization in the luminescent system. proteins as posttranslational evepts in both animal cells and viruses (Magee & Schlesinger, 1982; Schmidt & Schlesinger, 1979). Consequently, the isolation and study of enzymes responsible for acyl-protein formation are becoming of in- creasing biological importance. In Photobacterium phosphoreum, the reduction of long- chain fatty acids to the corresponding aldehydes required for the luminescent reaction has recently been shown to involve the formation of acylated proteins (Riendeau et al., 1982). This activity, designated as acyl-protein synthetase, was measured by the incorporation of fatty acid into material insoluble in chloroform/methanol/acetic acid in an assay similar to that developed for measuring acyl-ACP synthetase activity (Ray & Cronan, 1976). The acyl-protein synthetase activity is part of a fatty acid reductase complex which can be resolved into acyl-protein synthetase and acyl-CoA re- ductase activities (Riendeau et al., 1982). Although the ki- netics of formation of the acyl-protein were altered after 0 1983 American Chemical Society