Abstract
A double-tagging, dual affinity chromatographic procedure, which permits isolation of dimers independently mutated in each subunit, has been exploited to probe the functional topology of the animal fatty acid synthase. Dimers were engineered in which the chain-terminating thioesterase reaction was compromised by mutation of the (active-site) serine residue in both subunits; these dimers assembled two long-chain fatty acyl moieties, which remained covalently linked to the 4'-phosphopantetheine residues of the two acyl carrier protein domains. Significantly, dimers that contained an additional mutation that compromised the activity of either the beta-ketoacyl synthase or malonyl/acetyltransferase activity in only one subunit also assembled two long-chain acyl moieties. In contrast, in a control experiment, introduction of an additional mutation that compromised the function of the acyl carrier protein domain in only one subunit resulted in the assembly of only one long-chain acyl moiety per dimer. Because the beta-ketoacyl synthase and malonyl/acetyltransferase domains are located near the amino terminus of the polypeptide and the acyl carrier protein domain near the carboxyl terminus, these results support a modified model for the animal fatty acid synthase in which head-to-tail functional contacts are possible both within as well as between subunits.
Highlights
In the generally accepted model for the FAS, the two polypeptides lie side-by-side in a fully extended, antiparallel configuration such that each of the two centers for palmitate synthesis requires cooperation between catalytic domains located in the aminoterminal half of one subunit with those located in the carboxylterminal half of the adjacent subunit [7,8,9]
This unexpected finding raised the possibility that the malonyl/acetyltransferase domain may be able to deliver substrates to either of the two ACPs in the dimer and that the condensation reaction may be catalyzed by the cooperation of the -ketoacyl synthase with either of the two ACP domains
If the modified model is valid, it follows that FAS dimers containing only one inactive -ketoacyl synthase or malonyl/acetyltransferase domain ought to be capable of synthesizing fatty acids on both of the ACP domains
Summary
Materials—Anti-FLAG M2 monoclonal antibody and anti-FLAG M2 affinity gel were purchased from Eastman Kodak Co., and anti-His monoclonal antibody was from CLONTECH Laboratories Inc. (Palo Alto, CA). Construction of His6- and FLAG-tagged FAS cDNAs—Construction and expression of full-length wild-type rat FAS, and mutants thereof, defective in the thioesterase, S2302A [11], malonyl/acetyltransferase, S581A [12], ACP, S2151A [10], and -ketoacyl synthase, K326A [11], domains, has been described in detail previously. Isolation of Heterodimers by Affinity Chromatography—The subunits in a mixture of His6- and FLAG-tagged dimers were randomized, as described above, and the heterodimers containing one His6-tagged and one FLAG-tagged subunit were isolated by chromatography successively on an anti-FLAG column, using the FLAG octapeptide in the eluting buffer, and on a Ni-NTA column, by including imidazole in the eluting buffer [15]. SDS-Polyacrylamide Gel Electrophoresis and Western Analysis— FAS preparations were analyzed by SDS-polyacrylamide gel electrophoresis, using 7.5% polyacrylamide gels, and by Western analysis using either murine anti-FLAG M2 monoclonal antibody (10 g/ml) or murine anti-His monoclonal antibody (1:2000 dilution) as the primary antibody and alkaline phosphatase-coupled goat anti-mouse IgG antibody as the secondary antibody [15]
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