Complementation of mutations and nucleotide sequence of FAS1 gene encoding beta subunit of yeast fatty acid synthase.

Abstract

The yeast fatty acid synthase is a complex (alpha 6 beta 6) of two multifunctional proteins alpha and beta. The alpha subunit (Mr 212,000) contains two of the seven enzymatic activities required for the synthesis of fatty acids and the site for attachment of the prosthetic group 4'-phosphopantetheine. The beta subunit (Mr 203,000) contains the remaining five activities. Cloning of the genes encoding the alpha and beta proteins has been reported (Kuziora, M. A., Chalmers, J. H., Jr., Hitzeman, R. A., Douglas, M. G., and Wakil, S. J. (1983) J. Biol. Chem. 258, 11648-11653). In the present study it is shown that two of the clones containing the beta subunit gene, YEpFAS1 and YEp33F1, are not identical. The clone YEp33F1 contains the gene that codes for the entire beta subunit while YEpFAS1 is missing approximately half of the gene at the 3' end. Despite this loss, YEpFAS1 is still able to complement a fas1 mutation at the enoyl reductase domain. This complementation does not occur by recombination, rather a small mRNA is produced in cells transformed with YEpFAS1 and is translated into a protein of molecular weight of approximately 125,000 which is immunologically reactive with yeast fatty acid synthase antibodies. The data suggest that this truncated beta subunit interacts with the mutant alpha 6 beta 6 complex to restore fatty acid synthesis to the cell. The nucleotide sequence of the FAS1 gene cloned in YEp33F1 DNA, which encodes the beta subunit of fatty acid synthase, was determined. The coding region consists of 5940 base pairs (bp) and could encode a protein of 1980 amino acids with a calculated molecular weight of 220,077. A major transcriptional start point was mapped to a position of about 330 bp upstream from the first ATG codon. The termination of transcription was mapped at about 300 bp downstream from the first TGA stop codon. The sequence of the beta subunit protein does not appear to be similar to any other sequenced protein. The sites of the active seryl groups for the acetyltransacylase and malonyl/palmitoyl transacylase were identified from known amino acid sequences to be residues 274 and 1808, respectively. Putative binding sites for FMN and NADPH were suggested based on similarities with amino acid sequences of known flavin and pyridine nucleotide enzymes, respectively.