Cloning of the Fatty Acid Synthetase β Subunit from Fission Yeast, Coexpression with the α Subunit, and Purification of the Intact Multifunctional Enzyme Complex

Abstract We have cloned and sequenced the fission yeast ( Schizosaccharomyces pombe ) fas1 + gene, which encodes the fatty acid synthetase (FAS) β subunit, by applying a PCR technique to conserved regions in the β subunit of the α 6 β 6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids ( M r = 230,616), exhibits the 48.1% identity with the β subunit from the budding yeast ( Saccharomyces cerevisiae ). This subunit, with five different catalytic activities, bears four distinct domains, while the α subunit, the sequence of which was previously reported by Saitoh et al. (S. Saitoh et al., 1996, J. Cell Biol. 134, 949–961), carries three domains. We have developed a co-expression system of the FAS α and β subunits by cotransformation of two expression vectors, containing the lsd1 + / fas2 + gene and the fas1 + gene, into fission yeast cells. The isolated FAS complex showed quite high specific activity, of more than 4000 mU/mg, suggesting complete purification. Its molecular weight was determined by dynamic light scattering and ultracentrifugation analysis to be 2.1–2.4 × 10 6 , and one molecule of the FAS complex was found to contain approximately six FMN molecules. These results indicate that the FAS complex from S. pombe forms a heterododecameric α 6 β 6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.