CLONING OF THE FATTY-ACID SYNTHETASE BETA-SUBUNIT FROM FISSION YEAST,COEXPRESSION WITH THE ALPHA-SUBUNIT, AND PURIFICATION OF THE INTACT MULTIFUNCTIONAL ENZYME COMPLEX

We have cloned and sequenced the fission yeast (Schizosaccharomyces po mbe) fas1(+) gene, which encodes the fatty acid synthetase (FAS) beta subunit, by applying a PCR technique to conserved regions in the beta subunit of the alpha(6)beta(6) types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting o f 2073 amino acids (M-r = 230,616), exhibits the 48.1% identity with t he beta subunit from the budding yeast (Saccharomyces cerevisiae). Thi s subunit, with five different catalytic activities, bears four distin ct domains, while the alpha subunit, the sequence of which was previou sly reported by Saitoh ct al. (S. Saitoh ct at, 1996, J. Cell Biol. 13 4, 949-961), carries three domains. We have developed a co-expression system of the FAS alpha and beta subunits by cotransformation of two e xpression 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 sca ttering and ultracentrifugation analysis to be 2.1-2.4 x 10(6), and on e molecule of the FAS complex was found to contain approximately six F MN molecules. These results indicate that the FAS complex Rom S. pombe forms a heterododecameric alpha(6)beta(6), structure. Electron microg raphs of the negatively stained molecule suggest that the complex adop ts a unique barrel-shaped cage architecture. (C) 1998 Academic Press.