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 pombe) 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 alpha6beta6 types of FAS among different organisms. The deduced amino acid sequence of the Fas1 polypeptide, consisting of 2073 amino acids (Mr = 230,616), exhibits the 48.1% identity with the beta subunit from the budding yeast (Saccharomyces cerevisiae). This subunit, with five different catalytic activities, bears four distinct domains, while the alpha 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 alpha and beta 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 x 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 alpha6beta6 structure. Electron micrographs of the negatively stained molecule suggest that the complex adopts a unique barrel-shaped cage architecture.
Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka, 565-0874.
H
H
Niwa
E
E
Katayama
M
M
Yanagida
K
K
Morikawa
D000595:Amino Acid Sequence; D001483:Base Sequence; D001704:Biopolymers; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D064429:Fatty Acid Synthases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D012568:Schizosaccharomyces / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D001704:Biopolymers; D004274:DNA, Recombinant; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D064429:Fatty Acid Synthases