Overlapping cloned cDNAs representing the entire sequence of the rat fatty acid synthase mRNA have been isolated from a cDNA library and sequenced. Authenticity of the cDNA clones was supported by hybridization to fatty acid synthase mRNA and by amino-terminal sequencing of 39 fatty acid synthase CNBr fragments. The full-length fatty acid synthase mRNA is 9156 nucleotides long and includes an 84-nucleotide 5' noncoding region, a 7515-nucleotide coding sequence, and a 1537-nucleotide 3' noncoding region; a second mRNA species containing a shortened 3' noncoding sequence is also transcribed in the rat. The encoded fatty acid synthase subunit contains 2505 amino acids and has a molecular weight of 272,340. Active sites and substrate binding sites were located within the sequence, thus establishing the order of domains on the multifunctional animal fatty acid synthase as condensing enzyme-transferase-dehydrase-enoyl reductase-ketoreductase-acyl carrier protein-thioesterase.The synthesis of fatty acids from malonyl-CoA de novo requires several enzymatic activities (1). In most bacteria and plants the activities exist as discrete monofunctional polypeptides, whereas in animals they are integrated into a single multifunctional polypeptide (2). Partial sequences for animal fatty acid synthases have been reported (3-6). In this paper we report the complete amino acid sequencer of an animal fatty acid synthase and the ordering of the seven functional domains on the multifunctional subunit. MATERIALS AND METHODSIsolation and Sequencing of Fatty Acid Synthase cDNA Clones. Clones characterized in this study were isolated from Agtll cDNA libraries constructed from poly(A) RNA obtained from the mammary glands of lactating Long-Evans rats (7) or the livers of fasted-refed Long-Evans rats (8). To maximize the probability of including cDNA sequences corresponding to the 5' end of the fatty acid synthase in the liver library, we added a specific primer [nucleotides (nt) 3183-3200 of fatty acid synthase, antisense direction, 50 ng/ml] to the reaction mixture for first-strand synthesis.Asymmetrical adaptors with dephosphorylated EcoRI overhangs (Pharmacia) were used in the ligation reaction, eliminating the need for EcoRI methylase treatment. The liver library yielded, in Escherichia coli Y1090r-, 1.2 X 107 plaque-forming units/,ug of DNA and was amplified 7.6 x 104-fold (68% white plaques) before screening.Probes for library screening were derived from established fatty acid synthase cDNAs and were labeled with [32P]dCTP by random-priming (9). Inserts were subcloned (10) using pUC12 or pUC19 vectors and E. coli DH5a cells (Bethesda Research Laboratories). Nested deletions were constructed using BAL-31 nuclease (11). Double-stranded plasmid DNA was used directly in dideoxynucleotide sequencing reactions with purified synthetic oligonucleotide primers (3, 12).Amino Acid Sequencing. Fatty acid synthase was purified from the livers of Long-Evans rats. The thioesterase domains were removed with trypsin, and the core polypeptides...

The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/ acetyl transferase, residues 1630 -1850 to the enoyl reductase, residues 1870 -2100 to the oxyreductase, residues 21 14-2190 to the acyl-carrier protein and residues 2200 -2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.The de novo synthesis of fatty acids by the fatty-acid synthase requires the participation of several enzyme activities. In most bacteria and in plants, the activities exist as discrete monofunctional polypeptides. However, in fungi the functional components are distributed between two nonidentical polypeptides of 205 130 Da Preliminary mapping of the fatty-acid-synthase domain was based largely on analyses of limited proteolysis data. As amino acid sequence data became available, attempts were made to refine the domain map. However, there is disagreement as to the size of the polypeptide chain and the ordering of some of the domains [4,8, 91. In this paper we have re-evaluated the data pertaining to the exact size of the fatty-acid-synthase subunit and have refined the map by identifying the interdomain boundaries separating the various functional domains. The location of these linker regions has been established using several criteria which, although on their own may not be reliable indicators, collectively should give a good indication as to the boundary positions: (a) identifying si...

Rat genomic clones encompassing the entire fatty acid synthase gene have been isolated and characterized. The gene is present in a single copy of approx. 20 kb. Genomic DNA sequencing, direct RNA sequencing and S1 nuclease analysis showed that transcription is initiated primarily 1274 nucleotides upstream from the translation start site and that the 87-nucleotide-long 5'-untranslated mRNA sequence is the same in liver, lung and mammary gland. The 5'-flanking region and first intron contain several sequence elements which may be involved in the transcriptional regulation of this gene.

Enhanced phosphorylation of two specific protein bands accompanied catecholamine secretion from cultured bovine adrenal medulla cells stimulated by different secretagogues. Cells preincubated with 32Pi were treated with nicotine, veratridine, Ionomycin, or barium. Each of these secretagogues stimulated the phosphorylation of two protein bands with apparent molecular weights of 60,000 and 95,000. Phosphorylation of the 60,000 M. W. protein band was two- to threefold higher than that of the 95,000 M. W. band on stimulation with nicotine, veratridine, or barium, but Ionomycin stimulated phosphorylation of each protein band to the same extent. In general, the increase in phosphorylation was most rapid during the first minute of stimulation and occurred prior to detectable secretion. Phosphorylation reached a relatively constant level within 5 min after onset of stimulation at a time when catecholamine release was still proceeding at a rapid rate. Nicotine-stimulated phosphorylation and catecholamine secretion were calcium-dependent and blocked by d-tubocurarine, whereas tetrodotoxin inhibited veratridine-stimulated secretion and phosphorylation. We conclude that catecholamine secretion and protein phosphorylation occur under similar conditions and that Ca2+-dependent incorporation of phosphate into specific proteins may be a link in stimulus-secretion coupling.