Inhibition of Sporulation by Cerulenin and Its Reversion by Exogenous Fatty Acids in Saccharomyces cerevisiae

Abstract

Sporulation of Saccharomyces cerevisiae G2-2 was inhibited by the antibiotic cerulenin which is known to be a specific inhibitor of fatty acid and sterol synthesis. This inhibition was reversed by various fatty acids, especially by oleic acid (C18l:) and pentadecanoic acid (C15:0). Ergosterol showed only slight reversibility of this inhibition. When cerulenin was added to the sporulation medium later than 12 h after the start of incubation, the marked inhibition disappeared. When the fatty acid fraction extracted from the sporulated yeasts was added to the cells pretreated with cerulenin for more than 6 h, sporulation became evident 6 h after the fatty acid fraction addition. Therefore, sufficient synthesis of fatty acids required for sporulation was assumed to occur during the first 6 h in phase I of yeast sporulation. Illingworth et al. (11). Recently, Henry and Halvorson (10) tried precise analysis of this process and their study showed that much of the lipid synthesis is not sporulation specific, and that, if the necessary diploids from the auxotrophs for saturated (22) and unsaturated (23) fatty acids were constructed, these strains could be used to determine the lipid requirement during sporulation.Since it has been found that the antibiotic cerulenin is a specific inhibitor of the condensing enzyme in the fatty acid synthetase complex (24), f-ketoacyl-acyl carrier protein synthetase (1), and 3-hydroxy-3-methyl-coenzyme A synthase (19) in sterol synthesis, it was possible to examine the lipid requirement in autotrophic yeast by supplying the proper lipids in the medium containing cerulenin. This paper deals with the inhibition of sporulation of S. cerevisiae by cerulenin and its reversion by externally added fatty acids. I Present address: National Institute of RadiologicalSciences, 4-9-1, Anagawa, Chiba, Japan. 42 MATERIALS AND METHODS Culture conditions. The diploid strain of S. cerevisiae G2-2, which was kindly supplied by K. Tanaka of the Institute of Applied Microbiology, University of Tokyo, was grown on nutrient agar (14) at 27 C for 2 to 7 days. This was inoculated at a concentration of about 0.3 optical density units at 660 nm into 100 ml of the synthetic presporulation medium containing (per liter): potassium acetate, 10 g; (NH4)2SO4, 4 g; K2SO4, 4 g; Na2HPO4 12H2O, 4 g-, MgSO4-7H2O, 0.5 g; CuSO4 5H2O, 0.01 mg; ZnSO4, 7H20, 0.2 mg; FeSO4 7H2O, 20 mg; CaCl2 2H2O, 100 mg-, thiamine-hydrochloride, 0.4 mg; riboflavine, 0.2 mg; nicotinic acid, 5 mg; p-aminobenzoic acid, 0.3 mg; pyridoxine*hydrochloride, 1 mg; calcium pantothenate, 1 mg; inositol, 75 mg; biotin, 0.02 mg.Assay of sporulation. After 22 h of cultivation at 27 C on a reciprocating shaker, cells were collected by centrifugation, washed once with deionized water, and then inoculated into 6 ml of the sporulation medium (1% potassium acetate) (21) in a Monod-type tube to a population density of 0.5 optical density units at 660 nm (2.7 x 107 cells/ml), usually followed by incubation at 27 C for 24 h. Cells and two-spored (or more) asci wer...

The protein expression landscape of mitosis and meiosis in diploid budding yeast

“…It is not clear why Pry1 is present only at the onset of meiotic M-phase. However, the pattern we detect is consistent with work published 40 years ago showing that cerulenin, an inhibitor of fatty acid and sterol synthesis, could interfere with sporulation only during early but not late stages [108]. The tight post-transcriptional regulation of Pry1 is coherent with the fact that the phenomenon of dynamic changes in membrane lipid composition are conserved between yeast sporulation and mammalian spermatogenesis [109].…”

Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation

“…4 A ). Finally, the drug cerulenin, an inhibitor of fatty acid synthesis ( Ohno et al, 1976 ), blocked Acb1 secretion ( Fig. 4 A ).…”

“…This is consistent with the findings that proteins we show to be essential for Acb1 secretion, including several Atg proteins and Spo14, are also necessary for sporulation ( Neiman, 2005 ). Similarly, cerulenin treatment of starved yeast cells impairs sporulation ( Ohno et al, 1976 ). These facts establish beyond any doubt that P. pastoris cells secrete and produce SDF-2–like activity essentially like D. discoideum cells.…”

The antibiotic cerulenin, a novel tool for biochemistry as an inhibitor of fatty acid synthesis.

“…The inhibition of sporulation in S. cerevisiae by cerulenin and its reversion by externally added fatty acid were studied (60). Both palmitate and oleate reverse the inhibition of sporulation by cerulenin.…”