A Saccharomyces cerevisiae mutant strain defective in acetyl-CoA carboxylase arrests at the G2/M phase of the cell cycle

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The National Academy of Sciences

RESUMO

To elucidate the essential functions of acetyl-CoA carboxylase (ACC1/FAS3) in Saccharomyces cerevisiae, a temperature-sensitive mutant (acc1ts) was constructed. When the acc1ts cells were synchronized in G1 phase with α-factor at the permissive temperature of 24°C and then released from the blockade and incubated at the restrictive temperature of 37°C, 95% of the cell population became arrested at the G2/M phase of the cell cycle despite the presence of fatty acids (C14-C26) in the medium. These cells developed large undivided nuclei, and the spindles of the arrested mutant cells were short. Shifting the G2 arrested cells back to the permissive temperature resulted in a reversal of the cell-cycle arrest, with cells initiating mitosis. However, after 3 h of incubation at 37°C, G2 arrested mutant cells lost viability and displayed a uniquely altered nuclear envelope. Using [1-14C]acetate as a precursor for fatty acids synthesis, we identified the phospholipids and sphingolipids derived from acc1ts cells and wild-type cells at 24°C and 37°C, respectively. The levels of inositol-ceramides [IPC, MIPC, and M(IP)2C] and very long-chain fatty acids C24 and C26 declined sharply in the G2/M arrested cells because of ACC inactivation. Shifting the acc1ts cells to 24°C after 2 h of incubation at 37°C resulted in reactivation of the ACC and elevation of the ceramides and very long-chain fatty acid syntheses with normal cell-cycle progression. In contrast, synthesis of wild-type inositol-ceramides, C24 and C26, fatty acids were elevated on incubation at 37°C and declined when the cells shifted to the permissive temperature of 24°C.

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