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Vol. 18, Issue 9, 3568-3581, September 2007

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Geranylgeranyl Diphosphate Synthase in Fission Yeast Is a Heteromer of Farnesyl Diphosphate Synthase (FPS), Fps1, and an FPS-like Protein, Spo9, Essential for Sporulation

Yanfang Ye^*, Makoto Fujii, Aiko Hirata, Makoto Kawamukai, Chikashi Shimoda^*, and Taro Nakamura^*

*Department of Biology, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585, Japan; Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba 277-8562, Japan; and Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan

Submitted February 8, 2007; Revised May 23, 2007; Accepted June 20, 2007
Monitoring Editor: Akihiko Nakano

Both farnesyl diphosphate synthase (FPS) and geranylgeranyl diphosphate synthase (GGPS) are key enzymes in the synthesis of various isoprenoid-containing compounds and proteins. Here, we describe two novel Schizosaccharomyces pombe genes, fps1⁺ and spo9⁺, whose products are similar to FPS in primary structure, but whose functions differ from one another. Fps1 is essential for vegetative growth, whereas, a spo9 null mutant exhibits temperature-sensitive growth. Expression of fps1⁺, but not spo9⁺, suppresses the lethality of a Saccharomyces cerevisiae FPS-deficient mutant and also restores ubiquinone synthesis in an Escherichia coli ispA mutant, which lacks FPS activity, indicating that S. pombe Fps1 in fact functions as an FPS. In contrast to a typical FPS gene, no apparent GGPS homologues have been found in the S. pombe genome. Interestingly, although neither fps1⁺ nor spo9⁺ expression alone in E. coli confers clear GGPS activity, coexpression of both genes induces such activity. Moreover, the GGPS activity is significantly reduced in the spo9 mutant. In addition, the spo9 mutation perturbs the membrane association of a geranylgeranylated protein, but not that of a farnesylated protein. Yeast two-hybrid and coimmunoprecipitation analyses indicate that Fps1 and Spo9 physically interact. Thus, neither Fps1 nor Spo9 alone functions as a GGPS, but the two proteins together form a complex with GGPS activity. Because spo9 was originally identified as a sporulation-deficient mutant, we show here that expansion of the forespore membrane is severely inhibited in spo9 cells. Electron microscopy revealed significant accumulation membrane vesicles in spo9 cells. We suggest that lack of GGPS activity in a spo9 mutant results in impaired protein prenylation in certain proteins responsible for secretory function, thereby inhibiting forespore membrane formation.

Address correspondence to: Taro Nakamura (taronaka{at}sci.osaka-cu.ac.jp).

Abbreviations used: DMAPP, dimethylallyl diphosphate; FARM, first aspartate-rich motif; FPP, farnesyl diphosphate; FPS, farnesyl diphosphate synthase; GGPP, geranylgeranyl diphosphate; GGPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; SARM, second aspartate-rich motif; TLC, thin-layer chromatography.

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