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, ${dagger}$ Aiko Hirata, ${ddagger}$ Makoto Kawamukai, ${dagger}$ 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; Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan

Monitoring Editor: Akihiko Nakano

Both farnesyl diphosphatesynthase (FPS) and geranylgeranyl diphosphate synthase (GGPS)are key enzymes in the synthesis of various isoprenoid-containingcompounds and proteins. Here we describe two novel Schizosaccharomycespombe genes, fps1⁺ and spo9⁺, whose products are similar toFPS in primary structure, but whose functions differ from oneanother. Fps1 is essential for vegetative growth, whereas, aspo9 null mutant exhibits temperature-sensitive growth. Expressionof fps1⁺, but not spo9⁺, suppresses the lethality of a S. cerevisiaeFPS-deficient mutant and also restores ubiquinone synthesisin an E. coli ispA mutant which lacks FPS activity, indicatingthat S. pombe Fps1 in fact, functions as an FPS. In contrastto a typical FPS gene, no apparent GGPS homologues have beenfound in the S. pombe genome. Interestingly, while 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 associationof a geranylgeranylated protein, but not that of a farnesylatedprotein. Yeast two-hybrid and coimmunoprecipitation analysesindicate that Fps1 and Spo9 physically interact. Thus, neitherFps1 nor Spo9 alone functions as a GGPS, but the two proteinstogether form a complex with GGPS activity. Because spo9 wasoriginally identified as a sporulation-deficient mutant, weshow here that expansion of the forespore membrane is severelyinhibited in spo9 ${Delta}$ cells. Electron microscopy revealed significantaccumulation membrane vesicles in spo9 ${Delta}$ cells. We suggest thatlack of GGPS activity in a spo9 mutant results in impaired proteinprenylation in certain proteins responsible for secretory function,thereby inhibiting forespore membrane formation.

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

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