NATIONAL INSITUTE FOR BASIC BIOLOGY

National Institute for Basic Biology

Division of Cell Proliferation

(Adjunct)

Professor:
Masayuki Yamamoto
Research Associate:
Masuo Goto
Institute Research Fellow:
Takashi Kuromori
Visiting Scientist:
Satsuki Okamoto
Graduate Students:
Chikako Kitayama (from The University of Tokyo)
Masahiro Fujita (from The University of Tokyo)


The major goal of this Division is to elucidate regulatory mechanisms of meiosis. Meiosis is a crucial step in gamete formation and is essential for sexual reproduction. Meiotic steps are highly conserved among eukaryotic species. We have been screening for genes that may be relevant to the regulation of meiosis in animals and plants, by using transcomplementation methods. In the fission yeast Schizosaccharomyces pombe, which is a unicellular eukaryotic microorganism, genes controlling meiosis have been cloned and characterized extensively, and mutants defective in these genes are available. We have isolated animal and plant genes that can functionally complement such S. pombe mutants. Analysis of the cloned genes is in progress. In addition, efforts are also paid to in-depth analysis of regulatory mechanisms of meiosis in the fission yeast, to facilitate the above strategy.


I. Genes encoding farnesyl cysteine carboxyl methyltransferase in fission yeast and Xenopus

The mating pheromone signaling is essential for the induction of meiosis in Schizosaccharomyces pombe. The S. pombe mam4 mutation causes mating deficiency in cells of mating-type M but not in P. M cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides, but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homologue of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to give a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. These results will open the way to intensive studies of the role for methylation in a large body of proteins including ras-superfamily proteins and its relevance to meiosis. This work will be published shortly (Y. Imai et al. (1997) Mol. Cell. Biol., in press).


II. Phosphorylation of RNA-binding protein controls cell cycle switch from mitotic to meiotic in fission yeast

Little is known about the molecular controls over the switch from mitotic to meiotic cell cycles. In S. pombe, the switch from the mitotic to the meiotic cell cycle is controlled by the antagonistic effects of the Pat1 protein kinase and the Mei2 RNA-binding protein. We have shown that Mei2 is phosphorylated on Ser438 and Thr527 by Pat1 kinase, whose inactivation is necessary to induce meiosis. Mutant Mei2 carrying alanine in these two positions induces meiosis without inactivation of Pat1 kinase, establishing that Mei2 is the critical target of Pat1. Mei2 localizes mainly in the cytoplasm of proliferating cells, but is found in a single 'dot', closely opposed to the spindle pole body, in prophase nuclei during meiosis I (Fig. 1). Our results emphasize the crucial role of RNA-binding proteins in the initiation and execution of meiosis. This work has been published lately (Y. Watanabe et al. (1997) Nature 386, 187-190).

Fig. 1
A "dot" formed by the RNA-binding protein Mei2 in a meiotic prophase nuclei. Mei2 protein fused to GFP, which emits green fluorescence, was live-recorded. The yellow spot represents fluorescence of Mei2-GFP, whereas the red region represents nuclear DNA. The whole cell is faintly visible in red. Prophase nuclei of fission yeast move back and forth, assuming morphology like a horse tail. Mei2 appears to occupy a fixed position in the meiotic nucleus. See Watanabe et al. (Nature 386, 187-190, 1997) for more details. We were assisted by Y. Chikashige and Y. Hiraoka (Kansai Advanced Research Center, Communications Research Laboratory) in taking this photograph.


Publication List:
Hakuno, F. and Yamamoto, M. (1996). The Schizosaccharomyces pombe mra1 gene, which is required for cell growth and mating, can suppress the mating inefficiency caused by a deficit in the Ras1 activity. Genes Cells 1, 303-315.
Watanabe, Y. and Yamamoto, M. (1996). Schizosaccharomyces pombe pcr1+ encodes an ATF/CRE-binding protein involved in the regulation of gene expression for sexual development. Mol. Cell. Biol. 16, 704-711.
Yabana, N. and Yamamoto, M. (1996). Schizosaccharomyces pombe map1+ encodes a MADS-box-family protein required for cell-type-specific gene expression. Mol. Cell. Biol. 16, 3420-3428.
Yamamoto, M. (1996). The molecular control mechanisms of meiosis in fission yeast. Trends Biochem. Sci. 21, 18-22.
Yamamoto, M. (1996). Regulation of meiosis in fission yeast. Cell Struct. Funct. 21, 431-436.

nibb-adm@nibb.ac.jp
Last Modified: 12:00, June 27, 1997