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Formins are involved in diverse aspects of morphogenesis, and share two regions of homology: FH1 and FH2. We describe a new formin homology region, FH3. FH3 is an amino-terminal domain that differs from the Rho binding site identified in Bni1p and p140mDia. The Schizosaccharomyces pombe formin Fus1 is required for conjugation, and is localized to the projection tip in cells of mating pairs. We replaced genomic fus1 + with green fluorescent protein (GFP)- tagged versions that lacked either the FH1, FH2, or FH3 domain. Deletion of any FH domain essentially abolished mating. FH3, but neither FH1 nor FH2, was required for Fus1 localization. An FH3 domain–GFP fusion protein localized to the projection tips of mating pairs. Thus, the FH3 domain alone can direct protein localization. The FH3 domains of both Fus1 and the S. pombe cytokinesis formin Cdc12 were able to localize GFP to the spindle pole body in half of the late G2 cells in a vegetatively growing population. Expression of both FH3-GFP fusions also affected cytokinesis. Overexpression of the spindle pole body component Sad1 altered the distribution of both Sad1 and the FH3-GFP domain. Together these data suggest that proteins at multiple sites can interact with FH3 domains.

In Schizosaccharomyces pombe, the fus1 mutation blocks conjugation at a point after cell contact and agglutination. The cell walls separating the mating partners are not degraded, which prevents cytoplasmic fusion. In order to investigate the molecular mechanism of conjugation, we cloned the fus1 gene and found that it is capable of encoding a 1,372-amino-acid protein with no significant similarities to other known proteins. Expression of the fus1 gene is regulated by the developmental state of the cells. Transcription is induced by nitrogen starvation and requires a pheromone signal in both P and M cell types. Consequently, mutants defective in the pheromone response pathway fail to induce fus1 expression. The ste11 gene, which encodes a transcription factor controlling expression of many genes involved in sexual differentiation, is also required for transcription of fus1. Furthermore, deletion of two potential Ste11 recognition sites in the fus1 promoter region abolished transcription, and expression could be restored when we inserted a different Ste11 site from the mat1-P promoter. Since this element was inverted relative to the fus1 element, we conclude that activation of transcription by Ste11 is independent of orientation. Although the fus1 mutant has a phenotype very similar to that of Saccharomyces cerevisiae fus1 mutants, the two proteins appear to have different roles in the process of cell fusion. Budding yeast Fus1 is a typical membrane protein and contains an SH3 domain. Fission yeast Fus1 has no features of a membrane protein, yet it appears to localize to the projection tip. A characteristic proline-rich potential SH3 binding site may mediate interaction with other proteins.Nutritional starvation is the major signal that activates sexual differentiation in the fission yeast Schizosaccharomyces pombe (11). As long as the nutritional conditions are favorable, haploid cells will propagate vegetatively, but under conditions of nitrogen starvation, the cells exit from the mitotic cycle and undergo a differentiation process, which requires sexual agglutination, conjugation, nuclear fusion, meiosis, and spore formation to occur in an orderly fashion (see reference 13). The process of conjugation involves the action of diffusible pheromones secreted by P and M cell types in order to attract each other. When exposed to the opposite pheromone, the cells form projections toward each other (18, 37) and fuse upon cell-cell contact. Attachment at the projection tips between paired cells culminates in localized cell wall degradation and plasma membrane fusion. Nuclear fusion is coordinated with these events, resulting in the formation of a zygote (see reference 53).In the differentiation process, the pheromones act by binding to specific receptors on the surface of the opposite cell type (29, 60), thereby activating the pheromone response pathway. Transmission of the signal through the pathway involves the actions of the ras1 function and of three protein kinases encoded by byr2, byr1, and spk1 (19,45,47,49,58,6...

The fission yeast Schizosaccharomyces pombe switches mating type by transposition of a copy of DNA de. rived from either of the two storage cassettes, mat2-P and mat3-M, into the expression locus, mat]. The recombinational event of switching is initiated by a double-stranded DNA break present in approximately 20% of the molecules at mat]. Fiftythree mutants defective in switching of mating type have been isolated previously, and each has been assigned to 1 of 10 linkage groups. One group consists of cis-acting mutations at mat), which reduce the amount of the DNA double-strand cut.The remaining nine groups are mutations in genes that are unlinked to the mating-type locus and are studied here. Three (swil, -3, -7) are required for formation of the double-strand cut, whereas the others are not. Mutants of three genes (swi4, -8, -9) undergo high-frequency rearrangement of the matingtype locus indicative of errors of resolution of recombinational intermediates. The remaining three (swi2, -5, -6) have normal levels of cut, do not make errors of resolution, and possibly are required either for efficient utilization of the cut or determining the directionality of switching. The data suggest that the switching process can be dissected into genetically distinguishable steps.In wild-type (h90) strains of Schizosaccharomyces pombe, mating-type switching between h+ and h-occurs approximately once in every three cell divisions (1, 2). Under conditions of nutritional deprivation, cells of opposite mating type arrest in G1 phase and conjugate to form a temporary diploid zygote, which undergoes premeiotic S phase followed by meiosis to yield four haploid spores (3).Genetic analysis (4)(5)(6) indicates that mating type is controlled by a tightly linked cluster of genes (known as the mating-type locus) located in the long arm of chromosome II. Isolation of the DNA of this region (7-9) has confirmed that mating-type switching occurs by copy-transposition of information contained in stores of unexpressed plus (mat2-P) or minus (mat3-M) information into the expression locus mati (Fig. 1A). The phenotype of the cell is determined by the temporary presence of the P or M allele of matl. mati, mat2-P, and mat3-M are referred to as cassettes, by analogy with the situation described in Saccharomyces cerevisiae (10-12). The distance between each cassette is approximately 15 kilobases (kb) (9). The cassettes consist of a 1.1-kb plus (P)-or minus (M)-specific region bounded distally with respect to the centromere by a 90-base-pair (bp) region (H1) and proximally by a 190-bp region (H2) of homology common to each (ref. 8; Fig. 1A). The existence of these blocks of homology suggests that mating-type switching could occur by a mechanism similar to that of mitotic or meiotic gene conversion. However, mating-type switching is distinguishable from conventional gene conversion by several features.Mating-type switching occurs at very high frequency and shows extreme disparity. mati is always the recipient of a mating-type switch and never...