The yeast MER2 gene is required for chromosome synapsis and the initiation of meiotic recombination.

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.

SPO14 separation-of-function mutations define unique roles for phospholipase D in secretion and cellular differentiation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, phospholipase D (PLD), encoded by the SPO14 gene, catalyzes the hydrolysis of phosphatidylcholine, producing choline and phosphatidic acid. SPO14 is essential for cellular differentiation during meiosis and is required for Golgi function when the normal secretory apparatus is perturbed (Sec14-independent secretion). We isolated specific alleles of SPO14 that support Sec14-independent secretion but not sporulation. Identification of these separation-of-function alleles indicates that the role of PLD in these two physiological processes is distinct. Analyses of the mutants reveal that the corresponding proteins are stable, phosphorylated, catalytically active in vitro, and can localize properly within the cell during meiosis. Surprisingly, the separation-of-function mutations map to the conserved catalytic region of the PLD protein. Choline and phosphatidic acid molecular species profiles during Sec14-independent secretion and meiosis reveal that while strains harboring one of these alleles, spo14S-11, hydrolyze phosphatidylcholine in Sec14-independent secretion, they fail to do so during sporulation or normal vegetative growth. These results demonstrate that Spo14 PLD catalytic activity and cellular function can be differentially regulated at the level of phosphatidylcholine hydrolysis.

Meiosis-specific RNA splicing in yeast.

Previous studies have suggested that the differentiated state of meiosis in yeast is regulated primarily at the transcriptional level. This study reports a case of posttranscriptional regulation of a gene whose product is essential for meiosis. The MER2 gene is transcribed in mitosis as well as meiosis; however, the transcript is spliced efficiently to generate a functional gene product only in meiosis. Meiotic levels of splicing depend on the MER1 gene product, which is also essential for meiosis and which is produced only in meiotic cells. Therefore, at least one of the functions of the MER1 protein is to mediate splicing of the MER2 transcript. Genetic data suggest that the MER1 gene may also be responsible for splicing the transcript of at least one other gene.

ZIP1 is a synaptonemal complex protein required for meiotic chromosome synapsis.

ZIP1 is a novel meiosis-specific gene required for chromosome synapsis and cell cycle progression in S. cerevisiae. zip1 strains undergo homologous chromosome pairing, but are defective in synaptonemal complex (SC) formation. The zip1 mutation confers a uniform arrest in meiosis prior to the first division. zip1 strains display nearly wild-type levels of commitment to meiotic recombination; however, mature reciprocal recombinants are not formed until cells are released from meiotic arrest by return to growth medium. DNA sequence analysis of ZIP1 reveals structural homology to a number of proteins containing coiled coils. Immunofluorescence experiments using anti-ZIP1 antibodies demonstrate that the ZIP1 protein localizes to synapsed meiotic chromosomes but not to unsynapsed axial elements. Taken together, these data suggest that ZIP1 is a component of the central region of the SC. We propose a model in which ZIP1 acts as a molecular zipper to bring homologous chromosomes in close apposition.

Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects.

Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such 'bypass Sec14p' mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in 'bypass Sec14p' mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for 'bypass Sec14p', and yeast operating under 'bypass Sec14p' conditions are ethanol-sensitive. These data suggest that PLD supports 'bypass Sec14p' by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.