During meiosis II in the yeast Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body changes from a site of microtubule initiation to a site of de novo membrane formation. These membranes are required to package the haploid meiotic products into spores. This functional change in the spindle pole body involves the expansion and modification of its cytoplasmic face, termed the outer plaque. We report here that SPO21 is required for this modification. The Spo21 protein localizes to the spindle pole in meiotic cells. In the absence of SPO21 the structure of the outer plaque is abnormal, and prospore membranes do not form. Further, decreased dosage of SPO21 leaves only two of the four spindle pole bodies competent to generate membranes. Mutation of CNM67, encoding a known component of the mitotic outer plaque, also results in a meiotic outer plaque defect but does not block membrane formation, suggesting that Spo21p may play a direct role in initiating membrane formation.

Spore formation in Saccharomyces cerevisiae occurs via the de novo synthesis of the prospore membrane during the second meiotic division. Prospore membrane formation is triggered by assembly of a membraneorganizing center, the meiotic outer plaque (MOP), on the cytoplasmic face of the spindle pole body (SPB) during meiosis. We report here the identification of two new components of the MOP, Ady4p and Spo74p. Ady4p and Spo74p interact with known proteins of the MOP and are localized to the outer plaque of the SPB during meiosis II. MOP assembly and prospore membrane formation are abolished in spo74⌬/spo74⌬ cells and occur aberrantly in ady4⌬/ady4⌬ cells. Spo74p and the MOP component Mpc70p are mutually dependent for recruitment to SPBs during meiosis. In contrast, both Ady4p and Spo74p are present at SPBs, albeit at reduced levels, in cells that lack the MOP component Mpc54p. Our findings suggest a model for the assembled MOP in which Mpc54p, Mpc70p, and Spo74p make up a core structural unit of the scaffold that initiates synthesis of the prospore membrane, and Ady4p is an auxiliary component that stabilizes the plaque.Sporulation in Saccharomyces cerevisiae is a specialized form of cell division in which a single diploid cell produces four haploid spores within the cytoplasm of the original mother cell (10). The limiting membranes of the spores are synthesized de novo during meiotic segregation of nuclear DNA via the redirection of the secretory pathway (23). Golgi-derived vesicles that are destined for the plasma membrane during vegetative growth become targeted to the four spindle pole bodies (SPBs), the functional equivalents of centrosomes in higher eukaryotes, during sporulation. Fusion of these vesicles creates four discrete membrane compartments, the prospore membranes, which surround the nuclei upon the completion of meiosis to generate spores.Prospore membrane synthesis is initiated during the transition from the first to the second meiotic division. In yeast, both meiotic divisions occur within a single, continuous nuclear envelope, in which the SPBs are embedded. The cytoplasmic face of each SPB, termed the outer plaque, expands in early meiosis II and becomes a site for docking and fusion of secretory vesicles (20). Formation of the prospore membrane occurs via the fusion of these vesicles to form a flattened sac that abuts the meiotic outer plaque (MOP). Each prospore membrane grows toward the center of the spindle during anaphase II and engulfs the adjacent lobe of the nucleus. As nuclear division occurs at the end of meiosis II, each prospore membrane fuses with itself to enclose a haploid nucleus within two continuous membranes. Spore wall material is deposited into the lumen between the two new membranes to produce a mature spore (19).Regulation of SPB function during meiosis is critical for spore formation. The morphological expansion of the outer plaque during sporulation reflects a shift in its primary role from the anchoring of cytoplasmic microtubules to the initiation of prospore membrane ...

Yeast cells with mutations in BRO1 display phenotypes similar to those caused by deletion of BCK1, a gene encoding a MEK kinase that functions in a mitogen-activated protein kinase pathway mediating maintenance of cell integrity. bro1 cells exhibit a temperature-sensitive growth defect that is suppressed by the addition of osmotic stabilizers or Ca 2؉ to the growth medium or by additional copies of the BCK1 gene. At permissive temperatures, bro1 mutants are sensitive to caffeine and respond abnormally to nutrient limitation. A null mutation in BRO1 is synthetically lethal with null mutations in BCK1, MPK1, which encodes a mitogenactivated protein kinase that functions downstream of Bck1p, or PKC1, a gene encoding a protein kinase C homolog that activates Bck1p. Analysis of the isolated BRO1 gene revealed that it encodes a novel, 97-kDa polypeptide which contains a putative SH3 domain-binding motif and is homologous to a protein of unknown function in Caenorhabditis elegans.Cellular responses to a variety of environmental signals are mediated by modular protein kinase cascades whose ultimate targets are mitogen-activated protein (MAP) kinases (5). Roles for MAP kinases (also called ERKs) have been established in cells undergoing mitosis, meiosis, and differentiation (36). MAP kinase activation requires phosphorylation by a MAP kinase kinase, or MEK (for MAP kinase/ERK kinase). The MEK is, in turn, activated via phosphorylation by a member of the MEK kinase (MEKK) family, the upstream component of the MAP kinase functional unit. MAP kinase modules have been identified in a variety of eukaryotic species, ranging from mammals (46) to budding and fission yeasts (14).Maintenance of cell integrity in the budding yeast Saccharomyces cerevisiae requires a distinct MAP kinase pathway, which includes a MEKK, encoded by the BCK1/SLK1 gene (9, 23), two MEKs, products of the MKK1 and MKK2 genes (17), and a MAP kinase, encoded by the MPK1/SLT2 gene (22,30,47; reviewed in reference 12). Null mutants lacking any one of these functional units, the MAP kinase, the two MEKs, or the MEKK, display a cell lysis defect at elevated temperatures. Addition of osmotic stabilizing agents (e.g., 1 M sorbitol) to the growth medium suppresses this lysis defect.Genetic and biochemical data have demonstrated that signaling through the MAP kinase pathway that mediates maintenance of cell integrity is triggered by activation of yeast protein kinase C, product of the PKC1 gene (12, 25). Disruption of PKC1 results in a cell lysis defect at all temperatures as a result of defective cell wall construction (24, 37). Because loss of PKC1 results in a more severe phenotype than does loss of BCK1 or other downstream components of this cascade, one proposed model (23) suggests that the MAP kinase module constitutes one branch of a bifurcated pathway which is regulated by Pkc1p.Here we report the isolation of a yeast mutant, bro1, exhibiting a temperature-dependent, osmoremedial growth defect which is suppressed by Ca 2ϩ or by centromere-based plasmids cont...