Differential Requirement for Phospholipase D/Spo14 and Its Novel Interactor Sma1 for Regulation of Exocytotic Vesicle Fusion in Yeast Meiosis

Abstract

During sporulation and meiosis of budding yeast a developmental program determines the formation of the new plasma membranes of the spores. This process of prospore membrane (PSM) formation leads to the formation of meiotic daughter cells, the spores, within the lumen of the mother cell. It is initiated at the spindle pole bodies during meiosis II. Spore formation, but not meiotic cell cycle progression, requires the function of phospholipase D (PLD/Spo14). Here we show that PLD/Spo14 forms a complex with Sma1, a meiotically expressed protein essential for spore formation. Detailed analysis revealed that both proteins are required for early steps of prospore membrane assembly but with distinct defects in the respective mutants. In the ⌬spo14 mutant the initiation of PSM formation is blocked and aggregated vesicles of homogenous size are detected at the spindle pole bodies. In contrast, initiation of PSM formation does occur in the ⌬sma1 mutant, but the enlargement of the membrane is impaired. During PSM growth both Spo14 and Sma1 localize to the membrane, and localization of Spo14 is independent of Sma1. Biochemical analysis revealed that Sma1 is not necessary for PLD activity per se and that PLD present in a complex with Sma1 is highly active. Together, our results suggest that yeast PLD is involved in two distinct but essential steps during the regulated vesicle fusion necessary for the assembly of the membranous encapsulations of the spores.During cellular differentiation and development, tightly controlled cell polarity changes occur (1). These changes involve redirection of the secretory pathway and specify the site of exocytosis. For these processes, molecular mechanisms exist that ensure correct delivery of membranes and secretory proteins in response to cellular signals. During yeast gametogenesis (sporulation) so-called prospore membranes (PSMs) 5 form de novo at sites adjacent to the spindle pole bodies (SPBs), the centrosomes of yeast (2). This process involves redirection of the secretory machinery to the SPB (3) and results in formation of four haploid cells within the mother cell. The initiation of this process is a tightly regulated event that occurs exactly once per SPB at the onset of meiosis II. It is thought that PSM formation is initiated by the fusion of vesicles at the SPB that generate an initial compartment, which then subsequently becomes enlarged and grows around the haploid nucleus to which it is connected via the SPB. Only recently support for this model came from the analysis of a ⌬mso1 mutant in which a block of PSM formation on the level of vesicle fusion at the SPB was visible (39).The formation of PSMs appears to require several genes known to act in the exocytosis of post-Golgi vesicles in vegetatively growing (mitotic) cells. In temperature-sensitive mutants of sec1, sec4, and sec8 or cells deleted for the syntaxin SSO1, no PSM formation occurs at the restrictive temperature (3, 4). In addition, PSM formation requires Spo20, a sporulation-specific Sec9 target SNARE homologue (3...

Erv14 family cargo receptors are necessary for ER exit during sporulation inSaccharomyces cerevisiae

“…Of the 45 mutants, eight met these criteria: erv14⌬, gcs1⌬, mso1⌬, rcy1⌬, sma1⌬, sma2⌬, sec22⌬ and vps13⌬ (Table 1). Of these mutants, three, MSO1, SMA1 and GCS1, have recently been reported to have prospore membrane defects Riedel et al, 2005;Connolly and Engebrecht, 2006), validating our screen. The remaining mutants affecting prospore membrane growth were characterized further.…”

Phospholipase D and the SNARE Sso1p are necessary for vesicle fusion during sporulation in yeast

“…Thus, the catalytic activity of Spo14p appears to be necessary for additional aspects of membrane growth. A recent, independent study of Spo14p and its sporulation-specific binding partner Sma1p reported the same accumulation of SPB-associated vesicles in spo14 cells that we describe here (Riedel et al, 2005). Mutation of SMA1, however, resulted in formation of a membrane cap on the SPB, but a failure in continued expansion of the prospore membrane.…”

SSP2 and OSW1, Two Sporulation-Specific Genes Involved in Spore Morphogenesis in Saccharomyces cerevisiae

“…The phenotype of a spo14 null mutant is similar to that of the sso1 and sec9 spo20 mutants, indicating that Spo14 also participates in vesicle fusion (Riedel et al 2005;Nakanishi et al 2006). Studies of nonnull alleles of SPO14 and of mutations affecting the Spo14-interacting protein, Sma1, indicate that Spo14 acts at two discrete steps in PSM formation: the initial fusion of vesicles on the meiotic plaque and subsequent enlargement of the PSM to surround the adjoining nucleus (Riedel et al 2005;Nakanishi et al 2006).…”

“…Since these proteins are involved in vesicle fusion during PSM formation (Riedel et al 2005;Nakanishi et al 2006), it is likely that Ssp2 also plays a role in vesicle fusion. As noted in the Introduction, Spo14 in both initiation and expansion of the PSM (Riedel et al 2005;Nakanishi et al 2006). Similarly, Ssp2 could act at both steps.…”