Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

Abstract

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P 2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P 2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P 2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P 2 synthesis.

End3p-Mediated Endocytosis Is Required for Spore Wall Formation in Saccharomyces cerevisiae

“…In end3⌬, only weak or no signals for FM4-64 were detected at vacuoles during vegetative growth, and plasma membrane staining persisted for 60 min after removal of dye during sporulation. yeast PLD, which is essential for and marks PSMs (Rudge et al 1998(Rudge et al , 2004 almost exclusively by the action of Spo14p, as spo14⌬ strains did not generate any appreciable BODIPY-PA 6 hr; marked by arrow), which were not seen in wild- (Table 5). BODIPY-PC can be internalized by endocytotype cells.…”

“…The spore wall layers are laid down in a specific order in which the mannan, glucan, chitosan, and dityrosine layers overlay 1 quired for spore wall formation are transported to PSMs nositide 4,5 bisphosphate binding protein and has been proposed to recruit Sla1p, which is required for assemthrough vesicle trafficking. Dtr1p, a transporter for dityrosine (Felder et al 2002), is transported from the bly of the endocytic complex at the plasma membrane, and to regulate actin-dependent endocytic internalizaGolgi to PSMs (M. Morishita, unpublished data), indicating that vesicle trafficking between the Golgi and tion (Wesp et al 1997;Gourlay et al 2003;Sun et al 2005). END3 encodes a component of the endocytic PSMs is essential for spore formation (Neiman 1998;Moreno-Borchart and Knop 2003).…”

“…Spo14p localizes to, and is essential for, PSM formation nonfermentable carbon source (Kupiec et al 1997). During sporulation, two major processes, meiosis, which (Rudge et al 1998(Rudge et al , 2004. Furthermore, the enzymatic activity of Spo14p is required for PSM formation at the reduces the ploidy of the cell, and spore formation are SPBs (Rudge et al 1998).…”

Multiple Functions as Lipase, Steryl Ester Hydrolase, Phospholipase, and Acyltransferase of Tgl4p from the Yeast Saccharomyces cerevisiae

“…Also, in mammalian cells, PA plays key roles as an activator of cell growth and proliferation and in vesicular trafficking, secretion, and endocytosis (22)(23)(24). In Saccharomyces cerevisiae, the formation of PA by phospholipase D Pld1p/Spo14p is essential for the fusion of vesicles with the prospore membrane during sporogenesis (25,26). In addition, PA is important for cytokinesis in budding yeast (27) and for microvesicle formation from the plasma membrane.…”

Identification of Salt-Tolerant Sinorhizobium sp. Strain BL3 Membrane Proteins Based on Proteomics

“…In salt stressed cells, the outer membrane proteins provide an interface with the surrounding environment. Moreover, membrane proteins play an important role in maintaining cell volume and the intracellular ion balance (33), the transportation of nutrients to and from the cell (19), conjugation (21), controlling cell morphology (36), intercellular communication, and cell metabolism (37). Several rhizobial membrane proteins have been reported to respond to salt stress, including a glycine betaine/proline betaine transporter encoded by betS (5), a potassium uptake system protein encoded by kup (29), and an outer membrane lipoprotein encoded by omp10 (40).…”