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

Christian G Riedel,Massimiliano Mazza,Peter Maier,Roman Körner,Michael Knop

doi:10.1074/jbc.m504244200

Christian G Riedel, Massimiliano Mazza + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m504244200

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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 Deltaspo14 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 Deltasma1 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.

Highlights

Regulated event that occurs exactly once per SPB at the onset of meiosis II
The precise vesicle fusion machinery required for the initiation of membrane formation and the way that this machinery is regulated and how it correlates to the machinery required for subsequent membrane elongation during prospore membrane (PSM) growth are not known
The formation of these socalled prospore membranes is entirely different from mitotic bud membrane formation, as these membranes become assembled de novo inside the cell, discontinuous to the plasma membrane of the mother cell

Summary

Introduction

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 subsequently becomes enlarged and grows around the haploid nucleus to which it is connected via the SPB. PLD has been proposed to participate in cytoskeletal modeling and vesicular traffic in the secretory pathway (6 – 8) This regulation is thought to occur mainly through its capacity to hydrolyze phospholipids in order to generate the second messenger phosphatidic acid (PA). Both the Saccharomyces cerevisiae PLD/Spo and the mammalian phospholipase D associate with phosphatidyl inositol-4,5bisphosphate (PtdIns-4,5-P2), and PtdIns-4,5-P2 has been shown to be needed for efficient PLD function [9, 10] and for the localization of the mammalian PLD [11]. Our data are consistent with the idea that PLD plays several roles during this process, including one early Sma1-independent function during the initiation of membrane formation via homotypic vesicle fusion and a later function during elongation of the membrane by heterotypic membrane fusion in association with Sma

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