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Spore formation in Saccharomyces cerevisiae requires the de novo formation of prospore membranes. The coalescence of secretory vesicles into a membrane sheet occurs on the cytoplasmic surface of the spindle pole body. Spo14p, the major yeast phospholipase D, is necessary for prospore membrane formation; however, the specific function of Spo14p in this process has not been elucidated. We report that loss of Spo14p blocks vesicle fusion, leading to the accumulation of prospore membrane precursor vesicles docked on the spindle pole body. A similar phenotype was seen when the t-SNARE Sso1p, or the partially redundant t-SNAREs Sec9p and Spo20p were mutated. Although phosphatidic acid, the product of phospholipase D action, was necessary to recruit Spo20p to the precursor vesicles, independent targeting of Spo20p to the membrane was not sufficient to promote fusion in the absence of SPO14. These results demonstrate a role for phospholipase D in vesicle fusion and suggest that phospholipase D-generated phosphatidic acid plays multiple roles in the fusion process.

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Background : Phosphatidylinositol (3,5) bisphosphate, which is converted from phosphatidylinositol 3-phosphate by phosphatidylinositol 3-phosphate 5-kinase, is implicated in vacuolar functions and the sorting of cell surface proteins within endosomes in the endocytic pathway of budding yeast. A homologous protein, SpFab1p, has been found in the fission yeast Schizosaccharomyces pombe , but its role is not known.

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During sporulation in Saccharomyces cerevisiae, vesicles transported to the vicinity of spindle pole bodies are fused to each other to generate bilayered prospore membranes (PSMs). PSMs encapsulate the haploid nuclei that arise from the meiotic divisions and serve as platforms for spore wall deposition. Membrane trafficking plays an important role in supplying vesicles for these processes. The endocytosis-deficient mutant, end3⌬, sporulated poorly and the spores produced lost resistance to ether vapor, suggesting that END3-mediated endocytosis is important for sporulation. End3p-GFP localized to cell and spore peripheries in vegetative and sporulating cells and colocalized with actin structures. Correspondingly, the actin cytoskeleton appeared aberrant during sporulation in end3⌬. Analysis of meiosis in end3⌬ mutants revealed that the meiotic divisions occurred with wild-type kinetics. Furthermore, PSMs were assembled normally. However, the levels of proteins required for spore wall synthesis and components of the spore wall layers at spores were reduced, indicating that end3⌬ mutants are defective in spore wall synthesis. Thus, END3-mediated endocytosis is important for spore wall formation. Additionally, cytological analyses suggest that trafficking between the plasma membrane and PSMs is important earlier during sporulation.

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BackgroundHistone lysine methylation has a pivotal role in regulating the chromatin. Histone modifiers, including histone methyl transferases (HMTases), have clear roles in human carcinogenesis but the extent of their functions and regulation are not well understood. The NSD family of HMTases comprised of three members (NSD1, NSD2/MMSET/WHSC1, and NSD3/WHSC1L) are oncogenes aberrantly expressed in several cancers, suggesting their potential to serve as novel therapeutic targets. However, the substrate specificity of the NSDs and the molecular mechanism of histones H3 and H4 recognition and methylation have not yet been established.ResultsHerein, we investigated the in vitro mechanisms of histones H3 and H4 recognition and modifications by the catalytic domain of NSD family members. In this study, we quantified in vitro mono-, di- and tri- methylations on H3K4, H3K9, H3K27, H3K36, H3K79, and H4K20 by the carboxyl terminal domain (CTD) of NSD1, NSD2 and NSD3, using histone as substrate. Next, we used a molecular modelling approach and docked 6-mer peptides H3K4 a.a. 1-7; H3K9 a.a. 5-11; H3K27 a.a. 23-29; H3K36 a.a. 32-38; H3K79 a.a. 75-81; H4K20 a.a. 16-22 with the catalytic domain of the NSDs to provide insight into lysine-marks recognition and methylation on histones H3 and H4.ConclusionsOur data highlight the versatility of NSD1, NSD2, and NSD3 for recognizing and methylating several histone lysine marks on histones H3 and H4. Our work provides a basis to design selective and specific NSDs inhibitors. We discuss the relevance of our findings for the development of NSD inhibitors amenable for novel chemotherapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12900-014-0025-x) contains supplementary material, which is available to authorized users.