A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

The Sec14-like phosphatidylinositol transfer protein Sfh3 associates with bulk LDs in vegetative cells but targets to a neutral lipid hydrolase-rich LD pool during sporulation. Sfh3 inhibits LD utilization by a PtdIns-4-phosphate–dependent mechanism, and this inhibition prevents prospore membrane biogenesis in sporulating cells.Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches

A Highly Redundant Gene Network Controls Assembly of the Outer Spore Wall in <i>S. cerevisiae</i>

<div><p>The spore wall of <i>Saccharomyces cerevisiae</i> is a multilaminar extracellular structure that is formed <i>de novo</i> in the course of sporulation. The outer layers of the spore wall provide spores with resistance to a wide variety of environmental stresses. The major components of the outer spore wall are the polysaccharide chitosan and a polymer formed from the di-amino acid dityrosine. Though the synthesis and export pathways for dityrosine have been described, genes directly involved in dityrosine polymerization and incorporation into the spore wall have not been identified. A synthetic gene array approach to identify new genes involved in outer spore wall synthesis revealed an interconnected network influencing dityrosine assembly. This network is highly redundant both for genes of different activities that compensate for the loss of each other and for related genes of overlapping activity. Several of the genes in this network have paralogs in the yeast genome and deletion of entire paralog sets is sufficient to severely reduce dityrosine fluorescence. Solid-state NMR analysis of partially purified outer spore walls identifies a novel component in spore walls from wild type that is absent in some of the paralog set mutants. Localization of gene products identified in the screen reveals an unexpected role for lipid droplets in outer spore wall formation.</p></div

The effect of deletion of paralog sets on ether resistance of spores.

<p>Strains of the indicated genotypes were sporulated in liquid to >70% asci and then 10-fold serial dilutions were spotted onto YPD plates. Left panels, plates exposed to ether vapor for 45 minutes before incubation at 30°C. Right panels, no ether control. Multiple mutant diploids are indicated by the name of the set of genes deleted as listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen-1003700-t002" target="_blank">Table 2</a>.</p

Paralogous gene sets implicated in outer spore wall formation.

a<p>Indicates whether the transcript is induced during sporulation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen.1003700-Chu1" target="_blank">[55]</a>.</p>b<p>% similarity is based on comparison of the two sequences using BLASTP <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen.1003700-Altschul1" target="_blank">[56]</a>.</p

Electron microscopy of lipid droplets in sporulating cells.

<p>Electron micrographs of wild type cells. A) A cell in Meiosis II showing two prospore membranes (green arrows) engulfing nuclear lobes (N). Lipid droplets can be seen associated with the prospore membrane (yellow arrowheads). Scale bar = 500 nm. B) Higher magnification of one of the prospore membranes in A. Scale bar = 100 nm. C) Post meiotic cell showing dark-stained lipid droplets (yellow arrowhead) associated with the outer membrane (green arrow). Lipid droplets inside of the spore (yellow arrow) do not stain as darkly with permanganate. N = nucleus. Scale bar = 500 nm. D) Lipid droplets in the ascus (yellow arrowhead) associated with the outer spore wall (red arrow) of mature spores. Yellow arrow indicated a lipid droplet within the spore cytoplasm. N = nucleus. Scale bar = 500 nm.</p

Localization of the Lds proteins involved in outer spore wall synthesis.

<p>A) Diploids expressing GFP fusions to the indicated protein as well as mTag-BFP-Spo20^51–91 as a prospore membrane (PrM) marker were sporulated and examined by fluorescence microscopy. Examples of a cell in the middle of Meiosis II and a post-meiotic cell are shown. B) Sporulating wild-type cells expressing mTag-BFP-Spo20^51–91 were stained with the lipid droplet marker Bodipy TR. Arrowheads indicate lipid droplets associated with the ascal side of the prospore membrane. Arrows indicate lipid droplets within the prospore membrane C) Cells expressing both Lds2-GFP and mTag-BFP-Spo20^51–91 were stained with Bodipy TR. Arrowheads indicate localization of Lds2-GFP to a lipid droplet outside of the prospore membrane. For all experiments at least 20 cells at the indicated stage were scored and 100% of the cells displayed patterns similar to those shown here. Quantitation for each experiment is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen.1003700.s005" target="_blank">Table S2</a>. Scale bars = 1 micron.</p

Overview of spore wall formation.

<p>A) Each of the four nuclei (N) in a sporulating cell are engulfed by a prospore membrane (red). B) A single prospore membrane prior to closure. C) After closure of the prospore membrane, mannans and β-glucans (gray) are deposited in the lumen between the spore plasma membrane and outer membrane derived from the prospore membrane (both in red). D) The outer membrane disappears, exposing spore wall material to the ascal cytoplasm. E) Chitosan is synthesized and assembled as a discrete layer (green) on the outside of the β-glucan. F) The dityrosine layer (blue) is formed on the outside of the chitosan layer.</p

NMR analysis of outer spore walls in the paralog mutant strains.

<p>A) The dityrosine region of the ¹³C NMR spectrum is shown for spore walls from wild type, <i>dit1</i>Δ and the indicated paralog mutant strains. Positions of the dityrosine chemical shifts and chitin carbonyl chemical shifts are indicated above the wild type spectrum. The spectra have been scaled so the height of the carbonyl resonance is constant. B) The component χ region of the ¹³C NMR spectrum is shown for spore walls from wild type, <i>dit1</i>Δ and the indicated paralog mutant strains. Positions of the component χ chemical shifts and chitin methyl group shifts are indicated above the wild type spectrum. The spectra have been scaled to the C5/3 resonance as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen-1003700-g006" target="_blank">Figure 6</a>. Multiple mutant diploids are indicated by the name of the set of genes deleted as listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen-1003700-t002" target="_blank">Table 2</a>.</p

The effect of deletion of paralog sets on chitosan.

<p>Strains of the indicated genotype were sporulated and stained with both Eosin Y and Calcofluor White (CFW) to visualize the chitosan layer. The <i>cda1</i>Δ <i>cda2</i>Δ, <i>chs3</i>Δ, and <i>dit1</i>Δ strains provide standards for the conditions chitin/no chitosan/no dityrosine, no chitin/no chitosan/no dityrosine, and chitin/chitosan/no dityrosine, respectively. For all strains >30 cells were examined and >60% display the staining patterns shown here. Quantitation for each strain is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen.1003700.s005" target="_blank">Table S2</a>. DIC = differential interference contrast. Scale bar = 1 micron. Multiple mutant diploids are indicated by the name of the set of genes deleted as listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003700#pgen-1003700-t002" target="_blank">Table 2</a>.</p

NMR analysis of the outer spore wall.

<p>Outer spore walls were purified from wild type (AN120) or <i>dit1</i>Δ cells and then analyzed by solid state ¹³C NMR. Resonances assigned to chitosan carbons are designated by red labels and resonances from dityrosine are indicated by green labels. Unassigned chemical shifts from component χ are indicated in blue. Upper spectrum, spore wall from wild type. Lower spectrum, spore wall from <i>dit1</i>Δ. For comparison, spectra are scaled so the C5/3 resonance of chitosan is of constant height.</p