The yeast lysosome-like vacuole: endpoint and crossroads.

In response to nitrogen starvation in the presence of a poor carbon source, diploid cells of the yeast Saccharomyces cerevisiae undergo meiosis and package the haploid nuclei produced in meiosis into spores. The formation of spores requires an unusual cell division event in which daughter cells are formed within the cytoplasm of the mother cell. This process involves the de novo generation of two different cellular structures: novel membrane compartments within the cell cytoplasm that give rise to the spore plasma membrane and an extensive spore wall that protects the spore from environmental insults. This article summarizes what is known about the molecular mechanisms controlling spore assembly with particular attention to how constitutive cellular functions are modified to create novel behaviors during this developmental process. Key regulatory points on the sporulation pathway are also discussed as well as the possible role of sporulation in the natural ecology of S. cerevisiae.

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Sporulation in the Budding Yeast Saccharomyces cerevisiae

The exocyst complex is an evolutionarily conserved multisubunit protein complex implicated in tethering secretory vesicles to the plasma membrane. Originally identified two decades ago in budding yeast, investigations using several different eukaryotic systems have since made great progress toward determination of the overall structure and organization of the eight exocyst subunits. Studies point to a critical role for the complex as a spatiotemporal regulator through the numerous protein and lipid interactions of its subunits, although a molecular understanding of exocyst function has been challenging to elucidate. Recent progress demonstrates that the exocyst is also important for additional trafficking steps and cellular processes beyond exocytosis, with links to development and disease. In this review, we discuss current knowledge of exocyst architecture, assembly, regulation and its roles in a variety of cellular trafficking pathways.

/pdf/exorcising-the-exocyst-complex-5fbiku9tfq.pdf

Exorcising the Exocyst Complex

Estimates based on proteomic analyses indicate that a third of translated proteins in eukaryotic genomes enter the secretory pathway. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) selects folded cargo for export in membrane-bound vesicles. To accommodate the great diversity in secretory cargo, protein sorting receptors are required in a number of instances for efficient ER export. These transmembrane sorting receptors couple specific secretory cargo to COPII through interactions with both cargo and coat subunits. After incorporation into COPII transport vesicles, protein sorting receptors release bound cargo in pre-Golgi or Golgi compartments, and receptors are then recycled back to the ER for additional rounds of cargo export. Distinct types of protein sorting receptors that recognize carbohydrate and/or polypeptide signals in secretory cargo have been characterized. Our current understanding of the molecular mechanisms und...

Protein sorting receptors in the early secretory pathway.

RGG/RG Motif Regions in RNA Binding and Phase Separation.

The trans-Golgi network (TGN) is an important organelle for protein transport at the post-Golgi network, which functions as a sorting station that directs cargo proteins to a variety of destinations including post-Golgi compartments and the extracellular space. However, the functions and dynamics of the TGN in plant cells have not been well understood yet. To elucidate the dynamics of the plant TGN, we established transgenic plants expressing green fluorescent protein (GFP)-SYP43, the ortholog of Tlg2/syntaxin16, which is localized to the TGN in yeast and mammalian cells, under the control of the native promoter as a TGN marker. Observation by confocal laser scanning microscopy and super-resolution confocal live imaging microscopy revealed two types of TGN in Arabidopsis root: the GA-TGNs (Golgi-associated TGNs), located on the trans-side of the Golgi apparatus, and the GI-TGNs (Golgi-released independent TGNs), located away from the Golgi apparatus and behaving independently. The GI-TGNs is derived from a population of GA-TGNs by segregation, although the core of the GA-TGN remains even after the generation of the GI-TGN. We further found that the abundance of the GI-TGNs differs between observed tissues. Our results indicate that the dynamic features of the TGN in plant cells differ from those of animal and yeast cells.

/pdf/dynamic-behavior-of-the-trans-golgi-network-in-root-tissues-19x5v0xefv.pdf

Dynamic Behavior of the trans-Golgi Network in Root Tissues of Arabidopsis Revealed by Super-Resolution Live Imaging

The Golgi apparatus functions as the central station of membrane traffic in cells, where newly synthesized proteins moving along the secretory pathway merge with proteins recycled from subsequent membrane organelles such as endosomes. A series of Rab GTPases act consecutively and in concert with the maturation of cis- to-trans cisternae of the Golgi apparatus. Rab GTPases control various steps in intracellular membrane traffic by recruiting downstream effector proteins. Here, we report the dynamics of Ypt6, a yeast member of the Rab GTPase family, which mediates the fusion of vesicles from endosomes at the Golgi apparatus. Ypt6 resides temporarily at the Golgi and dissociates into the cytosol upon arrival of Ypt32, another Rab GTPase functioning in the late Golgi. We found that Gyp6, a putative GTPase-activating protein (GAP) for Ypt6, specifically interacts with Ypt32, most likely as an effector. Disruption of GYP6 or introduction of a Rab–GAP activity-deficient mutation in GYP6 resulted in continual residence of Ypt6 at the Golgi. We propose that Ypt32 acts to terminate endosome-to-Golgi traffic through a Rab–GAP cascade as it does for cis-to-trans intra-Golgi traffic. Simultaneous disruption of GAP for early-acting Rab proteins in the Golgi showed appreciable defects in post-Golgi trafficking, but did not significantly affect cell growth.

https://www.pnas.org/content/pnas/110/47/18976.full.pdf

Rab GAP cascade regulates dynamics of Ypt6 in the Golgi traffic

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are created within a single mother cell. During this process, the prospore membrane is generated de novo on the spindle pole body, elongates along the nuclear envelope and engulfs the nucleus. By screening previously identified sporulation-defective mutants, we identified additional genes required for prospore membrane formation. Deletion of either ERV14, which encodes a COPII cargo receptor, or the meiotically induced SMA2 gene resulted in misshapen prospore membranes. Sma2p is a predicted integral membrane that localized to the prospore membrane in wild-type cells but was retained in the ER in erv14 cells, suggesting that the prospore membrane morphology defect of erv14 cells is due to mislocalization of Sma2p. Overexpression of the ERV14 paralog ERV15 largely suppressed the sporulation defect in erv14 cells. Although deletion of ERV15 alone had no phenotype, erv14 erv15 double mutants displayed a complete block of prospore membrane formation. Plasma membrane proteins, including the t-SNARE Sso1p, accumulated in the ER upon transfer of the double mutant cells to sporulation medium. These results reveal a developmentally regulated change in the requirements for ER export in S. cerevisiae.

/pdf/erv14-family-cargo-receptors-are-necessary-for-er-exit-145z0rfi34.pdf

Erv14 family cargo receptors are necessary for ER exit during sporulation in Saccharomyces cerevisiae.

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.

Alternative Modes of Organellar Segregation during Sporulation in Saccharomyces cerevisiae

The oriented deposition of cellulose microfibrils in the plant cell wall plays a crucial role in various plant functions such as cell growth, organ formation and defense responses. Cellulose is synthesized by cellulose synthase complexes (CSCs) embedded in the plasma membrane (PM), which comprise the cellulose synthases (CESAs). The abundance and localization of CSCs at the PM should be strictly controlled for precise regulation of cellulose deposition, which strongly depends on the membrane trafficking system. However, the mechanism of the intracellular transport of CSCs is still poorly understood. In this study, we explored requirements for phosphoinositides (PIs) in CESA trafficking by analyzing the effects of inhibitors of PI synthesis in Arabidopsis thaliana expressing green fluorescent protein-tagged CESA3 (GFP-CESA3). We found that a shift to a sucrose-free condition accelerated re-localization of PM-localized GFP-CESA3 into the periphery of the Golgi apparatus via the clathrin-enriched trans-Golgi network (TGN). Treatment with wortmannin (Wm), an inhibitor of phosphatidylinositol 3- (PI3K) and 4- (PI4K) kinases, and phenylarsine oxide (PAO), a more specific inhibitor for PI4K, inhibited internalization of GFP-CESA3 from the PM. In contrast, treatment with LY294002, which impairs the PI3K activity, did not exert such an inhibitory effect on the sequestration of GFP-CESA3, but caused a predominant accumulation of GFP-CESA3 at the ring-shaped periphery of the Golgi apparatus, resulting in the removal of GFP-CESA3 from the PM. These results indicate that PIs are essential elements for localization and intracellular transport of CESA3 and that PI4K and PI3K are required for distinct steps in secretory and/or endocytic trafficking of CESA3.

Yasuyuki Suda

Papers

Dynamic Behavior of the trans-Golgi Network in Root Tissues of Arabidopsis Revealed by Super-Resolution Live Imaging

Rab GAP cascade regulates dynamics of Ypt6 in the Golgi traffic

Erv14 family cargo receptors are necessary for ER exit during sporulation in Saccharomyces cerevisiae.

Alternative Modes of Organellar Segregation during Sporulation in Saccharomyces cerevisiae

Phosphatidylinositol 3-Kinase and 4-Kinase Have Distinct Roles in Intracellular Trafficking of Cellulose Synthase Complexes in Arabidopsis thaliana