Mutants related to vacuolar function, biogenesis and inheritance.

<p>Mutants related to vacuolar function, biogenesis and inheritance.</p

Adaptin- and cytoskeleton-related mutants.

<p>Adaptin- and cytoskeleton-related mutants.</p

Identified mutants with strong defects.

<p>Identified mutants with strong defects.</p

Phenotype of mutants in the PI(3,5)P₂ pathway under screening conditions.

<p>(A) BY4741 <i>Δfab1</i> and BY4741 <i>Δfig4</i> cells were cultivated and subjected to vacuole fragmentation under screening conditions in diluted YPD. Pictures show an overlay of the fluorescence and brightfield channels. (B) Metabolic pathways leading to the synthesis of PI(3,5)P₂. Steps for which a gene deletion led to strong or moderate deficiency in vacuole fragmentation are indicated in red/bold.</p

Identified mutants affecting processes known to be involved in vacuole fragmentation: PI(3,5)P₂ metabolism, vacuole acidification and TOR signaling.

<p>Identified mutants affecting processes known to be involved in vacuole fragmentation: PI(3,5)P₂ metabolism, vacuole acidification and TOR signaling.</p

Identified mutants in triglyceride metabolism.

<p>Identified mutants in triglyceride metabolism.</p

The in vivo fragmentation assay under screening conditions.

<p>(A) The cells were grown overnight in 96-well plates in HC-Leu^- medium to an OD₆₀₀<2. They were diluted 10-fold in YPD, stained with 20 µM FM4-64 for 1 hour, centrifuged and resuspended in YPD, HC or YPD diluted 5-fold with water (diluted YPD). After shaking for 2 hrs at 27°C, cells were transferred into optical 96-well plates. Fragmentation was induced by supplementing the suspension with 0.4 M NaCl. After 10 min of incubation at room temperature, cells were analyzed by fluorescence microscopy. Note that the screen was performed on a non-confocal microscope. Fragmentation was easier to judge on the microscope than on the photos, due to the possibility to focus through the sample in the z-direction. (B) Examples illustrating the scoring of the fragmentation defect. Samples sho pictures from cells after incubation with salt as in A.</p

Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81

Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses

Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1

The size and copy number of an organelle depend on an equilibrium of membrane fusion and fission. In vitro reconstitution of yeast vacuole fission and fusion shows that TORC1 selectively stimulates fission but does not change fusion activity. This explains the morphological transitions of yeast vacuoles in response to nutrient availability