Sphingolipids containing very long-chain fatty acids regulate Ypt7 function during the tethering stage of vacuole fusion.

Sphingolipids are essential in membrane trafficking and cellular homeostasis. Here, we show that sphingolipids containing very long-chain fatty acids (VLCFAs) promote homotypic vacuolar fusion in Saccharomyces cerevisiae. The elongase Elo3 adds the last two carbons to VLCFAs that are incorporated into sphingolipids.

Lipid Rafts, Sphingolipids, and Ergosterol in Yeast Vacuole Fusion and Maturation.

The lysosome-like vacuole is a useful model for studying membrane fusion events and organelle maturation processes utilized by all eukaryotes. The vacuolar membrane is capable of forming micrometer and nanometer scale domains that can be visualized using microscopic techniques and segregate into regions with surprisingly distinct lipid and protein compositions. These lipid raft domains are liquid-ordered (L ) like regions that are rich in sphingolipids, phospholipids with saturated acyl chains, and ergosterol.

Phosphatidylinositol 3,5-bisphosphate regulates Ca transport during yeast vacuolar fusion through the Ca ATPase Pmc1.

The transport of Ca across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol-3-phosphate (PI3P) by the PI3P-5-kinase Fab1 to produce transient PI(3,5)P pools.

A small-molecule competitive inhibitor of phosphatidic acid binding by the AAA+ protein NSF/Sec18 blocks the SNARE-priming stage of vacuole fusion.

The homeostasis of most organelles requires membrane fusion mediated by oluble -ethylmaleimide-sensitive factor (NSF) ttachment protein ceptors (SNAREs). SNAREs undergo cycles of activation and deactivation as membranes move through the fusion cycle. At the top of the cycle, inactive -SNARE complexes on a single membrane are activated, or primed, by the hexameric ATPase associated with the diverse cellular activities (AAA+) protein, -ethylmaleimide-sensitive factor (NSF/Sec18), and its co-chaperone α-SNAP/Sec17.

Copper blocks V-ATPase activity and SNARE complex formation to inhibit yeast vacuole fusion.

The accumulation of copper in organisms can lead to altered functions of various pathways and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg , Cd and Cu are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations.

Phosphatidic acid induces conformational changes in Sec18 protomers that prevent SNARE priming.

Eukaryotic cell homeostasis requires transfer of cellular components among organelles and relies on membrane fusion catalyzed by SNARE proteins. Inactive SNARE bundles are reactivated by hexameric -ethylmaleimide-sensitive factor, vesicle-fusing ATPase (Sec18/NSF)-driven disassembly that enables a new round of membrane fusion. We previously found that phosphatidic acid (PA) binds Sec18 and thereby sequesters it from SNAREs and that PA dephosphorylation dissociates Sec18 from the membrane, allowing it to engage SNARE complexes.

Phosphatidylinositol 3,5-bisphosphate regulates the transition between trans-SNARE complex formation and vacuole membrane fusion.

Phosphoinositides (PIs) regulate a myriad of cellular functions including membrane fusion, as exemplified by the yeast vacuole, which uses various PIs at different stages of fusion. In light of this, the effect of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P) on vacuole fusion remains unknown. PI(3,5)P is made by the PI3P 5-kinase Fab1 and has been characterized as a regulator of vacuole fission during hyperosmotic shock, where it interacts with the TRP Ca channel Yvc1.

Deleting the DAG kinase Dgk1 augments yeast vacuole fusion through increased Ypt7 activity and altered membrane fluidity.

Diacylglycerol (DAG) is a fusogenic lipid that can be produced through phospholipase C activity on phosphatidylinositol 4,5-bisphosphate [PI(4,5)P ], or through phosphatidic acid (PA) phosphatase activity. The fusion of Saccharomyces cerevisiae vacuoles requires DAG, PA and PI(4,5)P , and the production of these lipids is thought to provide temporally specific stoichiometries that are critical for each stage of fusion. Furthermore, DAG and PA can be interconverted by the DAG kinase Dgk1 and the PA phosphatase Pah1.

The Central Polybasic Region of the Soluble SNARE (Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor) Vam7 Affects Binding to Phosphatidylinositol 3-Phosphate by the PX (Phox Homology) Domain.

The yeast vacuole requires four SNAREs to trigger membrane fusion including the soluble Qc-SNARE Vam7. The N-terminal PX domain of Vam7 binds to the lipid phosphatidylinositol 3-phosphate (PI3P) and the tethering complex HOPS (homotypic fusion and vacuole protein sorting complex), whereas the C-terminal SNARE motif forms SNARE complexes. Vam7 also contains an uncharacterized middle domain that is predicted to be a coiled-coil domain with multiple helices.

Phosphatidic Acid Sequesters Sec18p from cis-SNARE Complexes to Inhibit Priming.

Yeast vacuole fusion requires the activation of cis-SNARE complexes through priming carried out by Sec18p/N-ethylmaleimide sensitive factor and Sec17p/α-SNAP. The association of Sec18p with vacuolar cis-SNAREs is regulated in part by phosphatidic acid (PA) phosphatase production of diacylglycerol (DAG). Inhibition of PA phosphatase activity blocks the transfer of membrane-associated Sec18p to SNAREs.

Extracellular gluco-oligosaccharide degradation by Caulobacter crescentus.

The oligotrophic bacterium Caulobacter crescentus has the ability to metabolize various organic molecules, including plant structural carbohydrates, as a carbon source. The nature of β-glucosidase (BGL)-mediated gluco-oligosaccharide degradation and nutrient transport across the outer membrane in C. crescentus was investigated.