The SEC14 gene encodes an essential phosphatidylinositol (PtdIns) transfer protein required for formation of Golgi-derived secretory vesicles in yeast. Suppressor mutations that rescue temperature-sensitive sec14 mutants provide an approach for determining the role of Sec14p in secretion. One suppressor, sac1-22, causes accumulation of PtdIns(4)P. SAC1 encodes a phosphatase that can hydrolyze PtdIns(4)P and certain other phosphoinositides. These findings suggest that PtdIns(4)P is limiting in sec14 cells and that elevation of PtdIns(4)P production can suppress the secretory defect. Correspondingly, we found that PtdIns(4)P levels were decreased significantly in sec14-3 mutants shifted to 37°C and that sec14-3 cells could grow at an otherwise nonpermissive temperature (34°C) when carrying a plasmid overexpressing PIK1, encoding one of two essential PtdIns 4-kinases. This effect is specific because overexpression of the other PtdIns 4-kinase gene (STT4) or a PtdIns 3-kinase gene (VPS34) did not rescue sec14-3 cells. To further address Pik1p function in secretion, two different pik1 ts mutants were examined. Upon shift to restrictive temperature (37°C), the PtdIns(4)P levels dropped by about 60% in both pik1 ts strains within 1 h. During the same period, cells displayed a reduction (40 -50%) in release of a secreted enzyme (invertase). However, similar treatment did not effect maturation of a vacuolar enzyme (carboxypeptidase Y). These findings indicate that, first, PtdIns(4)P limitation is a major contributing factor to the secretory defect in sec14 cells; second, Sec14p function is coupled to the action of Pik1p, and; third, PtdIns(4)P has an important role in the Golgi-toplasma membrane stage of secretion.In eukaryotic cells, secreted proteins are synthesized on ribosomes targeted to the endoplasmic reticulum (ER), 1 translocated into the ER lumen, and transported through the secretory pathway (1). From the ER, secretory proteins are transported to the Golgi apparatus, through the subcompartments of the Golgi, and then to the cell surface or to certain intracellular organelles, all via small membrane-bound vesicles (transport vesicles) (2-4). Cargo proteins are packaged into transport vesicles that bud from one compartment and fuse with another. Mechanisms of vesicle budding and fusion are conserved from yeast to mammalian cells (3,5). Because of its tractability for genetic analysis, bakers' yeast (Saccharomyces cerevisiae) has proven to be a useful organism to identify gene products required for various events in secretion. Genetic screens, first applied by Schekman and co-workers (6), resulted in the isolation of temperature-sensitive sec mutants that displayed defects in different stages of secretion at the nonpermissive temperature. Characterization of the corresponding normal (SEC) genes has pinpointed many proteins necessary for secretory processes; and, a large number of gene products are now known to function at various steps in the secretory pathway (reviewed in Ref. 7). The SEC14 gene encodes a phosph...

The Saccharomyces cerevisiae gene SYR2, necessary for growth inhibition by the cyclic lipodepsipeptide syringomycin E, is shown to be required for 4-hydroxylation of long chain bases in sphingolipid biosynthesis. Four lines of support for this conclusion are presented: (a) the predicted Syr2p shows sequence similarity to diiron-binding membrane enzymes involved in oxygendependent modifications of hydrocarbon substrates, (b) yeast strains carrying a disrupted SYR2 allele produced sphingoid long chain bases lacking the 4-hydroxyl group present in wild type strains, (c) 4-hydroxylase activity was increased in microsomes prepared from a SYR2 overexpression strain, and (d) the syringomycin E resistance phenotype of a syr2 mutant strain was suppressed when grown under conditions in which exogenous 4-hydroxysphingoid long chain bases were incorporated into sphingolipids. The syr2 strain produced wild type levels of sphingolipids, substantial levels of hydroxylated very long chain fatty acids, and the full complement of normal yeast sphingolipid head groups. These results show that the SYR2 gene is required for the 4-hydroxylation reaction of sphingolipid long chain bases, that this hydroxylation is not essential for growth, and that the 4-hydroxyl group of sphingolipids is necessary for syringomycin E action on yeast.Syringomycin E is a member of a family of cyclic lipodepsipeptides produced by strains of the plant bacterium Pseudomonas syringae pv. syringae (1). Traditionally regarded as a virulence factor in a variety of bacterial necrotic diseases of plants (2), syringomycin E and its analogs also possess antifungal properties, and it has been suggested that these metabolites are fungal antagonists that aid survival of the producing bacteria on plants (3, 4).How these compounds produce their toxic effects is unknown, but past physiological studies have shown that syringomycin E targets primarily the plasma membrane (1, 5, 6). To further investigate the molecular mechanisms of action of this bioactive compound, resistant mutants of Saccharomyces cerevisiae were isolated to identify genes that encode proteins necessary for growth inhibition by syringomycin E (7). Several of the mutants were deficient in sterols, and one group was complemented by the gene SYR1 (identical to ERG3), which encodes sterol C-5,6 desaturase of the ergosterol biosynthetic pathway (8). These findings, when combined with results from binding (9) and lipid bilayer (10) studies, indicate that sterols influence the interaction of syringomycin E with the target plasma membrane.Syringomycin E action in yeast was more recently shown to require a second, nonsterol biosynthetic gene, SYR2 (11). SYR2 is identical to SUR2, which was identified in a screen for mutants that suppress the impaired recovery of rvs161 strains from nutritional starvation (12). Syringomycin E-resistant syr2 mutants showed altered glycerophospholipid levels, and the SYR2 gene product was localized to the endoplasmic reticulum (11). Nevertheless, the precise function of Syr2p was ...

By a combination of 1D and 2D 1H-and ~3C-NMR, FAB-MS, and chemical and enzymatic reactions carried out at the milligram level, it has been demonstrated that syringomycin E, the major phytotoxic antibiotic produced by Pseudomonas syringae pv. syringae, is a new lipodepsipcptide. Its amino acid sequence is Ser-Ser-Dab-Dab-Arg-Phe-Dhb-4(Cl)Thr-3(OH)Asp with the fl-carboxy group of the C-terminal residue closing a macrocyclic ring on the OH group of the N-terminal Scr, which in turn is N-acylated by 3-hydroxydodecanoic acid. Syringomycins A~ and G, two other metabolites of the same bacterium, differ from syringomycin E only in their fatty acid moieties corresponding, respectively, to 3-hydroxydecanoic and 3-hydroxytetradecanoic acid.

Highly reproducible ion channels of the lipopeptide antibiotic syringomycin E demonstrate unprecedented involvement of the host bilayer lipids. We find that in addition to a pronounced influence of lipid species on the open-channel ionic conductance, the membrane lipids play a crucial role in channel gating. The effective gating charge, which characterizes sensitivity of the conformational equilibrium of the syringomycin E channels to the transmembrane voltage, is modified by the lipid charge and lipid dipolar moment. We show that the type of host lipid determines not only the absolute value but also the sign of the gating charge. With negatively charged bilayers, the gating charge sign inverts with increased salt concentration or decreased pH. We also demonstrate that the replacement of lamellar lipid by nonlamellar with the negative spontaneous curvature inhibits channel formation. These observations suggest that the asymmetric channel directly incorporates lipids. The charges and dipoles resulting from the structural inclusion of lipids are important determinants of the overall energetics that underlies channel gating. We conclude that the syringomycin E channel may serve as a biophysical model to link studies of ion channels with those of lipidic pores in membrane fusion.

DmAMP1, an antifungal plant defensin from Dahlia merckii, was shown previously to require the presence of sphingolipids for fungicidal action against Saccharomyces cerevisiae. Sphingolipids may stabilize glycosylphosphatidylinositol (GPI)-anchored proteins, which interact with DmAMP1, or they may directly serve as DmAMP1 binding sites. In the present study, we demonstrate that S. cerevisiae disruptants in GPI-anchored proteins showed small or no increased resistance towards DmAMP1 indicating no involvement of these proteins in DmAMP1 action. Further, studies using an enzyme-linked immunosorbent assay (ELISA)-based binding assay revealed that DmAMP1 interacts directly with sphingolipids isolated from S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol. Therefore, DmAMP1 antifungal action involving membrane interaction with sphingolipids and ergosterol is proposed.