Identification of Triton X-100 Insoluble Membrane Domains in the Yeast Saccharomyces cerevisiae

Kübler, Eric; Dohlman, Henrik G.; Lisanti, Michael P.

doi:10.1074/jbc.271.51.32975

Cited by 94 publications

(72 citation statements)

References 35 publications

(43 reference statements)

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“…DIGs isolated by standard protocol [18] were collected, fixed, and embedded and stained thin-sections were examined by transmission electron microscopy. The electron micrographs of the wild-type DIGs showed membrane structures similar in appearance to those previously reported by Kubler et al [19]. These were closed vesicular structures with variable diameters ranging between 100 and 500 nm (Fig.…”

Section: Digs Morphologysupporting

confidence: 63%

“…Samples were prepared and analyzed as previously described [19] at the Advanced Microscopy and Imaging Laboratory of Auburn University (Auburn, Alabama).…”

Section: Electron Microscopy Of Digsmentioning

confidence: 99%

“…This property allows the extraction of detergent-insoluble glycolipid-enriched membranes (DIGs) that are considered to be the structural equivalent of functional lipid rafts [17]. DIGs have been isolated from many mammalian membranes and also from yeast [18,19]. Studies with lipid biosynthetic mutants of yeast showed that both sphingolipids and sterols are important for raft association [18,20,21].…”

Section: Introductionmentioning

confidence: 99%

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Sphingolipid C4 hydroxylation influences properties of yeast detergent‐insoluble glycolipid‐enriched membranes

2004

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Sphingoid base C4 hydroxylation is required for syringomycin E action on the yeast plasma membrane. Detergent-insoluble glycolipid-enriched membranes (DIGs) from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2D) are composed of linear membrane fragments instead of vesicular structures observed for wild-type DIGs, though they have similar lipid compositions and amounts of DIG marker proteins. Lightscattering bands collected from sur2D after centrifugation of Triton X-100-treated cell lysates in continuous density gradients have lower buoyant densities than that of the wild-type. The results show that C4 hydroxylation influences the physical and structural properties of DIGs and suggest that syringomycin E interacts with lipid rafts.

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Section: Digs Morphologysupporting

confidence: 63%

“…Samples were prepared and analyzed as previously described [19] at the Advanced Microscopy and Imaging Laboratory of Auburn University (Auburn, Alabama).…”

Section: Electron Microscopy Of Digsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Sphingolipid C4 hydroxylation influences properties of yeast detergent‐insoluble glycolipid‐enriched membranes

2004

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“…Lipid rafts in S. cerevisiae contain ergosterol and complex sphingolipids (9,10), whereas the rafts in higher eucaryotes contain cholesterol and sphingomyelin or glycosphingolipids (1). The complex sphingolipids in S. cerevisiae are inositolphosphoceramide (IPC), 1 mannose-inositol-phosphoceramide (MIPC), and mannose-(inositol phosphate) 2 -ceramide (M(IP) 2 C (11).…”

mentioning

confidence: 99%

The Uracil Transporter Fur4p Associates with Lipid Rafts

Hearn

Lester

Dickson

2003

Journal of Biological Chemistry

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Sphingolipids are abundant components of eucaryotic membranes, where they perform essential functions. To uncover new roles for sphingolipids, we studied Saccharomyces cerevisiae lcb1-100 cells, which have a temperature-sensitive block in the first step in sphingolipid synthesis. We find that the level of all five species of the sphingoid long chain base intermediates is reduced 2-7-fold in cells grown at a permissive temperature, and the level of complex sphingolipids is reduced 50%. In addition, lcb1-100 cells make no detectable phosphorylated sphingoid bases. After transfer to a restrictive temperature (a heat shock), the level of the major sphingoid bases drops rather than transiently rising, as in wild type cells. These changes affect lcb1-100 cells in multiple ways. Basal uracil transport by Fur4p is reduced 25%, and when cells are heat-shocked, uracil transport activity falls rapidly and is not restored as it is in wild type cells. Restoration requires a functional secretory pathway and synthesis of complex sphingolipids, leading us to hypothesize that Fur4p associates with lipid rafts. The finding that Fur4p is insoluble in TritonX-100 at 4°C and behaves like a raft-associated protein on a density gradient supports this hypothesis. Raft association may be essential for regulating breakdown of Fur4p in response to stresses and other factors that govern uracil transport activity. Our results show that long chain bases do not contribute to the inactivation of Fur4p transport activity after heat stress, but they are essential for some later, but unknown, process that leads to degradation of the protein. Further studies using lcb1-100 cells should reveal new roles of sphingolipids in nutrient uptake and other membrane-dependent processes.Sphingolipids are essential components of eucaryotic cells and are implicated in a growing number of cellular functions including signal transduction and association with sterols in membranes to form lipid rafts. To uncover new roles for sphingolipids we have studied Saccharomyces cerevisiae cells carrying a mutation that inhibits sphingolipid synthesis at a restrictive temperature. These studies demonstrate that delivery of the uracil transporter Fur4p to the plasma membrane requires synthesis of complex sphingolipids, which most likely reflects the need for de novo formation of lipid rafts, since we find that Fur4p is present in rafts.

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“…The pelleted membranes were treated with Triton X-100, which solubilized (S TX ) the TMD-containing syntaxins; but, syntaxin-11 and SNAP-23 were found in both Triton X-100 soluble and insoluble fractions (I TX ). The Triton X-100 insoluble fraction most likely contains actin cytoskeleton and lipid rafts (31,32). Since SNAP-23 has been found in detergent-resistant, lipid rafts in platelets (33), we fractionated the I TX material on a sucrose density gradient.…”

Section: Resultsmentioning

confidence: 99%

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

Zhang

Huang²,

Chen

et al. 2018

Journal of Biological Chemistry

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ABSTRACT:Platelets regulate vascular integrity by secreting a host of molecules that promote hemostasis and its sequelae. Given the importance of platelet exocytosis, it is critical to understand how it is controlled. The t-SNAREs, SNAP-23 and syntaxin-11, lack classical transmembrane domains (TMDs), yet both are associated with platelet membranes and redistributed into cholesterol-dependent, lipid rafts when platelets were activated. Using metabolic labeling and hydroxylamine/HCl (HA) treatment, we showed that both contain thioester-linked acyl groups. Mass spectrometry mapping further showed that syntaxin-11 was modified on Cysteine 275, 279, 280, 282, 283 and 285, was modified on Cysteine 79, 80, 83, 85, and 87. Interestingly, metabolic labeling studies showed incorporation of [ 3 H]-palmitate into the t-SNAREs increased though the protein levels were unchanged, suggesting that acylation turns over on the two t-SNAREs in resting platelets. Exogenously added fatty acids did compete with [ 3 H]-palmitate for t-SNARE labeling. To determine the effects of acylation, we measured aggregation, ADP/ATP release as well as P-selectin exposure in platelets treated with the acyl-transferase inhibitor, cerulenin, or the thioesterase inhibitor, palmostatin B. We found that cerulenin pretreatment inhibited t-SNARE acylation and platelet function in a dose-and time-dependent manner while palmostatin B had no detectable effect. Interestingly, pretreatment with palmostatin B blocked the inhibitory effects of cerulenin suggesting that maintaining the acylation state is important for platelet function. Thus, our work shows that t-SNARE acylation is actively cycling in platelets and suggests that the enzymes regulating protein acylation could be potential targets to control platelet exocytosis in vivo.Platelet exocytosis is critical for hemostasis; however, it is increasingly obvious that platelets secrete components with (8)(9)(10)(11)(12)(13)(14). Further studies defined specific SNARE regulators, which control when and where the v-and t-SNAREs interact. Aside from the role that I κ B Kinase-mediated phosphorylation plays in controlling SNAP-23/syntaxin-11 association (15), little is known about other post-translational modifications and their effects on the platelet exocytosis.Despite their importance to secretion, neither SNAP-23 nor syntaxin-11 contains a classical transmembrane domain (TMD), nor any identifiable CAAX motifs. Instead, both proteins contain cysteine-rich regions: human syntaxin-11 has 8 cysteines with 6 at its C-terminus, and SNAP-23 contains 6 cysteines with 5 in a central domain. These cysteine-rich regions have been shown, in some cells, to be important for membrane association and are potential sites for S-acylation (16)(17)(18)(19)(20) (27,28).Past studies have shown that acylation occurs in platelets (29). Dowal et al. characterized over 200 proteins in the platelet "palmitoylome", many of which appear to be involved in platelet signaling pathways (30). Consistently, Sim et al. showed that PAT-inhibit...

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Identification of Triton X-100 Insoluble Membrane Domains in the Yeast Saccharomyces cerevisiae

Cited by 94 publications

References 35 publications

Sphingolipid C4 hydroxylation influences properties of yeast detergent‐insoluble glycolipid‐enriched membranes

Sphingolipid C4 hydroxylation influences properties of yeast detergent‐insoluble glycolipid‐enriched membranes

The Uracil Transporter Fur4p Associates with Lipid Rafts

Dynamic cycling of t-SNARE acylation regulates platelet exocytosis

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