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Cover image for Vol. 17 Issue 8

Edited By: Michael S. Marks, Trina A. Schroer, Tom H. Stevens and Sharon A. Tooze

Online ISSN: 1600-0854

Highlights

  • ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes

    ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes

    Method validation. A) The outline of the QF-FRL method. (1) QF: Live cells are quickly frozen without ice crystal formation. High-pressure freezing was used in this study. In this method, samples are frozen using liquid nitrogen, while the nucleation and growth of ice crystal formation were slowed down by brief application of a pressure of 2100 bar. (2) Freeze-fracture: Frozen cells are fractured below −100°C in a high vacuum. Membranes are split into two leaflets and the hydrophobic interface (i.e. the acyl chain side of the phospholipid monolayer) is exposed. (3) Vacuum evaporation: By evaporation, thin layers of carbon and platinum are deposited onto the hydrophobic interface of membranes and physically stabilize membrane molecules. Because platinum is evaporated from an oblique angle to the surface of the specimen (45° in the present experiment), protruding structures block the evaporation to make ‘shadows’ behind them. The area thus being deficient in the platinum deposition appears electron-lucent under EM. Transmembrane proteins are observed as small bumps called IMPs. (4) SDS treatment. Specimens are thawed and treated with an SDS solution to dissolve materials other than a lipid monolayer and integral membrane proteins, which are in direct contact with the carbon and platinum layer. This makes lipid head groups accessible to probes for labeling. B) Freeze-fracture replicas of liposomes were treated with 25 nmGST-ATG18-4×FLAG in the absence or presence of 7.5 µm p40phoxPX domain. The combination with p40phoxPX was used for subsequent experiments unless described otherwise. Colloidal gold particles used in this experiment were 5 nm in diameter, whereas 10-nm gold was used except for double labeling shown in Figure 4E. The liposomes were prepared using 70 mol% phosphatidylcholine, 15 mol% phosphatidylserine and 15 mol% phosphatidylinositol or a phosphoinositide. C) The labeling density in liposome replicas. GST-ATG18-4×FLAG alone bound to PtdIns(3)P and PtdIns(3,5)P2, but in the presence of p40phoxPX domain, the labeling was virtually limited to PtdIns(3,5)P2. GST-ATG18FTTG-4×FLAG did not bind to PtdIns(3,5)P2. Quantification of samples from one representative experiment is shown. N indicates the number of analyzed liposomes. Steel–Dwass nonparametric test. D) Diagram of the method to label PtdIns(3,5)P2. E) PtdIns(3,5)P2 labeling in atg18Δ treated with 0.9 mNaCl for 10 min and vac7Δ. Intense labeling was observed in the P face (cytoplasmic leaflet) of the vacuole in atg18Δ, but not in vac7Δ. F) The labeling density in atg18Δ and vac7Δ treated with or without 0.9 mNaCl for 10 min. Three independent experiments were performed, each analyzing 11–37 vacuoles. N indicates the total number of analyzed vacuoles. GST-ATG18FTTG-4×FLAG showed only negligible labeling even in atg18Δ treated with 0.9 mNaCl. Steel–Dwass test.

  • ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25

    ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25

    A schematic model of the protein–protein interactions leading to SNARE complex assembly. A) The molecular machinery controlling yeast SNARE complex assembly. Prior to SNARE complex formation Sec9p (N marking the N-terminus, 1 the first SNARE motif and 2 the second SNARE motif) can form a complex with Sro7p and Sec4p. Interaction between Sec9p and the Sec1p groove (yellow) mediates a shift of this complex toward the plasma membrane and facilitates SNARE complex formation. B) The molecular machinery controlling mammalian SNARE complex assembly. Interaction between SNAP-25 and the Munc18 groove (yellow) allows priming of SNAP-25, enabling interaction with Syntaxin1, followed by formation of ternary SNARE complexes mediating membrane fusion. Alternatively, Tomosyn can displace Munc18 from SNAP-25 and inhibit SNARE complex formation.

  • ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

    ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii

    TuneableαSNAPphosphomutant expression impedes theToxoplasmalytic cycle. A) Schematic of αSNAP overexpression constructs regulated by Shld-1 through the DD and tagged with myc epitope tag. i) DD-myc-αSNAPWT-wildtype and (ii) DD-myc-αSNAPS6A-phosphonull mutant. Bi) Induction of protein expression through titration of Shld-1 by western blot as detected by probing for αmyc (loading control MIC2). Bii) Quantitation of αmyc fluorescence intensity by IFA in response to Shld-1. Equivalent protein expression between DD-myc-αSNAPWT and DD-myc-αSNAPS6A is defined at 0.75 µm Shld-1. ±SD. C) Gross morphology of DD-myc-αSNAPWT and DD-myc-αSNAPS6A-expressing tachyzoites under increasing concentration of Shld-1. αmyc (green) and cell periphery marker αGAP45 (red). Scale bar = 10 µm. D) Plaque assays of DD-myc-αSNAPWT and DD-myc-αSNAPS6A-expressing parasites under increasing concentrations of Shld-1.

  • REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

    REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

    Trafficking pathways regulating synaptic growth signaling at theDrosophilaNMJ. A) Drosophila motor neuron showing long-range versus local targets of BMP signaling involved in regulating synaptic growth. Trio, Dad and Twit are direct transcriptional targets of the BMP cascade, while effects on dFMRP and Futsch expression may occur downstream of primary targets. B) Regulation of the BMP ligand Gbb (Glass Bottom Boat, yellow stars) by membrane traffic. Release of postsynaptically expressed Gbb into the synaptic cleft is positively regulated by the BAR domain protein dRich and negatively regulated by the F-BAR domain protein dCip4. This pool of Gbb binds to its receptors Tkv and Wit (pink) and Sax (not shown) on the motor neuron to control synaptic growth. In contrast, presynaptically expressed Gbb is packaged into DCVs by Cmpy, and released in an activity-dependent manner to control synaptic transmission but not synaptic growth. C) Regulation of presynaptic BMP signaling via local endosomal traffic of its receptors. Upon internalization, receptors bound to the ligand (Gbb) signal from the early endosomal compartment (EE). Receptors can be downregulated by recycling to the plasma membrane (blue arrows) via recycling endosomes (RE) or by endosomal degradation (red arrows) via late endosomes/MVB and acidified lysosomal compartments. Drosophila mutants with defects associated with receptor recycling (blue) or degradation (red) are listed. D) Traffic of presynaptic Wg (orange) into exosomes. Wg is released from neurons via exosomes along with its carrier protein Evi (purple) and binds its receptor dFz2 on the muscle to regulate synaptic growth.

  • ORIGINAL ARTICLE: Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes
  • ORIGINAL ARTICLE: The Sec1/Munc18 Protein Groove Plays a Conserved Role in Interaction with Sec9p/SNAP-25
  • ORIGINAL ARTICLE: Phosphorylation of αSNAP is Required for Secretory Organelle Biogenesis in Toxoplasma gondii
  • REVIEW: The Crossroads of Synaptic Growth Signaling, Membrane Traffic and Neurological Disease: Insights from Drosophila

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Recently Published Articles

  1. The HOPS/Class C Vps Complex Tethers High-Curvature Membranes via a Direct Protein–Membrane Interaction

    Ruoya Ho and Christopher Stroupe

    Version of Record online: 15 JUL 2016 | DOI: 10.1111/tra.12421

    Thumbnail image of graphical abstract

    Here, we show that the HOPS membrane tethering complex can tether highly curved membranes by binding to these membranes via the amphipathic lipid packing sensor (ALPS) motif in its Vps41p subunit. We propose that this protein–membrane interaction directs the localization of HOPS to the highly curved ‘vertex ring’ at the edge of the flattened zone of contact between tethered yeast vacuoles.

  2. Vesicles are persistent features of different plastids

    Emelie Lindquist, Katalin Solymosi and Henrik Aronsson

    Accepted manuscript online: 12 JUL 2016 09:40PM EST | DOI: 10.1111/tra.12427

    Thumbnail image of graphical abstract

    Vesicles in plastids have been observed repeatedly, suggested to function in thylakoid biogenesis. Previous observations have mainly concerned proplastids and chloroplasts, often being pre-treated to induce formation or inhibit fusion of vesicles. Here we present vesicle-like structures in etio-, etio-chloro-, leuco-, chromo- and desiccoplasts, in addition to both proplastids and chloroplasts. They are here shown without any pre-treatment of plants aiming to enhance vesicle appearance, and in different species (including both C3 and C4 plants), cell types and organs.

  3. Rab11 Regulates the Mast Cell Exocytic Response

    Joshua D. Wilson, Sarah A. Shelby, David Holowka and Barbara Baird

    Version of Record online: 11 JUL 2016 | DOI: 10.1111/tra.12418

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    Mast cell exocytosis causes allergy through the release of mediators from secretory lysosomes; costimulated exocytosis of recycling endosomes (REs) provides a source of additional membrane and may play other roles. Here, we show that a dominant negative form of the RE protein Rab11 (S25N) interferes with both of these exocytic processes. Inhibition mediated by S25N Rab11 can be bypassed using compounds that block actin polymerization. Furthermore, inhibition of stimulated exocytosis by the F-actin stabilizer, jasplakinolide, is not additive with S25N Rab11, implicating Rab11 in actin remodeling necessary for exocytosis.

  4. BMP2 Transfer to Neighboring Cells and Activation of Signaling

    Hamed Alborzinia, Marjan Shaikhkarami, Peter Hortschansky and Stefan Wölfl

    Version of Record online: 10 JUL 2016 | DOI: 10.1111/tra.12420

    Thumbnail image of graphical abstract

    BMP signaling plays a central role in development and organ homeostasis, and is tightly regulated at various levels. Internalization of BMP2 by cells can provide another layer of regulation. We show that internalized BMP2 is transferred to neighboring cells and can induce BMP signaling. Cell–cell contact increased transfer and enhanced signaling. Noggin not only blocked signaling but also enhanced transfer. Inhibition of vesicular transport blocked transfer without clear interference with signaling, suggesting that BMP2 signaling occurs independent of internalization and transfer.

  5. Identification of new fungal peroxisomal matrix proteins and revision of the PTS1 consensus

    Christopher Nötzel, Thomas Lingner, Heiner Klingenberg and Sven Thoms

    Accepted manuscript online: 8 JUL 2016 08:30AM EST | DOI: 10.1111/tra.12426

    Thumbnail image of graphical abstract

    The peroxisomal targeting signal type 1 (PTS1) is the prevalent peroxisomal targeting signal. Its current definition, however, is largely incomplete. We have modelled PTS1 by a machine learning approach in yeast. Based on this, we identified two conserved genes encoding novel peroxisomal proteins and we updated the consensus motif to now include all PTS1 proteins in yeast.

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