Membrane fusion mediated by peptidic SNARE protein analogues: Evaluation of FRET‐based bulk leaflet mixing assays

Protein synthesis begins at free ribosomes or ribosomes attached with the endoplasmic reticulum (ER). Newly synthesized proteins are transported to the plasma membrane for secretion through conventional or unconventional pathways. In conventional protein secretion, proteins are transported from the ER lumen to Golgi lumen and through various other compartments to be secreted at the plasma membrane, while unconventional protein secretion bypasses the Golgi apparatus. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins are involved in cargo vesicle trafficking and membrane fusion. The ER localized vesicle associated SNARE (v-SNARE) protein Sec22 plays a major role during anterograde and retrograde transport by promoting efficient membrane fusion and assisting in the assembly of higher order complexes by homodimer formation. Sec22 is not only confined to ER–Golgi intermediate compartments (ERGIC) but also facilitates formation of contact sites between ER and plasma membranes. Sec22 mutation is responsible for the development of atherosclerosis and symptoms in the brain in Alzheimer’s disease and aging in humans. In the fruit fly Drosophila melanogaster, Sec22 is essential for photoreceptor morphogenesis, the wingless signaling pathway, and normal ER, Golgi, and endosome morphology. In the plant Arabidopsis thaliana, it is involved in development, and in the nematode Caenorhabditis elegans, it is in involved in the RNA interference (RNAi) pathway. In filamentous fungi, it affects cell wall integrity, growth, reproduction, pathogenicity, regulation of reactive oxygen species (ROS), expression of extracellular enzymes, and transcriptional regulation of many development related genes. This review provides a detailed account of Sec22 function, summarizes its domain structure, discusses its genetic redundancy with Ykt6, discusses what is known about its localization to discrete membranes, its contributions in conventional and unconventional autophagy, and a variety of other roles across different cellular systems ranging from higher to lower eukaryotes, and highlights some of the surprises that have originated from research on Sec22.

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Differential Regulation of α-Granule and Dense Granule Secretion by an Actin Cytoskeletal Barrier.

Blood ◽

10.1182/blood.v104.11.3528.3528 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3528-3528

Author(s):

Robert Flaumenhaft ◽

James R. Dilks ◽

Nataliya Rozenvayn ◽

Rita A. Monahan-Earley ◽

Dian Feng ◽

...

Keyword(s):

Membrane Fusion ◽

Actin Polymerization ◽

Dense Granule ◽

Snare Protein ◽

Latrunculin A ◽

Cytochalasin E ◽

Dense Granule Secretion ◽

Granule Secretion ◽

Cytoskeletal Disruption ◽

Granule Release

Abstract Platelet granule secretion is an essential component of normal arterial thrombus formation. Stimulation of platelets with strong agonists results in centralization of cytoplasmic organelles and loss of granules. These observations have lead to the supposition that cytoskeletal contraction facilitates granule secretion. Yet, the influence of the actin cytoskeleton in controlling membrane fusion events required for granule secretion remains largely unknown. Initial studies using electron microscopy revealed that the actin disrupting agents latrunculin A (4 μM) or cytochalasin E (4 μM) prevented pseudopod formation and granule centralization in platelets exposed to SFLLRN or PMA, but did not prevent degranulation. We next determined the effects of disruption of the actin cytoskeleton on α-granule secretion by monitoring P-selectin expression and β-thromboglobulin release. Incubation of platelets with either latrunculin A or cytochalasin E failed to stimulate α-granule secretion, but increased the rate of SFLLRN-induced α-granule secretion by 3.5-fold. Cytoskeletal disruption also augmented the degree of SFLLRN-induced α-granule secretion by 41±18% and reduced the amount of SFLLRN required to cause half-maximal stimulation by 2-fold. Incubation with latrunculin A stimulated α-granule secretion by the weak secretogues epinephrine or ADP by 7.6-fold and 5.4-fold, respectively. Cytoskeletal disruption also facilitated β-thromboglobulin release in response to SFLLRN, epinephrine, or ADP. In platelets permeabilized in the absence of ATP, exposure to 2 μM latrunculin A resulted in a 6.5- and 3.5-fold increase in α-granule release induced by Ca2+- or GTP-γ-S, respectively. Antibodies directed at a SNARE protein termed vesicle-associated fusion protein (VAMP) inhibited latrunculin A-dependent α-granule secretion. Thus, disruption of the actin cytoskeletal barrier by latrunculin A supports SNARE protein-dependent membrane fusion. Since actin acts as a barrier to α-granule secretion, we evaluated α-granules purified by subcellular fractionation for the presence of F-actin. Purified α-granules, but not phospholipid micelles, bound the F-actin probe FITC-phalloidin as determined by flow cytometry. FITC-phalloidin binding was inhibited in a dose-dependent manner by latrunculin A. These data indicated that α-granules are coated with F-actin that could serve a barrier function. We next evaluated the effects of cytoskeletal disruption on dense granule secretion by monitoring ADP/ATP release using a luciferin-luciferase based assay and by quantifying [3H]serotonin release. Cytoskeletal disruption by 4 μM latrunculin A failed to affect the degree of dense granule secretion from platelets stimulated by either SFLLRN, epinephrine, or ADP. Yet, 200 μM latrunculin A stimulated substantial dense granule release in the absence of agonist exposure and augmented SFLLRN-induced dense granule release by 2-fold. In contrast, 200 μM latrunculin A abolished SFLLRN-induced α-granule secretion. These observations indicate that the cytoskeleton differentially regulates α-granule and dense granule secretion. Our results also suggest that while some degree of actin polymerization is required for α-granule secretion, dense granule secretion is not dependent on actin polymerization.

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Faculty Opinions recommendation of Resolving the function of distinct Munc18-1/SNARE protein interaction modes in a reconstituted membrane fusion assay.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.12242957.13414056 ◽

2011 ◽

Author(s):

David Tareste

Keyword(s):

Membrane Fusion ◽

Protein Interaction ◽

Snare Protein

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α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

Journal of Biological Chemistry ◽

10.1074/jbc.m114.556803 ◽

2014 ◽

Vol 289 (23) ◽

pp. 16326-16335 ◽

Cited By ~ 26

Author(s):

Yongsoo Park ◽

Wensi Vennekate ◽

Halenur Yavuz ◽

Julia Preobraschenski ◽

Javier M. Hernandez ◽

...

Keyword(s):

Membrane Fusion ◽

Snare Protein ◽

Protein Membrane

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SNARE protein analog-mediated membrane fusion

Journal of Peptide Science ◽

10.1002/psc.2773 ◽

2015 ◽

Vol 21 (8) ◽

pp. 621-629 ◽

Cited By ~ 15

Author(s):

Pawan Kumar ◽

Samit Guha ◽

Ulf Diederichsen

Keyword(s):

Membrane Fusion ◽

Snare Protein

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Mutations in Membrin/GOSR2 reveal stringent secretory pathway demands of dendritic growth and synaptic integrity

10.1101/142679 ◽

2017 ◽

Author(s):

Roman Praschberger ◽

Simon A. Lowe ◽

Nancy T. Malintan ◽

Henry Houlden ◽

Dimitri M. Kullmann ◽

...

Keyword(s):

Nervous System ◽

Membrane Fusion ◽

Dendritic Growth ◽

Secretory Pathway ◽

Synaptic Function ◽

Golgi Membrane ◽

Snare Protein ◽

Progressive Myoclonus Epilepsy ◽

Myoclonus Epilepsy ◽

Axonal Trafficking

AbstractMutations in the Golgi SNARE protein Membrin (encoded by the GOSR2 gene) cause progressive myoclonus epilepsy (PME). Membrin is a ubiquitously important protein mediating ER-to-Golgi membrane fusion, and hence it is unclear how these mutations result in a disorder restricted to the nervous system. Here we use a multi-layered strategy to elucidate the consequences of Membrin mutations from protein to neuron. We show that the pathogenic mutations cause partial reductions in SNARE-mediated membrane fusion. Importantly, these alterations were sufficient to profoundly impair dendritic growth in novel Drosophila models of GOSR2-PME. We also observed axonal trafficking abnormalities in this model, as well as synaptic malformations, trans-synaptic instability and hyperactive synaptic transmission. Our study highlights how dendritic growth is vulnerable even to subtle secretory pathway deficits, uncovers a previously uncharacterized role for Membrin in synaptic function, and provides a comprehensive explanatory basis for genotype-phenotype relationships in GOSR2-PME.

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Analysis of Sec22p in Endoplasmic Reticulum/Golgi Transport Reveals Cellular Redundancy in SNARE Protein Function

Molecular Biology of the Cell ◽

10.1091/mbc.e02-04-0204 ◽

2002 ◽

Vol 13 (9) ◽

pp. 3314-3324 ◽

Cited By ~ 84

Author(s):

Yiting Liu ◽

Charles Barlowe

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Fusion ◽

Protein Function ◽

Secretory Pathway ◽

Snare Proteins ◽

Snare Protein ◽

Intracellular Membrane ◽

Protein Receptor ◽

Intracellular Membrane Fusion

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. Insec22Δ strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of thesec22Δ mutation with a conditionalykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization.

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