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Sequential N‐ to C‐terminal SNARE complex assembly drives priming and fusion of secretory vesicles
During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding...
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| Published in: | The EMBO journal 2006-03, Vol.25 (5), p.955-966 |
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| Main Authors: | , , , , , , , |
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| container_end_page | 966 |
| container_issue | 5 |
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| container_title | The EMBO journal |
| container_volume | 25 |
| creator | Sørensen, Jakob B Wiederhold, Katrin Müller, Emil M Milosevic, Ira Nagy, Gábor de Groot, Bert L Grubmüller, Helmut Fasshauer, Dirk |
| description | During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C‐terminal end affected fusion triggering
in vivo
and led to two‐step unfolding of the SNARE complex
in vitro
, indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state. |
| doi_str_mv | 10.1038/sj.emboj.7601003 |
| format | article |
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in vivo
and led to two‐step unfolding of the SNARE complex
in vitro
, indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.</description><identifier>ISSN: 0261-4189</identifier><identifier>EISSN: 1460-2075</identifier><identifier>DOI: 10.1038/sj.emboj.7601003</identifier><identifier>PMID: 16498411</identifier><identifier>CODEN: EMJODG</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Binding Sites ; capacitance measurements ; Cell Membrane - metabolism ; chromaffin cells ; Chromaffin Cells - cytology ; Chromaffin Cells - metabolism ; Circular Dichroism ; Electrophysiology ; EMBO20 ; Exocytosis ; Fusion ; Membrane Fusion ; Mice ; Mice, Knockout ; Molecular Sequence Data ; Mutation ; Proteins ; Qa-SNARE Proteins - metabolism ; R-SNARE Proteins - metabolism ; Secretory Vesicles - chemistry ; Secretory Vesicles - metabolism ; Sequence Homology, Amino Acid ; SNAP‐25 ; SNARE proteins ; Synaptosomal-Associated Protein 25 - metabolism ; Upstream</subject><ispartof>The EMBO journal, 2006-03, Vol.25 (5), p.955-966</ispartof><rights>European Molecular Biology Organization 2006</rights><rights>Copyright © 2006 European Molecular Biology Organization</rights><rights>Copyright Nature Publishing Group Mar 8, 2006</rights><rights>Copyright © 2006, European Molecular Biology Organization 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1409717/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1409717/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,725,778,782,883,27911,27912,53778,53780</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16498411$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sørensen, Jakob B</creatorcontrib><creatorcontrib>Wiederhold, Katrin</creatorcontrib><creatorcontrib>Müller, Emil M</creatorcontrib><creatorcontrib>Milosevic, Ira</creatorcontrib><creatorcontrib>Nagy, Gábor</creatorcontrib><creatorcontrib>de Groot, Bert L</creatorcontrib><creatorcontrib>Grubmüller, Helmut</creatorcontrib><creatorcontrib>Fasshauer, Dirk</creatorcontrib><title>Sequential N‐ to C‐terminal SNARE complex assembly drives priming and fusion of secretory vesicles</title><title>The EMBO journal</title><addtitle>EMBO J</addtitle><addtitle>EMBO J</addtitle><description>During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C‐terminal end affected fusion triggering
in vivo
and led to two‐step unfolding of the SNARE complex
in vitro
, indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.</description><subject>Amino Acid Sequence</subject><subject>Amino Acid Substitution</subject><subject>Animals</subject><subject>Binding Sites</subject><subject>capacitance measurements</subject><subject>Cell Membrane - metabolism</subject><subject>chromaffin cells</subject><subject>Chromaffin Cells - cytology</subject><subject>Chromaffin Cells - metabolism</subject><subject>Circular Dichroism</subject><subject>Electrophysiology</subject><subject>EMBO20</subject><subject>Exocytosis</subject><subject>Fusion</subject><subject>Membrane Fusion</subject><subject>Mice</subject><subject>Mice, Knockout</subject><subject>Molecular Sequence Data</subject><subject>Mutation</subject><subject>Proteins</subject><subject>Qa-SNARE Proteins - metabolism</subject><subject>R-SNARE Proteins - metabolism</subject><subject>Secretory Vesicles - chemistry</subject><subject>Secretory Vesicles - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>The EMBO journal</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sørensen, Jakob B</au><au>Wiederhold, Katrin</au><au>Müller, Emil M</au><au>Milosevic, Ira</au><au>Nagy, Gábor</au><au>de Groot, Bert L</au><au>Grubmüller, Helmut</au><au>Fasshauer, Dirk</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Sequential N‐ to C‐terminal SNARE complex assembly drives priming and fusion of secretory vesicles</atitle><jtitle>The EMBO journal</jtitle><stitle>EMBO J</stitle><addtitle>EMBO J</addtitle><date>2006-03-08</date><risdate>2006</risdate><volume>25</volume><issue>5</issue><spage>955</spage><epage>966</epage><pages>955-966</pages><issn>0261-4189</issn><eissn>1460-2075</eissn><coden>EMJODG</coden><abstract>During exocytosis a four‐helical coiled coil is formed between the three SNARE proteins syntaxin, synaptobrevin and SNAP‐25, bridging vesicle and plasma membrane. We have investigated the assembly pathway of this complex by interfering with the stability of the hydrophobic interaction layers holding the complex together. Mutations in the C‐terminal end affected fusion triggering
in vivo
and led to two‐step unfolding of the SNARE complex
in vitro
, indicating that the C‐terminal end can assemble/disassemble independently. Free energy perturbation calculations showed that assembly of the C‐terminal end could liberate substantial amounts of energy that may drive fusion. In contrast, similar N‐terminal mutations were without effects on exocytosis, and mutations in the middle of the complex selectively interfered with upstream maturation steps (vesicle priming), but not with fusion triggering. We conclude that the SNARE complex forms in the N‐ to C‐terminal direction, and that a partly assembled intermediate corresponds to the primed vesicle state.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>16498411</pmid><doi>10.1038/sj.emboj.7601003</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Amino Acid Sequence Amino Acid Substitution Animals Binding Sites capacitance measurements Cell Membrane - metabolism chromaffin cells Chromaffin Cells - cytology Chromaffin Cells - metabolism Circular Dichroism Electrophysiology EMBO20 Exocytosis Fusion Membrane Fusion Mice Mice, Knockout Molecular Sequence Data Mutation Proteins Qa-SNARE Proteins - metabolism R-SNARE Proteins - metabolism Secretory Vesicles - chemistry Secretory Vesicles - metabolism Sequence Homology, Amino Acid SNAP‐25 SNARE proteins Synaptosomal-Associated Protein 25 - metabolism Upstream |
| title | Sequential N‐ to C‐terminal SNARE complex assembly drives priming and fusion of secretory vesicles |
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