Negative regulation of syntaxin4/SNAP-23/VAMP2-mediated membrane fusion by Munc18c in vitro.
BACKGROUND
Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.


RESULTS
Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.


CONCLUSIONS
Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.
Brandie
Fiona M
FM
Henry Wellcome Laboratory of Cell Biology, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK.
Aran
Veronica
V
Verma
Avani
A
McNew
James A
JA
Bryant
Nia J
NJ
Gould
Gwyn W
GW
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D008561:Membrane Fusion / Q000502:physiology*; D051938:Munc18 Proteins / Q000378:metabolism*; D050765:Qa-SNARE Proteins / Q000378:metabolism*; D050767:Qc-SNARE Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism*
D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D050767:Qc-SNARE Proteins; D050988:Vesicle-Associated Membrane Protein 2