Abrogating Munc18-1-SNARE complex interaction has limited impact on exocytosis in PC12 cells.
Neuronal communication relies on the fusion of neurotransmitter-containing vesicles with the plasma membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins initiate membrane fusion through the formation of the SNARE complex, a process tightly regulated by Sec1/Munc18-1 (SM) proteins. The emerging trend is that SM proteins promote SNARE-mediated membrane fusion by binding to a Syntaxin N-terminal motif. Here we report that mutations in the hydrophobic pocket of Munc18-1 (F115E and E132A), predicted to disrupt the N-terminal Sx1a interaction have a modest effect on binding to Sx1a in its free state, but abolish binding to the SNARE complex. Overexpression of the Munc18-1 mutant in PC12 cells lacking Munc18-1 rescues both neuroexocytosis and the plasma membrane localization of Syntaxin. However, total internal reflection fluorescence microscopy analysis reveals that expression of a Munc18-1 double mutant reduces the rate of vesicle fusion, an effect only detectable at the onset of stimulation. The Munc18-1 hydrophobic pocket is therefore critical for SNARE complex binding. However, mutations abrogating this interaction have a limited impact on Ca(2+)-dependent exocytosis in PC12 cells.
Malintan
Nancy T
NT
Queensland Brain Institute and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland 4072, Australia.
Nguyen
Tam H
TH
Han
Liping
L
Latham
Catherine F
CF
Osborne
Shona L
SL
Wen
Peter J
PJ
Lim
Siew Joo Tiffany
SJ
Sugita
Shuzo
S
Collins
Brett M
BM
Meunier
Frederic A
FA
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis; D008954:Models, Biological; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D051938:Munc18 Proteins / Q000502:physiology*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D050600:SNARE Proteins / Q000378:metabolism*
D051938:Munc18 Proteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; D050600:SNARE Proteins; C497939:Stxbp1 protein, rat; D002118:Calcium