Abrogating Munc18-1-SNARE Complex Interaction Has Limited Impact on Exocytosis in PC12 Cells

作者：Malintan Nancy T; Nguyen Tam H; Han Liping; Latham Catherine F; O**orne Shona L; Wen Peter J; Lim Siew Joo Tiffany; Sugita Shuzo; Collins Brett M; Meunier Frederic A^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2009, 284(32): 21637-21646.

DOI：10.1074/jbc.M109.013508

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.

• 出版日期2009-8-7