Alternative splicing of SNAP-25 regulates secretion through nonconservative substitutions in the SNARE domain.
The essential membrane fusion apparatus in mammalian cells, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consists of four alpha-helices formed by three proteins: SNAP-25, syntaxin 1, and synaptobrevin 2. SNAP-25 contributes two helices to the complex and is targeted to the plasma membrane by palmitoylation of four cysteines in the linker region. It is alternatively spliced into two forms, SNAP-25a and SNAP-25b, differing by nine amino acids substitutions. When expressed in chromaffin cells from SNAP-25 null mice, the isoforms support different levels of secretion. Here, we investigated the basis of that different secretory phenotype. We found that two nonconservative substitutions in the N-terminal SNARE domain and not the different localization of one palmitoylated cysteine cause the functional difference between the isoforms. Biochemical and molecular dynamic simulation experiments revealed that the two substitutions do not regulate secretion by affecting the property of SNARE complex itself, but rather make the SNAP-25b-containing SNARE complex more available for the interaction with accessory factor(s).
Nagy
Gábor
G
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Milosevic
Ira
I
Fasshauer
Dirk
D
Müller
E Matthias
EM
de Groot
Bert L
BL
Lang
Thorsten
T
Wilson
Michael C
MC
Sørensen
Jakob B
JB
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019439:Chromaffin Cells / Q000502:physiology; D002942:Circular Dichroism; D003198:Computer Simulation; D005091:Exons / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D050600:SNARE Proteins / Q000737:chemistry / Q000235:genetics*; D050825:Synaptosomal-Associated Protein 25 / Q000737:chemistry / Q000172:deficiency / Q000235:genetics*
D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25