Passage
Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1) .
Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer
An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF) , which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2.
Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin
Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5) :2213-7.
-Wild-type syntaxin (Synt-WT) and mutant syntaxins containing a deletion of the N-terminal sequence (SyntΔA) or C-terminal sequence (SyntΔB) were separated and incubated with α-SNAP. After washing away unbound molecules, which of the following is the most likely result of protein immunoblotting for α-SNAP?

Question

Passage
Presynaptic nerve terminals release neurotransmitters via synaptic vesicle exocytosis. This action is mediated by the membrane-bound target-SNARE (t-SNARE) proteins syntaxin and SNAP-25, and the vesicle-associated SNARE (v-SNARE) protein synaptobrevin. Membrane fusion is initiated when the t-SNAREs and v-SNAREs form an α-helix bundle known as the core-trans-complex, achieved by zipping SNARE proteins together to form a more stable cis-complex (Figure 1) .

Figure 1 Formation of the core-trans-complex and subsequent membrane fusionThe cis-complex contains the zero ionic layer, the main site of interaction between complexed proteins. Within the zero ionic layer, arginine on synaptobrevin coordinates with carbonyl groups present on the other residues shown in Table 1. The zero ionic layer is buried within leucine zipper domains, which act as a shield to solvent molecules.Table 1 Composition of cis-Complex Zero Ionic Layer

An early step in cis-complex disassembly is destabilization by the ATPase N-ethylmaleimide-sensitive factor (NSF) , which initiates unzipping by breaking flanking leucine zipper regions, leading to vesicle reuptake. Movement of NSF to the cell membrane is mediated by the cytoplasmic protein α-SNAP, which must first bind the N-terminal domain of syntaxin.To determine the effect of vesicle-bound synaptobrevin on the binding of α-SNAP to syntaxin, increasing amounts of recombinant His-tagged α-SNAP were added to a constant amount of syntaxin affixed to glutathione-agarose beads with or without synaptobrevin. After incubation, bound proteins were recovered and quantitatively analyzed by immunoblotting. Results are shown in Figure 2.

Figure 2 α-SNAP binding to syntaxin in the absence (square) or presence (circle) of synaptobrevin
Adapted from Mcmahon HT, Südhof TC. Synaptic core complex of synaptobrevin, syntaxin, and SNAP25 forms high affinity alpha-SNAP binding site. J Biol Chem. 1995;270(5) :2213-7.
-Wild-type syntaxin (Synt-WT) and mutant syntaxins containing a deletion of the N-terminal sequence (SyntΔA) or C-terminal sequence (SyntΔB) were separated and incubated with α-SNAP. After washing away unbound molecules, which of the following is the most likely result of protein immunoblotting for α-SNAP?

Quizplus · Accepted Answer

11ecba2d_0dc1_9e00_a3bf_87c441b24ce1_MD0008_00 Western blot (protein immunoblotting) is a protein separation and identification technique that uses labeled antibodies to detect specific proteins after gel electrophoresis. In this example, the antibody is designed to bind α-SNAP. Therefore, after unbound α-SNAP is washed away, protein samples will be visualized on western blot in proportion to α-SNAP binding. The passage indicates that α-SNAP binds syntaxin at its N-terminal domain. The mutant syntaxin SyntΔA described in the question lacks the N-terminal domain and is unlikely to bind α-SNAP in large quantities, leading to little α-SNAP-bound syntaxin for this mutation. In contrast, deletion of the C-terminal domain (SyntΔB) is not expected to affect α-SNAP binding compared to binding of the WT protein. Therefore, α-SNAP in the WT protein and SyntΔB samples will be visualized as large bands whereas less α-SNAP will be visualized in the SyntΔA sample. (Choice B) α-SNAP poorly binds the mutant syntaxin SyntΔA. The quantity (band intensity) of α-SNAP-bound SyntΔA should be less than WT and SyntΔB. (Choice C) According to the passage, α-SNAP binds syntaxin's N-terminal domain. Modification of the C-terminal domain in SyntΔB should not impact α-SNAP binding, so the SyntΔB lane should look similar to the WT lane. (Choice D) Synt-WT binds α-SNAP effectively, so the western blot should show a full band in the WT lane. Educational objective: Western blot (protein immunoblotting) uses antibodies to detect specific proteins. Greater quantities of the protein of interest will manifest as larger bands on the resultant blot.

Passage Presynaptic Nerve Terminals Release Neurotransmitters Via Synaptic Vesicle Exocytosis. This