Syntaxin-1A modulates vesicle fusion in mammalian neurons via juxtamembrane domain dependent palmitoylation of its transmembrane domain
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Evaluation Summary:
Exocytosis of synaptic vesicles is mediated by synaptic SNARE proteins that overcome the energy barrier for membrane fusion by assembling into a helical bundle, thus pulling the membranes together. Here the authors have introduced mutations into the membrane-proximal region and transmembrane domain of one of the SNAREs (syntaxin 1a) showing that not only charge reversal but also palmitoylation of the transmembrane domain influence both spontaneous and evoked neurotransmitter release. The results add important details to our understanding of the late steps in SNARE-mediated exocytosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
SNAREs are undoubtedly one of the core elements of synaptic transmission. Contrary to the well characterized function of their SNARE domains bringing the plasma and vesicular membranes together, the level of contribution of their juxtamembrane domain (JMD) and the transmembrane domain (TMD) to the vesicle fusion is still under debate. To elucidate this issue, we analyzed three groups of STX1A mutations in cultured mouse hippocampal neurons: (1) elongation of STX1A’s JMD by three amino acid insertions in the junction of SNARE-JMD or JMD-TMD; (2) charge reversal mutations in STX1A’s JMD; and (3) palmitoylation deficiency mutations in STX1A’s TMD. We found that both JMD elongations and charge reversal mutations have position-dependent differential effects on Ca 2+ -evoked and spontaneous neurotransmitter release. Importantly, we show that STX1A’s JMD regulates the palmitoylation of STX1A’s TMD and loss of STX1A palmitoylation either through charge reversal mutation K260E or by loss of TMD cysteines inhibits spontaneous vesicle fusion. Interestingly, the retinal ribbon specific STX3B has a glutamate in the position corresponding to the K260E mutation in STX1A and mutating it with E259K acts as a molecular on-switch. Furthermore, palmitoylation of post-synaptic STX3A can be induced by the exchange of its JMD with STX1A’s JMD together with the incorporation of two cysteines into its TMD. Forced palmitoylation of STX3A dramatically enhances spontaneous vesicle fusion suggesting that STX1A regulates spontaneous release through two distinct mechanisms: one through the C-terminal half of its SNARE domain and the other through the palmitoylation of its TMD.
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Evaluation Summary:
Exocytosis of synaptic vesicles is mediated by synaptic SNARE proteins that overcome the energy barrier for membrane fusion by assembling into a helical bundle, thus pulling the membranes together. Here the authors have introduced mutations into the membrane-proximal region and transmembrane domain of one of the SNAREs (syntaxin 1a) showing that not only charge reversal but also palmitoylation of the transmembrane domain influence both spontaneous and evoked neurotransmitter release. The results add important details to our understanding of the late steps in SNARE-mediated exocytosis.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
Here the authors studied the function of the linker domain of syntaxin 1 (the relatively short stretch connecting the SNARE domain with the transmembrane domain) as well as the possible role of palmitoylation by (i) elongating the linker at two different positions by inserting three additional amino acids (GSG), (ii) by substituting individual basic residues with glutamate residues in the basic cluster directly upstream of the TMD, and (iii) by substituting the two adjacent cysteine residues in the middle of the TMD with valines. The results are rather complex, with different effects on spontaneous and calcium-evoked release dependent on the position of the exchanges or insertions. A major part of the manuscript deals with the role of cysteine palmitoylation in the TMD, which intriguingly appears to be …
Reviewer #1 (Public Review):
Here the authors studied the function of the linker domain of syntaxin 1 (the relatively short stretch connecting the SNARE domain with the transmembrane domain) as well as the possible role of palmitoylation by (i) elongating the linker at two different positions by inserting three additional amino acids (GSG), (ii) by substituting individual basic residues with glutamate residues in the basic cluster directly upstream of the TMD, and (iii) by substituting the two adjacent cysteine residues in the middle of the TMD with valines. The results are rather complex, with different effects on spontaneous and calcium-evoked release dependent on the position of the exchanges or insertions. A major part of the manuscript deals with the role of cysteine palmitoylation in the TMD, which intriguingly appears to be prevented by one of the K-E substitutions in the linker region (K260E). The authors also carry out some experiments with Syntaxin 1/Syntaxin 3B chimeras as well as some point mutations in Syx3. While wt Syx3B does not rescue release, converting a glutamate to a lysine residue in the polybasic stretch (i.e. rendering it more similar to Syx1) rescued both spontaneous and evoked release. Thus, one of the main findings is that palmitoylation is essential for spontaneous vesicle release: it is abolished when palmitoylation is blocked, either by direct replacement of the cysteines or indirectly by the K260E substitution.
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Reviewer #2 (Public Review):
In this study, Vardar and colleagues demonstrate that juxtamembrane domain (JMD) and transmembrane domain (TMD) of syntaxin play critical roles in mediating neurotransmitter release by employing loss-of-function mutant forms of syntaxin 1A as well as gain-of-function mutant forms of syntaxin 3B/A. Only 3 residues (GSG) insertion in the JMD of syntaxin 1A altered neurotransmitter release in a location-dependent manner. In particular, K260 residue in the JMD of syntaxin 1A was important for the palmitoylation of C271/C272 residues in the TMD of syntaxin 1A. The neurotransmitter release that is mediated by palmitoylation-deficient syntaxin 1A significantly decreased Pvr (%) and mEPSC frequency. Therefore, the authors concluded that both JMD and TMD of syntaxin play important roles in mediating neurotransmitter …
Reviewer #2 (Public Review):
In this study, Vardar and colleagues demonstrate that juxtamembrane domain (JMD) and transmembrane domain (TMD) of syntaxin play critical roles in mediating neurotransmitter release by employing loss-of-function mutant forms of syntaxin 1A as well as gain-of-function mutant forms of syntaxin 3B/A. Only 3 residues (GSG) insertion in the JMD of syntaxin 1A altered neurotransmitter release in a location-dependent manner. In particular, K260 residue in the JMD of syntaxin 1A was important for the palmitoylation of C271/C272 residues in the TMD of syntaxin 1A. The neurotransmitter release that is mediated by palmitoylation-deficient syntaxin 1A significantly decreased Pvr (%) and mEPSC frequency. Therefore, the authors concluded that both JMD and TMD of syntaxin play important roles in mediating neurotransmitter release.
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Reviewer #3 (Public Review):
Using a detailed mutational analysis of the SNARE protein syntaxin 1, Rosenmund and colleagues addressed the question of whether the juxtamembrane domain (JMD) and transmembrane domain (TMD) of the protein have an effect on neurotransmitter release from hippocampal neurons. The results show that insertions into the juxtamembrane domain of syntaxin strongly reduce synaptic strength, an observation that is consistent with previous studies on the mode of action of vesicular SNARE proteins, such as synaptobrevin. Furthermore, the authors show that some but not all charge reversal mutations in the JMD of syntaxin (e.g. K256, K260 and K263) reduce the pool of releasable vesicles while affecting palmitoylation of the protein. Palmitoylation mutants (C/V exchange), on the other hand, have little effect on evoked …
Reviewer #3 (Public Review):
Using a detailed mutational analysis of the SNARE protein syntaxin 1, Rosenmund and colleagues addressed the question of whether the juxtamembrane domain (JMD) and transmembrane domain (TMD) of the protein have an effect on neurotransmitter release from hippocampal neurons. The results show that insertions into the juxtamembrane domain of syntaxin strongly reduce synaptic strength, an observation that is consistent with previous studies on the mode of action of vesicular SNARE proteins, such as synaptobrevin. Furthermore, the authors show that some but not all charge reversal mutations in the JMD of syntaxin (e.g. K256, K260 and K263) reduce the pool of releasable vesicles while affecting palmitoylation of the protein. Palmitoylation mutants (C/V exchange), on the other hand, have little effect on evoked transmitter release but reduce spontaneous transmitter release, leading the authors to speculate that palmitoylation reduces the energy barrier of the actual fusion process.
Taken together, the findings suggest that the JMD and TMD of syntaxin 1 play an important yet unrecognized role in the fusion of small synaptic vesicles.
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