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  1. 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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  2. 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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  3. 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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  4. 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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