A Pivotal Role for Pro-335 in Balancing the Dual Functions of Munc18-1 Domain-3a in Regulated Exocytosis
Bibliographic record
Abstract
Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".