Munc18-1: Sequential Interactions with the Fusion Machinery Stimulate Vesicle Docking and Priming

Jiang

et al. 2009

MBoC

130

Munc18-1 binds to syntaxin-1A via two distinct sites referred to as the "closed" conformation and N terminus binding. The latter has been shown to stimulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis, whereas the former is believed to be inhibitory or dispensable. To precisely define the contributions of each binding mode, we have engineered Munc18-1/-2 double knockdown neurosecretory cells and show that not only syntaxin-1A and -1B but also syntaxin-2 and -3 are significantly reduced as a result of Munc18-1 and -2 knockdown. Syntaxin-1 was mislocalized and the regulated secretion was abolished. We next examined the abilities of Munc18-1 mutants to rescue the defective phenotypes. Mutation (K46E/E59K) of Munc18-1 that selectively prevents binding to closed syntaxin-1 was unable to restore syntaxin-1 expression, localization, or secretion. In contrast, mutations (F115E/ E132A) of Munc18-1 that selectively impair binding to the syntaxin-1 N terminus could still rescue the defective phenotypes. Our results indicate that Munc18-1 and -2 act in concert to support the expression of a broad range of syntaxins and to deliver syntaxin-1 to the plasma membrane. Our studies also indicate that the binding to the closed conformation of syntaxin is essential for Munc18-1 stimulatory action, whereas the binding to syntaxin N terminus plays a more limited role in neurosecretory cells.

Section: Discussionmentioning

confidence: 91%

Rescue of Munc18-1 and -2 Double Knockdown Reveals the Essential Functions of Interaction between Munc18 and Closed Syntaxin in PC12 Cells

Jiang

et al. 2009

MBoC

130

“…Our analysis of the previously alleged 'priming mutants' (e.g. E59K, D34V/M38V) (Deák et al, 2009;Gulyás-Kovács et al, 2007) in the surface of domain 1 cleft suggested that their phenotypes are better explained by their reduced syntaxin-1 chaperoning activity. Therefore, the Munc18-1 mutant that selectively loses priming function is yet to be identified (Han et al, 2011).…”

Section: Introductionmentioning

confidence: 81%

The domain-3a of Munc18-1 plays a crucial role at the priming stage of exocytosis

Journal of Cell Science

Bin

Kang

et al. 2013

SummaryMunc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.

Proc. Natl. Acad. Sci. U.S.A.

“…28 and 29). Although PMA potentiates release from embryonic chromaffin cells (21,30), potentiation persists after inhibition of PKC (22), deletion of Munc18-1 (30), or rescue of Syt1-KO with Syt T112A (21). In addition, the relative contribution of PKC to DAG-induced potentiation differs among synapses, as PKC activation is strictly required in hippocampal (6,8), but not in Calyceal synapses (7,11).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of synaptotagmin-1 controls a post-priming step in PKC-dependent presynaptic plasticity

Jong

Meijer

Saarloos

et al. 2016

Presynaptic activation of the diacylglycerol (DAG)/protein kinase C (PKC) pathway is a central event in short-term synaptic plasticity. Two substrates, Munc13-1 and Munc18-1, are essential for DAGinduced potentiation of vesicle priming, but the role of most presynaptic PKC substrates is not understood. Here, we show that a mutation in synaptotagmin-1 (Syt1 T112A ), which prevents its PKCdependent phosphorylation, abolishes DAG-induced potentiation of synaptic transmission in hippocampal neurons. This mutant also reduces potentiation of spontaneous release, but only if alternative Ca 2+ sensors, Doc2A/B proteins, are absent. However, unlike mutations in Munc13-1 or Munc18-1 that prevent DAG-induced potentiation, the synaptotagmin-1 mutation does not affect pairedpulse facilitation. Furthermore, experiments to probe vesicle priming (recovery after train stimulation and dual application of hypertonic solutions) also reveal no abnormalities. Expression of synaptotagmin-2, which lacks a seven amino acid sequence that contains the phosphorylation site in synaptotagmin-1, or a synaptotagmin-1 variant with these seven residues removed (Syt1 Δ109-116 ), supports normal DAG-induced potentiation. These data suggest that this seven residue sequence in synaptotagmin-1 situated in the linker between the transmembrane and C2A domains is inhibitory in the unphosphorylated state and becomes permissive of potentiation upon phosphorylation. We conclude that synaptotagmin-1 phosphorylation is an essential step in PKC-dependent potentiation of synaptic transmission, acting downstream of the two other essential DAG/PKC substrates, Munc13-1 and Munc18-1.P resynaptic strength changes rapidly during repetitive stimulation [short-term plasticity (STP)] and activation of intracellular signal transduction pathways (1, 2). The diacylglycerol (DAG)/protein kinase C (PKC) cascade is one of the most potent pathways at the presynaptic terminal. Its activation leads to 50-100% potentiation of spontaneous and action potential (AP)-evoked release (3-5), and is critical for multiple forms of presynaptic plasticity (6-8). DAG directly activates the vesicle priming factor Munc13-1 (9, 10) and indirectly activates downstream effectors via PKC. Activation of both Munc13-1 and PKC is essential for this pathway to operate (Fig. 1A) (8, 11). We previously identified Munc18-1 as an essential PKC substrate, because a nonphosphorylatable Munc18-1 mutant completely inhibits PKC-dependent STP (8, 12). Importantly, a phosphomimetic mutation of Munc18-1 cannot fully bypass the requirement for PKC activation, indicating that other PKC substrates must contribute to this form of plasticity (8). These substrates have not been identified to date.Among other presynaptic PKC substrates is synaptotagmin-1 (Syt1, ref. 13). Syt1 is the vesicular Ca 2+ sensor that mediates fast AP-evoked release in the hippocampus (14) and drives a large fraction of spontaneous release (15). In the latter case, Syt1 competes with alternative sensors, in particular Doc2s, for SNARE binding ...