A Pivotal Role for Pro-335 in Balancing the Dual Functions of Munc18-1 Domain-3a in Regulated Exocytosis

Han, Gayoung A.; Park, Seungmee; Bin, Na-Ryum; Jung, Chang Hun; Kim, Byungjin; Chandrasegaram, Prashanth; Matsuda, Maiko; Riadi, Indira; Han, Liping; Sugita, Shuzo

doi:10.1074/jbc.m114.584805

Cited by 32 publications

(54 citation statements)

References 36 publications

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“…3d). These results, together with functional studies of Vps33 12 and Munc18 109,110 , imply that SM proteins use their helical hairpins to facilitate SNARE complex formation. At least in the case of Vps33, the SM protein functions as a template upon which an early SNARE complex assembly intermediate, resembling a half-zippered complex between the Qa- and R-SNAREs, can form.…”

Section: Sec1-munc18 Proteins and Snare Assemblymentioning

confidence: 83%

Chaperoning SNARE assembly and disassembly

Baker

Hughson

2016

Nat Rev Mol Cell Biol

228

238

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Intracellular membrane fusion is mediated in most cases by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Yet in vitro, the assembly of such complexes is inefficient, and their uncatalyzed disassembly is undetectably slow. Here, we focus on the cellular machinery that orchestrates SNARE complex assembly and disassembly, thereby regulating processes ranging from vesicle trafficking to organelle fusion to neurotransmitter release. Rapid progress is being made on many fronts, including the development of more realistic cell-free reconstitutions, the application of single-molecule biophysics, and the elucidation of x-ray and high-resolution electron microscopy structures of the SNARE assembly and disassembly machineries ‘in action’.

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Section: Sec1-munc18 Proteins and Snare Assemblymentioning

confidence: 83%

Chaperoning SNARE assembly and disassembly

Baker

Hughson

2016

Nat Rev Mol Cell Biol

228

238

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“…The neuronal SNARE complex, consisting of syntaxin-1, SNAP-25, and synaptobrevin/VAMP-2, plays a central role in mediating neurotransmitter release through membrane fusion (exocytosis; Söllner et al, 1993). This process is regulated by key proteins such as Munc18-1 (Hata et al, 1993;, Munc13 (Brose et al, 1995;Augustin et al, 1999), and Tomosyn (Fujita et al, 1998;McEwen et al, 2006). However, the precise regulatory mechanisms of exocytosis remain largely unclear.…”

Section: Introductionmentioning

confidence: 99%

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans

Park¹,

Bin²,

et al. 2017

J. Neurosci.

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Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in , as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to null mutants, () mutants partially bypass the requirement of UNC-13. Moreover, () and null mutations confer a strong synergy in suppressing the phenotypes of mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1. At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using as an model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.

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“…The essential function of Munc18-1 in neuronal exocytosis (Verhage et al, 2000) is known to be mediated by its complex interactions with syntaxin-1 and/or the neuronal SNARE complex (Han et al, 2010). At least two important functions of Munc18-1 have been proposed: (1) priming of membrane fusion through direct interaction with the SNARE complex (Han et al, 2013;Han et al, 2014;Hu et al, 2010;Ma et al, 2013;Parisotto et al, 2014;Rodkey et al, 2008;Südhof and Rothman, 2009;Tareste et al, 2008;Xu et al, 2010); (2) molecular chaperone function of syntaxin-1 regulating appropriate intracellular localization and expression of syntaxin-1 (Arunachalam et al, 2008;Han et al, 2009;Malintan et al, 2009;McEwen and Kaplan, 2008;Medine et al, 2007;Rowe et al, 2001;Rowe et al, 1999;Shen et al, 2007). A similar chaperoning function (regulation of expression level as well as intracellular trafficking of its cognate syntaxin partner) of Munc18-2 has also been discovered; some FHL5 patients, who lack functional Munc18-2 protein, present with strongly reduced syntaxin-11 expression levels in their immune cells (Côte et al, 2009;zur Stadt et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

Bin

Jung

Kim

et al. 2015

Journal of Cell Science

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Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation -whose levels and intracellular localization are regulated by Munc18-2 -remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.

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A Pivotal Role for Pro-335 in Balancing the Dual Functions of Munc18-1 Domain-3a in Regulated Exocytosis

Cited by 32 publications

References 36 publications

Chaperoning SNARE assembly and disassembly

Chaperoning SNARE assembly and disassembly

UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis

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