Munc18a: Munc-y business in mediating exocytosis.

Abstract

The precise sequence of molecular events underlying release of neurotransmitter in neurons is yet to be fully understood. This process, called exocytosis, is tightly controlled by a number of protein-protein and protein-lipid interactions. One such regulatory factor is Munc18a, a cytosolic protein characterized by its interaction with the molecular machinery of exocytosis, primarily with the target SNARE protein, syntaxin1a. While Munc18a interactions have been extensively investigated for more than a decade, the role of Munc18a in vesicular fusion is still not fully defined. In this review, we discuss: (i) the recent analysis of the role of Munc18a in tethering and docking, (ii) the known structural and (iii) functional data surrounding Munc18a interactions with numerous other proteins of the exocytic machinery. Integration of Munc18a regulation by phosphorylation and lipids and the apparent complexity of its pleiotropic functional interactions is critical to deciphering Munc18a's role in exocytosis.

Full text

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The International Journal of Biochemistry & Cell Biology 39 (2007) 1576–1581
Molecules in focus
Munc18a: Munc-y business in mediating exocytosis
Catherine F. Latham, Frederic A. Meunier∗
Molecular Dynamics of Synaptic Function Laboratory, School of Biomedical Sciences,
The University of Queensland, St. Lucia, Qld 4072, Australia
Received 7 November 2006; received in revised form 16 November 2006; accepted 18 November 2006
Available online 30 November 2006
Abstract
The precise sequence of molecular events underlying release of neurotransmitter in neurons is yet to be fully understood. This
process, called exocytosis, is tightly controlled by a number of protein–protein and protein–lipid interactions. One such regulatory
factor is Munc18a, a cytosolic protein characterized by its interaction with the molecular machinery of exocytosis, primarily with
the target SNARE protein, syntaxin1a. While Munc18a interactions have been extensively investigated for more than a decade, the
role of Munc18a in vesicular fusion is still not fully defined. In this review, we discuss: (i) the recent analysis of the role of Munc18a
in tethering and docking, (ii) the known structural and (iii) functional data surrounding Munc18a interactions with numerous other
proteins of the exocytic machinery. Integration of Munc18a regulation by phosphorylation and lipids and the apparent complexity
of its pleiotropic functional interactions is critical to deciphering Munc18a’s role in exocytosis.
© 2006 Elsevier Ltd. All rights reserved.
Keywords: Munc18a; SNARE; Vesicle trafficking; SM protein
1. Introduction
Neuronal communication relies on the fusion of
neurotransmitter-containing vesicles with the neuronal
plasma membrane in a tightly regulated process
called exocytosis. During exocytosis, a number of
well-orchestrated protein–protein and protein–lipid
interactions occur. This sequence of interactions allows
vesicles to approach the plasma membrane (tethering
Abbreviations: APP, amyloid precursor protein; Cdk5, cyclin-
dependent kinase 5; DOC2, double C2-domain; FRET, fluorescence
resonance energy transfer; LDCV, large dense core vesicle; Mint,
Munc18-interacting protein; PKC, protein kinase C; SM protein,
Sec1/Munc18; SNARE, soluble N-ethylmaleimide-sensitive attach-
ment protein receptor; TIRF, total internal reflection fluorescence
∗Corresponding author. Tel.: +61 7 3365 3506;
fax: +61 7 3365 1766.
E-mail address: f.meunier@uq.edu.au (F.A. Meunier).
anddocking),toundergoprimingand,uponCa2+influx,
to trigger vesicle fusion with the plasma membrane
thereby releasing neurotransmitter in the synaptic cleft.
Understanding the mechanism of vesicular exocytosis
has focussed on the role played by the exocytic machin-
ery, namely the soluble N-ethylmaleimide-attachment
protein receptor (SNARE) proteins and a cytosolic
regulatory protein, Munc18a (reviewed in Rizo &
S¨ udhof, 2002). While the function of the SNARE
proteins in mediating the latest step of exocytosis is
well established, defining the precise role of Munc18a
in the process of exocytosis is still under debate.
Munc18a (n-sec1 or Munc18-1) belongs to the
Sec1/Munc18 (SM) family of proteins that are involved
in mediating membrane trafficking events (Toonen &
Verhage,2003).ThefirstneuronalSMprotein,UNC-18,
wasdescribedintheearly1990saspartofaCaenorhab-
ditis elegans screen of uncoordinated phenotypes, and
wasfoundtobeverysimilartotheyeast(Saccharamyces
1357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2006.11.015

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1577
cerevisiae) secretory regulatory protein, Sec1p (Garcia,
Gatti, Butler, Burton, & De Camilli, 1994). Subsequent
studies identified neuronal homologs in Drosophila
melanogaster (Rop), a mammalian neuronal homolog,
Munc18a (Garcia et al., 1994) and numerous non-
neuronal SM proteins also involved in various vesicular
trafficking pathways (Toonen & Verhage, 2003).
Munc18a was first described as a mammalian brain-
specific protein tightly bound to syntaxin1a, a neuronal
plasma membrane t-SNARE protein (Garcia et al.,
1994). While the biochemical characterisation of the
Munc18a:syntaxin1ainteractionpointstoanegativereg-
ulatory role for Munc18a (Misura, Scheller, & Weis,
2000),thefunctionalanalysisofMunc18aandofitsneu-
ronal counterparts in D. melanogaster and C. elegans is
somewhat conflicting (Rizo & S¨ udhof, 2002). Here, we
summarisethecurrenthypothesessurroundingMunc18a
regulation of vesicular exocytosis.
2. Structure
Initial studies of Munc18a-syntaxin1a molecular
interactions by Yang and colleagues revealed that
Munc18arequirestheentirecytoplasmicdomainofsyn-
taxin1a for binding (Yang, Steegmaier, Gonzalez, &
Scheller, 2000). Syntaxin1a is known to assume two
major conformations: (i) open, where the SNARE bind-
ing domain is free to interact with the other SNARE
proteins, SNAP25 and VAMP2, to form the fusogenic
SNARE complex, and (ii) closed, where an intramolec-
ular interaction between the two domains of syntaxin1a
prevents SNARE complex assembly (Rizo & S¨ udhof,
2002). Consequently, it was hypothesized that only a
closed conformation of syntaxin1a binds to Munc18a,
precluding SNARE complex formation (Yang et al.,
2000). The crystal structure of the complex between
syntaxin1aandMunc18aconfirmedthatMunc18ainter-
acts with the closed syntaxin1a (Misura et al., 2000)
(PDB code 1DN1) (Fig. 1A). Munc18a is a horse-shoe
shaped protein consisting of three domains (Fig. 1A–C).
Closed-syntaxin1a binds in the cavity created by the
three domains and forms contacts with domain 1 and
3a. The crystal structure of the uncomplexed form of
neuronal Munc18a (from squid, Loligo pealei) has also
been determined (Bracher, Perrakis, Dresbach, Betz,
& Weissenhorn, 2000) (Fig. 1B) (PDB code 1FVH).
Comparison of the crystal structures of monomeric
Munc18a and complexed Munc18a:syntaxin1a revealed
few changes in Munc18a upon syntaxin1a bind-
ing, mainly some secondary structure changes in
Fig. 1. Structural organization of Munc18a (n-sec1) and the Munc18a:syntaxin1a complex. (A) The crystal structure of the rat (Rattus norvegicus)
Munc18a:syntaxin1a complex (Misura et al., 2000) (PDB code 1DN1) where Munc18a binds to the closed conformation of syntaxin1a. Domains
1 (blue), 2 (orange), 3a and b (green) of Munc18a and syntaxin1a (yellow) are indicated. Phosphorylation sites for protein kinase C (Ser306 and
Ser313)andcyclin-dependentkinase5(Thr574)areshowninredandlabelledaccordingly.(B)Thecrystalstructureoftheneuronalsquid(L.pealei)
homolog of Munc18a reveals a conserved structure with few conformational differences from the Munc18a:syntaxin1a complex (Bracher et al.,
2000) (PDB code 1FVH). (C) Schematic representation of Munc18a domains, indicating the residues contributing to each domain. Phosphorylation
sites are highlighted in red. Domain 2 comprises two segments in the polypeptide sequence but folds as a single domain. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of the article.)

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Fig. 2. Munc18a interactions and their effects on exocytosis. Munc18a is associated with syntaxin1a in the basal state (centre panel). (A) Phospho-
rylation of Munc18a at Ser306 and Ser313 by protein kinase C and at Thr574 by cyclin-dependent kinase 5 prevents binding to syntaxin1a (Liu
et al., 2004) and increases the replenishment of the ready releasable pool (RRP) (Nili et al., 2006). (B) Mints bind to Munc18a independently of
syntaxin1a binding and could tether Munc18a to the membrane by binding to phosphatidylinositol (4,5)-bisphosphate (Okamoto & S¨ udhof, 1997).
(C) DOC2 could be involved in disassembly of the Munc18a:syntaxin1a complex, as it competes with syntaxin1a for binding to Munc18a (Verhage
et al., 1997). On addition of syntaxin1a-binding SNARE proteins such as SNAP25, DOC2 binding to Munc18a is enhanced resulting in and increase
in exocytosis. (D) Granuphilin binds to the Munc18a:syntaxin1a complex in PC12 cells (Tsuboi & Fukuda, 2006) and Munc18a in ?-cells (Coppola
et al., 2002), and mediates interactions between Munc18a and Rab proteins, inhibiting exocytosis. (E) Arachidonic acid allows Munc18a binding to
the SNARE complex (Latham et al., 2006). The relevance of this binding to the observed potentiation of exocytosis is yet to be established.
domain 3. Elucidation of the crystal structure of the
Munc18a:syntaxin1a complex represents a landmark in
our understanding of Munc18a-SNARE interactions, as
it provides a scaffold to complement existing functional
studies of the role of Munc18a (Misura et al., 2000).
3. Expression and activation
Munc18a is one mammalian counterpart for the
exocytic regulators Sec1 (S. cerevisiae), Rop (D.
melanogaster) and UNC-18 (C. elegans) (Garcia et al.,
1994).NorthernblotanalysisrevealedthatMunc18awas
only present in the brain (Garcia et al., 1994). However,
more recent studies have identified Munc18a expression
andexocyticfunctioninothercelltypessuchasneurose-
cretory cells (Tsuboi & Fukuda, 2006) and pancreatic
?-cells (Coppola et al., 2002). The closely-related SM
proteins, Munc18b and c are ubiquitously expressed in
mammals (Toonen & Verhage, 2003) and further stud-
ies may reveal that Munc18a expression might be more
widespread than previously thought.
ThecurrentviewisthatMunc18aexertsitsinhibitory
function through binding to closed syntaxin1a and that
the Munc18a:syntaxin1a complex can be dissociated to
allow syntaxin1a to interact with the other SNAREs – a
process deemed to facilitate exocytosis (Rizo & S¨ udhof,
2002). In the basal state, cytosolic Munc18a is associ-
ated with syntaxin1a at the plasma membrane (Rizo &
S¨ udhof, 2002). Syntaxin1a release from Munc18a could
be caused by a currently unknown effector, that might
involve either phosphorylation of Munc18a (Fletcher et
al., 1999; Liu et al., 2004), polyunsaturated fatty acids

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(Rickman & Davletov, 2005) or other protein inter-
actions either in a SNARE dependent or independent
manner (Ciufo, Barclay, Burgoyne, & Morgan, 2005;
Toonen & Verhage, 2003) (see Fig. 2).
4. Biological function
4.1. Role of Munc18a in docking and priming
There is strong genetic evidence of a critical role
for Munc18a in the early stages of exocytosis since
a Munc18a knockout mouse exhibits a total block of
spontaneous and evoked release (Verhage et al., 2000).
Ultrastructural analysis of neurons isolated from knock-
out mice revealed a usual proportion of docked synaptic
vesicles, suggesting a role for Munc18a downstream
of tethering and docking (Verhage et al., 2000). The
situation is clearly different in chromaffin cells where
large dense core vesicle (LDCV) docking is com-
pletely impaired in the Munc18a knockout (Voets et
al., 2001). A recent, elegant study using total internal
reflection fluorescence (TIRF) microscopy reveals that
Munc18a acts as a strong tethering factor, following
the initial binding of the LDCV to the plasma mem-
brane (Toonen et al., 2006). Interestingly, this study also
demonstrates a restoration of (non-functional) docking
following latrunculin-A treatment, which is suggestive
ofapossiblesyntaxin1a-independentroleofMunc18ain
facilitating docking. This could occur by controlling the
amount of cortical actin through interaction with F-actin
as previously suggested (Bhaskar et al., 2004).
4.2. Munc18a inhibition by phosphorylation
Protein kinase C (PKC)-dependent in vivo phospho-
rylation of Munc18a on Ser313 has been shown to occur
upon stimulation of exocytosis in chromaffin cells and
synaptosomes (reviewed in Morgan et al., 2005). Phos-
phorylation of Munc18a alters its affinity for syntaxin1a
both in vitro (Morgan et al., 2005) and in vivo (Liu
et al., 2004). In vitro, phosphorylation of Munc18a by
PKC at Ser313 and Ser306 prevents Munc18a from
forming a complex with syntaxin1a (Fujita et al., 1996)
(Fig. 2A). In addition, Munc18a cannot be phosphory-
lated once this Munc18a:syntaxin1a complex is formed
(Fujita et al., 1996). Cyclin-dependent kinase 5 (Cdk5)
can also phosphorylate Munc18a in vitro at Thr574,
whichpromotesdisassemblyoftheMunc18a:syntaxin1a
complex (Fletcher et al., 1999). More recent studies
using fluorescence resonance energy transfer (FRET)
in HEK293-S3 and bovine chromaffin cells show that
Munc18a phosphomimetic mutants of all three phos-
phorylation sites (see Fig. 1A) had ∼70% reduction in
binding to syntaxin1a (Liu et al., 2004). Inhibition of
Munc18a–syntaxin1a binding by PKC phosphorylation
at Ser306 and Ser313 residues is consistent with the
structural features of the Munc18a:syntaxin1a complex
interaction (Misura et al., 2000).
The functional relevance of Munc18a phosphoryla-
tion is highlighted by recent studies (Barclay et al.,
2003). Ser313 phosphorylation has recently been shown
to potentiate the replenishment of the readily releasable
pool (Nili et al., 2006). A negative effect of Munc18a
phosphorylation on the kinetics of the exocytic events
was demonstrated by amperometry in chromaffin cells
(Barclay et al., 2003) but this result has not been con-
firmed by capacitance measurements (Nili et al., 2006).
The latter study suggests that Munc18a phosphorylation
does not affect its docking activity (Nili et al., 2006).
4.3. Munc18a and other effectors
SeveralothereffectorscaninfluenceMunc18amolec-
ular interactions such as Mints (Munc18-interacting
proteins; also called X11 proteins) which are predom-
inantly expressed in the brain (Okamoto & S¨ udhof,
1997). Mint proteins could help association of Munc18a
with the plasma membrane via their interaction with
phosphatidylinositol (4,5)-bisphosphate (Okamoto &
S¨ udhof, 1997). In a recent study, Munc18a mutants that
displayed a reduced ability to bind to the Mints but
maintainedwild-typesyntaxin1abindingaffinitygreatly
potentiated catecholamine release when expressed in
chromaffin cells (Ciufo et al., 2005) (Fig. 2B).
Munc18a-Mintinteractionshavealsobeenimplicatedin
Alzheimer’s disease. Mint1 over-expression suppresses
?-secretase processing of amyloid precursor protein
(APP), an important precursor in the pathology of
Alzheimer’sdisease.Mint1-inducedsuppressionofAPP
cleavage is greatly enhanced by co-expression of Mint1
withMunc18a(Hoetal.,2002)andtheseproteins,along
with syntaxin1a, Mint2 and Cdk5 are up-regulated in
Alzheimer’s disease positive cortex (Jacobs, Williams,
& Francis, 2006).
Other protein effectors of Munc18a are the DOC2
(double C2-domain) proteins, DOC2A and DOC2B.
DOC2 binds Munc18a via its internal C2-domain and
that interaction competes with Munc18a binding to
syntaxin1a (Verhage et al., 1997). Interestingly, Ver-
hage and colleagues have shown that DOC2 binding
to Munc18a is greatly increased by the addition of the
other SNARE proteins that bind to syntaxin1a (Verhage
et al., 1997) (Fig. 2C). This suggests that the DOC2
interaction with Munc18a could be involved in an early

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stage of exocytosis by facilitating disassembly of the
Munc18a:syntaxin1a complex (Verhage et al., 1997).
Disassembly of this complex would remove Munc18a-
mediated inhibition of syntaxin1a and, in turn, lead to
SNARE complex formation and subsequent vesicular
fusion.
Granuphilin is a Rab-interacting protein that selec-
tively binds the Munc18a:syntaxin1a complex via
Munc18a (Fig. 2D). In PC12 cells, granuphilin has been
shown to promote (presumably unfunctional) docking
of vesicles and to inhibit exocytosis (Tsuboi & Fukuda,
2006).Boththeseeffectsresultedfromthedirectinterac-
tion of granuphilin with Munc18a. In pancreatic ?-cells,
granuphilin expression has been shown to inhibit exo-
cytosis via molecular interactions mainly with Rab3 but
also with Munc18a (Coppola et al., 2002). The precise
contribution of Munc18a to this negative effect requires
clarification.
Certainspecificpolyunsaturatedfattyacidshavebeen
implicated in Munc18a function in synaptic vesicle exo-
cytosis. Two recent studies have shown that arachidonic
acid can influence Munc18a interactions with SNARE
proteins as well as potentiating exocytosis (Latham et
al.,2006;Rickman&Davletov,2005).Arachidonicacid
allows SNARE complex formation when SNAP25 and
VAMP2 are combined with Munc18a:syntaxin1a pre-
formed complex in an in vitro binding assay (Rickman
& Davletov, 2005). Arachidonic acid was also shown
to induce Munc18a binding to the SNARE complex
(Latham et al., 2006) (Fig. 2E). This novel interaction
could link the dynamic molecular events of exocytosis
to the observed potentiating effect of arachidonic acid
oncatecholaminereleasefromchromaffincells(Latham
et al., 2006) and glutamate release from synaptosomes
(Latham & Meunier, unpublished data). Although the
mechanistic consequences of lipid-induced changes in
Munc18a interactions/functions are not yet fully under-
stood, studying Munc18a in a lipid context could reveal
novel roles in exocytosis. In this view, it is interesting
to note that using fusion-competent membrane sheets,
Munc18a allows the t-SNARE complex (syntaxin1a
and SNAP25) to form as an acceptor intermediate for
vesicle-bound VAMP2 (Zilly, Sorensen, Jahn, & Lang,
2006).
Further analysis of known and yet to be discov-
ered effectors of Munc18a could help to decode the
pleiotropic roles of Munc18a in exocytosis.
Acknowledgements
WewouldliketothankDr.ShonaOsborneforcritical
reading of the manuscript and the National Health &
Medical Research Council for financial support (grant
#351434).
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