(Received for publication, June 13, 1995; and in revised form, August 14, 1995)
From the
The unc-13 gene in Caenorhabditis elegans is
essential for normal presynaptic function and encodes a large protein
with C- and C
-domains. In protein kinase C and
synaptotagmin, C
- and/or C
-domains are
regulatory domains for Ca
, phospholipids, and
diacylglycerol, suggesting a role for unc-13 in regulating
neurotransmitter release. To determine if a similar protein is a
component of the presynaptic machinery for neurotransmitter release in
vertebrates, we studied unc-13 homologues in rat. Molecular
cloning revealed that three homologues of unc-13 called
Munc13-1, -13-2, and -13-3 are expressed in rat
brain. Munc13s are large, brain-specific proteins with divergent N
termini but conserved C termini containing C
- and
C
-domains. Specific antibodies demonstrated that
Munc13-1 is a peripheral membrane protein that is enriched in
synaptosomes and localized to plasma membranes but absent from synaptic
vesicles. Our data suggest that the function of unc-13 in C. elegans is conserved in mammals and that Munc13s act as
plasma membrane proteins in nerve terminals. The presence of
C
- and C
-domains in these proteins and the
phenotype of the C. elegans mutants raise the possibility that
Munc13s may have an essential signaling role during neurotransmitter
release.
In presynaptic nerve terminals, synaptic vesicles store
neurotransmitters and release them by exocytosis. Major progress has
been made in recent years in the identification of proteins important
for synaptic vesicle functions and in the understanding of the
molecular interactions that underlie exocytosis. At this point, the
molecular composition of synaptic vesicles is well described, and the
functions for key proteins in membrane fusion have been explored (for
review, see (1) and (2) ). However, the identification
of the proteins that mediate synaptic vesicle functions is far from
complete. Even in the case of the most intensely studied proteins, our
understanding is limited. For example, synaptotagmin I is an intrinsic
membrane protein of synaptic vesicles that is essential for
Ca-triggered fast neurotransmitter
release(3) . Structurally, synaptotagmin I contains two
C
-domains, binds phospholipids as a function of
Ca
, and interacts with several synaptic proteins in
vitro(4, 5, 6, 7, 8, 9, 10, 11) .
However, the exact reactions performed by synaptotagmin I in the nerve
terminal are unknown, as are the number of protein-protein interactions
it performs physiologically. It seems likely that many more proteins
will ultimately be found to be required for neurotransmitter release
than the few proteins that are currently being studied.
Pioneering experiments by Sydney Brenner identified a collection of mutants in Caenorhabditis elegans called unc mutants that are completely or partially paralyzed(12) . A subgroup of these unc mutants is characterized by the presence of high levels of acetylcholine, resistance to acetylcholine esterase inhibitors such as aldicarb, and normal activities of acetylcholine esterase and choline acetyltransferase(13, 14) . These characteristics suggest that in this subgroup of unc genes, presynaptic neurotransmitter release is impaired, a hypothesis that is supported by the molecular identification of several unc genes of this subgroup. unc-18 encodes a protein whose mammalian homologue, Munc18, stoichiometrically binds to syntaxin, a component of the synaptic vesicle fusion complex that is essential for release (15, 16, 17) . The protein encoded by another gene, unc-17, encodes the vesicular transporter for acetylcholine(18) . Finally, mutants in the gene encoding the C. elegans homologue of synaptotagmin have the same phenotype as the unc mutants that accumulate acetylcholine and are aldicarb-resistant(19) .
Although putative functions of
several of the unc genes have been identified, many remain to
be studied. unc-13 is a particularly interesting member of
this group because of its severe phenotype(14) .
Characterization of the unc-13 gene (20) revealed that
it encodes a large protein (1734 amino acids) with no homology to other
proteins except for two regions: a C-domain homologous to
the phorbol ester- and diacylglycerol binding region of protein kinase
C that as a recombinant protein also binds phorbol esters (21, 22) and a C
-domain that in
synaptotagmin constitutes a Ca
-binding and a
protein-protein interaction
domain(4, 5, 6, 7, 8, 9, 10, 11) .
These characteristics suggested a potential role for unc-13 in
neurotransmitter release that involves a diacylglycerol-dependent and
Ca
-dependent step. No such step has yet been
identified in synaptic vesicle exocytosis, but phorbol esters have
profound effects on neurotransmitter release that cannot be wholly
attributed to protein kinase C(23) . (
)In order to
determine if unc-13 and its presumptive function is conserved
in vertebrates and to gain insight into its nature, we have now cloned
and characterized multiple mammalian homologues for unc-13 that we have named Munc13-1, -13-2, and -13-3.
Our data show that these proteins form a highly conserved family of
plasma membrane proteins with a probable function in neurotransmitter
release.
Figure 2:
Domain structure of Munc13s: Comparison
with other C-domain proteins. A, diagram of the
domain structures of Munc13-1 and other representative proteins
containing C
-domains. The domain structures of the other
Munc13s and of unc-13 are similar to that of Munc13-1
except that the N-terminal C
-domain is absent. Below
Munc13-1, representative members of other protein families
containing C
-domains are depicted: synaptotagmins, protein
kinase C isozymes (PKC), phospholipase C (PLC), and
phospholipase A
(PLA2), rabphilin-3A and its C. elegans homologue f37a4.7, and a number of databank entries
of sequences of unknown function. C
- and
C
-domains are labeled C1 and C2,
respectively. Kinase domains, kinase; pleckstrin homology
regions, PH; and transmembrane regions, TM. X denotes a conserved region in phospholipase C of unknown
significance, and catal denotes their catalytic domain; SH2 and SH3 stands for src-homology regions 2 and 3; rabph is for the rabphilin homology domain; and r and URE-B1 for the rsp5- and the
URE-B1-domains(40) . B, sequence alignment of
C
-domains from representative members of the protein
families depicted in A. Sequences are aligned for maximal
homology. Dots indicate gaps, and numbers in brackets are residues that are absent from other
C
-domains and have been deleted from the alignment.
Conserved residues are shown on a blackbackground.
Sequences are identified on the left; C
-domains
from proteins with multiple domains are labeled A, B, C, and D. C, dendrogram of the
C
-domains aligned in B. The length of the lines
connecting the different C
-domains is a measure for their
sequence distance. The tree was calculated by the neighbor joining
method (29) and tested by bootstrapping, which recalculates the
tree for random subsets of the data (100 identical alignments in which
random sequences were substituted by Xs). Bifurcation points
that were confirmed by bootstrapping analysis in 80-100
replicates are marked by filledtriangles, in
50-80 replicates by filledcircles, in
20-50 replicates by opencircles, and in less
than 20 replicates by no label. Note that the different
C
-domains can be divided in subclasses based on the
sequence alignment and the dendrogram. One subclass contains the
different C
-domains from synaptotagmins,
Ca
-dependent protein kinase C isozymes, and
rabphilins; a second subclass contains the phospholipases, and a third
contains the Munc13s, with the reliability of the tree being the
highest at bifurcation points between closely related
proteins.
Figure 1:
Primary structures of Munc13-1,
-13-2, and -13-3. The amino acid sequences of the rat
proteins were deduced from the nucleotide sequence assembled from
multiple overlapping cDNA clones (GenBank accession numbers U24070,
U24071, and U24072). Sequences are shown in single letter amino acid
code and are aligned for maximal homology with the C. elegans
unc-13 gene product. Residues that are identical in at least 50%
of the sequences are shown on a blackbackground, and
residues that are similar are shaded (similarity groups: F, Y,
W; I, L, V, M; H, R, K; D, E; G, A; T, S; N, Q). C-domains
are marked by a dottedline, and
C
-domains are marked by continuouslines.
Sequences are identified and numbered on the left. For
Munc13-3, only a partial cDNA sequence was obtained. Two sequence
stretches encoding residues 1434-1456 and residues
1541-1559, respectively, were either present or absent in
Munc13-1 cDNAs, suggesting that these sequences are alternatively
spliced. Note that the C. elegans sequence contains gaps at
the corresponding positions.
Munc13s are large proteins; Munc13-1 has 1735 residues, and Munc13-2 has 1985 residues, resulting in sizes of 196 and 222 kDa respectively. Munc13s are highly homologous to each other in their C-terminal two-thirds, but dissimilar in their N-terminal third (Fig. 1). 50% of all residues of the C-terminal two-thirds of unc-13 and Munc13-1, -13-2, and -13-3 are invariant (621 of 1243 residues). Blocks of almost 100% identical residues are separated by small islands of nonconserved sequences. At three positions, a 13-19 amino acid gap is present in the C. elegans sequence compared with the Munc13-1 sequence. At two of these positions, overlapping Munc13-1 cDNA clones either contained or lacked the corresponding sequence, suggesting that these regions are subject to alternative splicing.
We systematically
analyzed the sequence homology between different C-domains
of Munc13s and various proteins in order to examine their relationship.
Databank searches showed that C
-domains are found in a
large number of proteins, with a total of more than 50
C
-domains. Many of these proteins are involved in signal
transduction or membrane trafficking pathways, but others have only
been defined at the sequence level. All Munc13s contain a single
C
-domain immediately N-terminal to the middle
C
-domain; only protein kinase C isoforms contain a similar
arrangement of C
- and C
-domains (Fig. 2A). Several proteins in addition to Munc13s,
including synaptotagmins and rabphilins, contain multiple
C
-domains. The sequences of the C
-domains of
Munc13s are aligned with each other and with the sequences of the most
conserved C
-domains in the databanks in Fig. 2B, revealing that some residues are conserved in
all C
-domains, others only in subgroups of
C
-domains, and others vary between almost all
C
-domains.
The crystal structure of the first
C-domain of synaptotagmin I showed that it is composed of 8
-sheets that are arranged in a compact greek key
motif(37) . Synaptotagmin I probably contains multiple
Ca
binding sites, at least one of which was
identified in the crystal as formed by two loops connecting
-sheets. When the sequence of the first C
-domain of
synaptotagmin I is compared with those of the other
C
-domains, the sequences corresponding to the
-strands
in synaptotagmin I are the most conserved sequences of the
C
-domains. The sequences corresponding to loops connecting
the
-strands are less conserved except for the Ca
binding loops (see (37) ). However, even here some of the
residues are replaced in the C
-domains from Munc13s,
particularly the C-terminal and N-terminal C
-domains. The
biggest sequence variations are observed in the loops between the
fourth, fifth, and sixth
-strands that contain insertions of up to
24 amino acids (Fig. 2B).
Quantitation of the
similarities between different C-domains demonstrated that
they fall into distinct classes (Fig. 2C). The
dendrogram revealed that the middle C
-domain (referred to
as C
-B in all Munc13s because Munc13-1 contains an
additional N-terminal C
-domain called C
-A) and
the last C
-domain of Munc13s (C
-C) form
separate groups that are most similar to the corresponding domains from
other isoforms of the same gene family. It seems likely that the
different C
-domains originated from a single evolutionary
ancestor but evolved and duplicated independently in precursor proteins
to gene families such as the synaptotagmins and Munc13s. Their
independent evolution suggests that C
-domains perform
specialized functions in different proteins.
Figure 3:
Ca-dependent
phospholipid binding to the middle C
-domain of
Munc13-1, -13-2, and -13-3. Glutathione S-transferase-fusion proteins of the middle
C
-domains of Munc13s and the first C
-domain of
synaptotagmin I were analyzed for phospholipid binding as a function of
divalent cations as indicated. Divalent cation-induced changes are
expressed as percent of the EGTA-control. Errorbars show standard deviations from triplicate
determinations.
In contrast to the first C-domain of
synaptotagmin I, no Ca
-dependent phospholipid binding
was observed for the Munc13 C
-domains. Interestingly,
background binding of phospholipids in the absence of Ca
was reproducibly reduced by both Ca
and
Mg
in all three Munc13 C
-domains tested.
This finding suggests that divalent cations affect the conformation of
the C
-domains from Munc13s. The C-terminal
C
-domain from Munc13-1 also failed to bind
phospholipids as a function of Ca
(data not shown).
The absence of Ca
-dependent phospholipid binding to
the middle C
-domains of Munc13s supports the conclusion
from the dendrogram (Fig. 2C) that the group of
C
-domains of protein kinase C, rabphilin, and
synaptotagmins forms a separate group from that of the Munc13s.
Figure 4:
Expression of Munc13-1, -13-2,
and -13-3 in rat tissues analyzed by RNA blotting. Blots
containing poly(A)-enriched RNA from the indicated rat
tissues were hybridized at high stringency with uniformly labeled
probes from the coding regions of Munc13-1, -13-2, and
-13-3 and exposed to film for 24 h. The same blots were
rehybridized with probes for GAPDH and cyclophilin as loading controls
as shown in a representative autoradiogram at the bottom.
Figure 5: Expression of Munc13-1 and -13-2 in transfected COS cells and characterization by specific antibodies. COS cells transfected with full-length Munc13-1 and -13-2 expression vectors (firsttwolanes), skeletal muscle homogenate, and synaptic plasma membranes (SPM) (20 µg/lane) were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting using affinity-purified polyclonal antibodies to Munc13-1 or Munc13-2 as indicated. Immunoreactive bands were visualized by ECL. Numbers on the left indicate positions of molecular weight standards. Note the lack of cross-reactivity between Munc13-1 and Munc13-2 transfected COS cells; the lowerband in the Munc13-1 transfected COS cells presumably represents a proteolytic product in the COS cells.
We next examined the distribution of Munc13 proteins. Munc13-1 was only detected in the nervous system, confirming the RNA blotting data (Fig. 6). The size of the protein in brain corresponds to that of the protein expressed by transfection in COS cells, suggesting that the expression vector encodes full-length Munc13-1 (Fig. 5). By contrast, antibodies to Munc13-2 failed to identify an immunoreactive protein in all tissues tested, possibly because of low protein levels and/or low antibody affinity.
Figure 6: Tissue distribution of Munc13-1 protein. Homogenates from the indicated tissues (20 µg/lane) were analyzed by immunoblotting using affinity-purified antibodies and ECL detection.
Figure 7:
Membrane association of Munc13-1. A, Munc13-1 is associated with particulate material in
the presence or absence of Ca. Rat brains were
homogenized Tris-buffered saline containing 1 mM free
Ca
or 1 mM EGTA and separated into soluble
and particulate fractions by centrifugation. Equivalent amounts of the
pellet and the supernatant were analyzed by immunoblotting and compared
with the total homogenate. B, the association of
Munc13-1 with membranes is salt-resistant. Membranes were washed
with Tris-buffered salt solutions of the indicated concentrations, and
pellets and supernatants were analyzed by immunoblotting. C, Munc13-1 can be solubilized from membranes by carbonate
buffer at pH 11. Synaptic plasma membranes were washed with 0.1 M NaHCO
, 1 M NaCl, and the pellet and
supernatant were analyzed by immunoblotting with antibodies to
Munc13-1. Analysis of the same samples for intrinsic synaptic
vesicle membrane proteins showed that they remained particulate (data
not shown).
In
order to examine the nature of the association of Munc13-1 with
membranes, synaptic plasma membranes that are enriched for
Munc13-1 (see below) were treated with increasing amounts of NaCl
in the presence and absence of Ca (Fig. 7B). Even at high salt concentrations,
Munc13-1 remained membrane-bound. Some peripheral membrane
proteins that are tightly attached to membranes can be removed by
chaotropic agents or alkaline carbonate. We therefore washed the
synaptic plasma membranes with carbonate buffer at pH 11. Now the
majority of Munc13-1 was removed from the membranes, whereas
intrinsic membrane proteins remained attached (Fig. 7C and data not shown). This result demonstrates that Munc13-1
is tightly bound to membranes. Treatment of membranes from transfected
COS cells gave an identical result, suggesting that membrane binding of
Munc13-1 is an intrinsic property of the protein and not
dependent on nerve-terminal specific proteins (data not shown).
Figure 8:
Subcellular distribution of
Munc13-1. Equal amounts of subcellular fractions from rat brain
(20 µg/lane) were analyzed by immunoblotting with an
affinity-purified Munc13-1 antibody. Antibodies to synaptophysin
and the NMDA receptor subunit NMDA R1 were used as controls for
synaptic vesicle proteins and postsynaptic membrane proteins,
respectively. Subcellular fractions are designated as follows: HOM, homogenate; P, nuclear
pellet; P
, crude synaptosomal pellet; P
, light membrane pellet; S
, cytosolic fraction; LP
, lysed synaptosomal
membranes; LP
, crude synaptic vesicle
fraction; LS
, cytosolic synaptosomal
fraction; SPM, synaptic plasma
membranes.
The C. elegans unc-13 gene belongs to a subgroup of unc genes that encode proteins with putative functions in
neurotransmitter release(13, 14) . Worms with
mutations in this subgroup are characterized by elevated levels of
acetylcholine, resistance to the acetylcholinesterase inhibitor
aldicarb, normal acetylcholine agonist responses, and wild-type levels
of choline acetyltransferase and acetylcholinesterase. Several genes in
this subgoup of unc mutants have been identified, including unc-17 as the vesicular acetylcholine
transporter(18) , unc-18 as a component of the
synaptic vesicle docking-fusion
complex(15, 16, 17) , and
synaptotagmin(19) . However, the functions of many unc genes in this group of presynaptic mutants are unknown. Of these, unc-13 is remarkable because of its particularly severe
phenotype(14) . Cloning revealed that the unc-13 gene
encodes a protein with C- and C
-domains similar
to protein kinase C, suggesting that it is involved in
Ca
- and diacylglycerol-signaling(20) .
However, little is known about the properties of this protein, its
localization, its functions in the nerve terminal, and its general
presence in organisms other than C. elegans. In order to
address these questions, we have now examined the presence and
properties of unc-13 homologues in mammalian brain.
cDNA
clones encoding three rat brain proteins that are homologues to unc-13 were isolated and characterized. The high degree of
sequence homology between the rat proteins and unc-13 suggests
that the newly identified proteins are true mammalian homologues of the
nematode protein, and therefore we named them Munc13-1,
-13-2, and -13-3 (Fig. 1). RNA blotting experiments
demonstrated that all Munc13s are expressed only in brain. Similar to
other mammalian homologues of C. elegans unc genes with
neuronal functions, unc-13 is highly conserved in evolution
but expressed in multiple isoforms instead of the single form observed
in C. elegans. Munc13s are large proteins with an interesting
domain structure (Fig. 2B): an N-terminal region that
is not conserved between the different family members and is followed
by a domain doublet composed of adjacent C- and
C
-domains, a large middle segment that is highly conserved
between different Munc13s but exhibits no homologies to other proteins
in the current databanks, and a C-terminal C
-domain that is
also present in all family members. Biochemical experiments
demonstrated that Munc13-1 is a peripheral membrane protein that
is tightly bound to the plasma membrane and enriched in synaptic plasma
membranes. Considering the homology between the different family
members, it is likely that all Munc13s are plasma membrane proteins and
have similar functions. Since unc-13 probably has an essential
presynaptic function based on its phenotype, Munc13s probably also
function in neurotransmitter release, a role that would fit very well
with its localization to the presynaptic plasma membrane.
The
presence of a C-domain in Munc13s and the demonstrated
binding of phorbol esters to the C
-domain of unc-13(21, 22) suggest that these proteins are
regulated by phorbol esters and diacylglycerol. Phorbol esters have
multiple effects on synaptic transmission that have largely been
attributed to their effects on protein kinase C. Interestingly, part of
the effect of phorbol esters on synaptic transmission cannot be
inhibited by protein kinase C inhibitors(23) ,
suggesting that there may be additional phorbol ester receptors
at a synapse. It is possible that some of these effects are mediated by
Munc13s. In addition to C
-domains, the
C
-domains of Munc13s are also likely to participate in
intracellular signaling reactions. The sequence comparisons showed that
they form a subgroup of C
-domains distinct from those of
synaptotagmin and protein kinase C isoforms (Fig. 2). Binding
measurements revealed that the middle C
-domains of Munc13s
do not bind phospholipids as a function of Ca
(Fig. 3). However, this does not imply that the Munc13
C
-domains are not Ca
-binding modules
since recent experiments on C
-domains of synaptotagmins
have discovered Ca
-dependent activities that are
independent of phospholipid binding (11) . (
)
The identification of mammalian homologues of unc-13 now allows a biochemical investigation into their functions in neurotransmitter release. Based on the similarity between the phenotypes of the unc-13 and unc-18 mutants in C. elegans, it has been proposed that unc-13 may be involved in synaptic vesicle docking like unc-18(39) . Future experiments will have to determine if Munc13s are components of the active zone that function in docking and what its interacting partners are in this function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U24070[GenBank], U24071[GenBank], and U24072[GenBank].