Phosphorylation-regulated axonal dependent transport
of syntaxin 1 is mediated by a Kinesin-1 adapter
John Jia En Chuaa, Eugenia Butkevichb, Josephine M. Worseckc, Maike Kittelmannd, Mads Grønborga,
Elmar Behrmanna, Ulrich Stelzlc, Nathan J. Pavlosa, Maciej M. Lalowskie,1, Stefan Eimerd, Erich E. Wankere,
Dieter Robert Klopfensteinb,f,2, and Reinhard Jahna,2
aDepartment of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany;bGeorg-August-Universität Göttingen, Drittes
Physikalisches Institut-Biophysik, 37077 Göttingen, Germany;fBiochemistry II, Georg-August-Universität Göttingen, 37073 Göttingen, Germany;cMax-Planck-
Institute for Molecular Genetics, 14195 Berlin, Germany;dEuropean Neuroscience Institute Göttingen and German Research Foundation Research Center for
Molecular Physiology of the Brain, 37077 Göttingen, Germany; andeMax Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany
Edited by Ronald D. Vale, University of California, San Francisco, CA, and approved March 5, 2012 (received for review August 22, 2011)
Presynaptic nerve terminals are formed from preassembled vesicles
that are delivered to the prospective synapse by kinesin-mediated
axonal transport. However, precisely how the various cargoes are
linked to the motor proteins remains unclear. Here, we report
a transport complex linking syntaxin 1a (Stx) and Munc18, two pro-
teins functioning in synaptic vesicle exocytosis at the presynaptic
plasma membrane, to the motor protein Kinesin-1 via the kinesin
adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabdi-
that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phos-
phorylation, with a conserved site (serine 58) being essential for bind-
deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude
that FEZ1 operates as a kinesin adaptor for the transport of Stx, with
cargo loading and unloading being regulated by protein kinases.
fasciculation and elongation protein zeta 1|transport defect|
intricate but highly efficient processes during which the ma-
chinery for exocytosis and recycling of synaptic vesicles is as-
sembled from preformed units. These units are delivered to the
nascent synapse via molecular motor proteins of the kinesin
superfamily (reviewed in Ref. 1).
Although Kinesin-3 appears to be the main motor transporting
synaptic vesicle precursors, recent evidence suggests that Kinesin-
1 (KIF5) is also involved. In Drosophila, deletion of UNC-76/
fasciculation and elongation protein zeta 1 (FEZ1), a specific
adaptor for Kinesin-1, left synaptic vesicles stranded in the axon
(2, 3), showing that Kinesin-1 is needed at least during later
phases of axonal transport. Transport of the synaptic vesicle
protein synaptotagmin by the UNC-76/Kinesin-1 complex re-
quires phosphorylation of UNC-76 by the UNC-51/ATG1 ki-
nase, a prerequisite for UNC-76 to bind synaptotagmin (3). Dele-
tion of this kinase phenocopies deletion of UNC-76. Indeed,
phosphorylation-regulated interactions between cargo, adaptors,
and kinesins have also been observed for other transport com-
plexes such as the kinesin light chain/JIP1 (c-Jun N-terminal ki-
nase-interacting protein 1) complex (4). This suggests that
phosphorylation is a common mechanism for the regulation of
kinesin-based transport complexes (5).
of otherclasses of synapticprecursorvesicles. Transport ofsyntaxin
residing in the presynaptic plasma membrane, is clearly distinct
from synaptic vesicle precursors and appears to involve a complex
between Kinesin-1 and the Stx-binding protein syntabulin (6, 7).
Down-regulation or expression of dominant-negative syntabulin
reduces but does not abolish membrane delivery of Stx, indicating
the existence of other transport mechanisms (6). Moreover, proper
intracellulartraffickingof Stx andits functioninexocytosisdepends
on Munc18 coexpression (8–14). Stx trafficking defects were ob-
served in unc-18 knockouts in Caenorhabditis elegans (14), Munc18
knockdowns in PC12 cells (9, 13) but not in mouse Munc18-1
he formation and maintenance of presynaptic boutons are
Munc18 isoforms cannot be excluded. These defects were attrib-
uted to a need for Stx to be stabilized by Munc18 in the inactive
conformation during transport to prevent it from being trapped in
nonproductive SNARE complexes (10) but Munc18 could addi-
tionally participate in loading Stx onto kinesin. Here, we identify
FEZ1, and the Kinesin-1 family member KIF5C.
FEZ1 Interacts with Stx and Munc18. We recently initiated an effort
to systematically identify interaction partners of established
presynaptic proteins using an automated yeast two-hybrid (Y2H)
screen. Bait proteins corresponding to defined regions of these
proteins were tested against an arrayed matrix containing human
full-length ORF prey constructs. As part of the data stemming
from this screen, we discovered that the Kinesin-1 adaptor FEZ1
binds both to Stx and Munc18 (Fig. 1A). These results suggested
an alternative, Munc18-dependent, transport route for plasma
membrane delivery of Stx in addition to the reported syntabulin-
To validate the interactions, lysates of HEK 293 cells transiently
expressing GFP-FEZ1 and Myc-Stx, or FLAG-Munc18 constructs
were immunoprecipitated with a GFP antibody and analyzed by
immunoblotting. FEZ1 readily precipitated both Stx (Fig. 1B) and
Munc18 (Fig. 1C). FEZ1 also precipitated Munc18 in reciprocal
coimmunoprecipitations (Fig. S1). These results validate the Y2H
identified interactions and show that FEZ1 specifically interacts
with both Munc18 and Stx in mammalian cells.
The Y2H screening revealed that the N terminus of FEZ1
mediates interaction with neither presynaptic protein as a FEZ1
prey containing amino acids 8–91 tested did not yield viable
colonies (Fig. 1A). Additionally, FEZ1 binds amino acids 246–
449 of Munc18. To determine which FEZ1 region binds Stx and
Munc18, GFP-FEZ1 deletion mutants (Fig. S2A) were tested in
coimmunoprecipitations as described above. Munc18 coprecipi-
tated with both full-length FEZ1 and its N-terminal fragment
(amino acids 1–310) (Fig. S2B). A further deletion of amino
acids 221–310 of FEZ1 abolished Munc18 binding, indicating
that the region is primarily responsible for this interaction. The
region contains a coiled-coil domain reported to bind other
proteins and is evolutionarily conserved (16). An even shorter
Author contributions: J.J.E.C., S.E., E.E.W., D.R.K., and R.J. designed research; J.J.E.C.,
E. Butkevich, J.M.W., M.K., M.G., and E. Behrmann performed research; M.M.L. contrib-
uted new reagents/analytic tools; J.J.E.C., E. Butkevich, J.M.W., M.K., M.G., U.S., N.J.P., and
D.R.K. analyzed data; and J.J.E.C. and R.J. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1Present address: Protein Chemistry/Proteomics/Peptide Synthesis and Array Unit, Biome-
dicum Helsinki, University of Helsinki, FI-00014 Helsinki, Finland.
2To whom correspondence may be addressed. E-mail: email@example.com or rjahn@gwdg.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| April 10, 2012
| vol. 109
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FEZ1 peptide (amino acids 131–210) also did not bind Munc18.
Additional mapping of the Munc18 binding domain of FEZ1
with peptides containing only its coiled-coil domain or additional
flanking segments (Fig. S2A) confirmed that the coiled-coil do-
main suffices to bind Munc18 (Fig. S2B). However, the in-
teraction is considerably weaker compared with the N-terminal
fragment (amino acids 1–310). Because the N-terminal domain
of FEZ1 mediates its dimerization (17), we speculate that FEZ1
dimerization enhances its binding to Munc18.
Likewise, binding of Stx to FEZ1 progressively decreased
when the coiled-coil domain was removed (Fig. S2C; compare
amino acids 1–310 with amino acids 1–220). Only background
binding was observed when amino acids 131–210 of FEZ1 were
used to coimmunoprecipitate Stx (see also Fig. 2).
FEZ1 Forms a Transport Complex with Stx, Munc18, and Kinesin-1.
The Sec1/Munc18 protein binds to Stx (12). Thus, the three pro-
teins may form a tripartite complex in vivo. Indeed, immunopre-
cipitation of FEZ1 in HEK 293 cells expressing all three proteins
efficiently pulls down both Munc18 and Stx (Fig. 2A). Deletion of
the coiled-coil domain of FEZ1 responsible for binding to Munc18
does notabolish formation ofthis complex,suggesting that, at least
under these conditions, binding of Munc18 to FEZ1 is indirect,
being mediated by binding to Stx. This finding also excludes the
possibility that coprecipitation of both Munc18 and Stx is attrib-
utable to the isolation of two distinct FEZ1 binary complexes (i.e.,
FEZ1-Munc18 versus FEZ1-Stx complexes).
FEZ1 binds Kinesin-1 and is involved in synaptic vesicle
transport (2, 3, 18). We wondered whether FEZ1 might also
cation of FEZ1 as an interactor of Munc18 and Stx using
a Y2H assay. Bait constructs containing fragments of
Munc18 and Stx (without its transmembrane domain) were
used to screen preys containing different FEZ1 fragments
[amino acid residues (aa) given in parentheses]. (B and C)
Validation of FEZ1 interactions by coimmunoprecipitation.
HEK 293 cells expressing tagged FEZ1 with either Stx or
Munc18 (black circles on top of the lanes) were immuno-
precipitated (IP) using tag-specific antibodies (anti-GFP)
and analyzed by immunoblotting (IB) for the presence of
GFP-FEZ1, Myc-Stx, and FLAG-Munc18, respectively. FEZ1
interacts with both Munc18 and Stx. Input corresponds to
1% of the starting material used for immunoprecipitation.
Molecular mass markers indicated are in kilodaltons.
FEZ1 interacts with Munc18 and Stx. (A) Identifi-
versions of FEZ1, Munc18, KIF5C, or Stx were immunoprecipitated (IP) using anti-GFP antibodies and immunoblotted (IB) using tag-specific antibodies (anti-
FLAG, anti-GFP, anti-Myc, or anti-V5). Molecular mass markers are indicated are in kilodaltons. *Ig heavy chain. (A) FEZ1 forms a trimeric complex with Munc18
and Stx. Full-length and FEZ1 (amino acids 1–220) efficiently immunoprecipitate Stx and Munc18 indicating that direct binding of Munc18 to FEZ1 is not
necessary for trimeric complex formation. (B) KIF5C, FEZ1, Stx, and Munc18 can be concurrently isolated as a complex. In addition, binding of Stx to KIF5C can
occur without Munc18 but is dependent on the presence of the coiled-coil domain of FEZ1. (C) The FEZ1/Kinesin-1 transport complex comprising FEZ1, Stx,
Munc18, and Kinesin-1 can be immunoisolated from rat brain postnuclear supernatants using anti-FEZ1 or anti-Kinesin-1 antibodies.
Protein complexes formed by FEZ1 reveal its function as cargo adaptor for Kinesin-1 (KIF5C). (A and B) HEK 293 cells expressing combinations of tagged
Chua et al.PNAS
| April 10, 2012
| vol. 109
| no. 15
similarly transport Stx and/or Munc18. We first investigated how
these proteins interact with Kinesin-1. Binding of Stx to Kinesin-
1 is dependent on syntabulin, another motor adapter protein (6).
Immunoprecipitation of Munc18 from cell lysates after cotrans-
fection of Munc18 with Kinesin-1 (KIF5C) revealed both pro-
teins do not directly interact (Fig. S3A). As reported, FEZ1
coprecipitates with Kinesin-1 (KIF5C) (Fig. S3B, lane 1) (18). In
triple transfections with FEZ1, KIF5C, and either Munc18 or
Stx, immunoprecipitation of FEZ1 resulted in efficient copreci-
pitation of either set of proteins, demonstrating that FEZ1 in-
deed functions as a kinesin adaptor for Munc18 and Stx (Fig. 2B,
right lane, and Fig. S3B).
Importantly, immunoprecipitation of FEZ1 in quadruple-
transfected cells with all four proteins resulted in the isolation of
the Munc18-Stx-FEZ1-KIF5C quaternary complex (Fig. 2B, lane
3). As a further control, we replaced full-length FEZ1 with the
truncated version (amino acids 1–220) that is unable to bind
Kinesin-1 and Munc18. Here, FEZ1 (amino acids 1–220), Stx,
and Munc18 can still be concomitantly isolated, but, as expected,
KIF5C does not coprecipitate (Fig. 2B, lane 2).
To confirm that FEZ1 actually binds Kinesin-1 to Stx and
Munc18 in neurons, we prepared postnuclear supernatants from
the rat brain and then immunoisolated FEZ1-containing cargo-
motor complexes using anti-FEZ1 antibody-coupled magnetic
microbeads. Kinesin-1, Munc18, and Stx were readily detected in
immunoisolated FEZ1 complexes (Fig. 2C). Reciprocal coim-
munoprecipitation using anti-Kinesin-1 antibody also coprecipi-
tated FEZ1, Munc18, and Stx. In summary, the experiments here
support the existence of a FEZ1/Kinesin-1 transport complex
containing Stx and Munc18 in vivo, with FEZ1 serving as a cen-
tral scaffold to connect cargo and motor using overlapping yet
distinct binding domains.
Mutation of UNC-76 in C. elegans Impairs Axonal Transport of Stx.
During axonal outgrowth, Stx is not transported together with
not beencharacterizedtodate.Our results indicate thatFEZ1may
serve as a Kinesin-1 motor adaptor for Stx and Munc18. In view of
the role of FEZ1 in neuritogenesis and microtubule-based trans-
port (21), we hypothesized that FEZ1-dependent transport of both
proteins may already function during early axonogenesis. Indeed,
FEZ1 is present and localizes well with α-tubulin in neuronal
growth cones of young neurons (Fig. 3A). As previously reported,
Stx and Munc18 are also present in growth cones (22, 23), where
they extensively colocalize as expected from their tight association
with FEZ1, indicating they are likely to be transported by FEZ1 in
young neurons (Fig. 3 D–F).
Although the data indicate these proteins association in neu-
rons, the approach is unable to determine whether FEZ1/Kine-
sin-1 is responsible for their transport in vivo. We, therefore,
tested whether the distributions of Stx and Munc18 in axons are
affected if FEZ1 is absent. Because all proteins under in-
vestigation are highly conserved evolutionarily, these experi-
ments were conducted in C. elegans, an organism that has been
extensively used to study axonal transport (24). Mutations in
each of the four proteins are known to result in an uncoordinated
phenotype typical for most defects in synaptic transmission (25–
29), with the orthologs being UNC-18 (Munc18), UNC-76
(FEZ1), UNC-64 (Stx), and UNC-116 (Kinesin-1).
Coimmunoprecipitation of tagged C. elegans variants expressed
from HEK 293 cells confirmed that interactions between FEZ1,
Stx and Munc18 are conserved in worms (Fig. S5). In transgenic
worm strains expressing GFP-UNC-64 or GFP-UNC-18, both
proteins show diffuse cytoplasmic distribution in processes of
ventral nerve cord (VNC) neurons (Fig. 4A, a and f). If UNC-76
is involved in anterograde trafficking of either protein, lack of
UNC-76 should leave them stranded in the cell body and/or the
axons. Indeed, in unc-76 mutants, the distribution of GFP-UNC-
64 was more irregular than in wild-type controls, with clusters
becoming clearly visible in axons and sometimes also observable
within cell bodies (Fig. 4A, a vs. b; also Fig. S6A). Quantification
by quantitative image analyses confirmed significantly greater
clustering of GFP-UNC-64 in ventral cord neurons of unc-76
mutant animals (Fig. 4B). This phenotype is similar in appear-
ance to axonal aggregates of synaptic vesicles seen in Drosophila
lacking FEZ1 or Kinesin-1, which was attributed to defects in
axonal transport following loss of either protein (2, 3). Impor-
tantly, GFP-UNC-64 distribution anomalies were completely
rescued by pan-neuronal expression of wild-type UNC-76 in these
mutants (Fig. 4A, d, and 4B). This result demonstrates that loss of
UNC-76/FEZ1 in neurons specifically cause formation of GFP-
UNC-64 aggregates. In contrast, no major changes were seen in
the distribution of GFP-UNC-18 in neither unc-76 nor unc-116
mutants (Fig. 4A, f–h, and 4B; also Fig. S6B). Nevertheless, it is
plausible that the soluble pool of UNC-18 occludes changes of
UNC-18 associated with the Stx transport vesicles.
Deletion of UNC-116 also phenocopied the transport defect
of Stx-containing vesicles in unc-76 mutants (Fig. 4A, c, and
Fig. S6A). Quantification of clustering indicates that unc-116
mutants exhibited an even more pronounced phenotype (Fig. 4B)
cones. Two to three DIV neurons were fixed and stained for endogenous
FEZ1, Stx, Munc18, or α-tubulin. (A) Numerous FEZ1 puncta are observed in
neuronal growth cones that are strongly microtubule-associated (e.g.,
arrowheads). (B) Stx and Munc18 colocalizes in growth cones as expected.
FEZ1 colocalizes with Stx (D) and Munc18 (E) in growth cones. Line scans of
regions of interest indicated are shown in C and F. (Scale bars, 10 μm.) Cor-
relation coefficients for each colocalization pair are 0.88 ± 0.02 (Munc18+
Stx), 0.802 ± 0.02 (FEZ1+Stx), and 0.75 ± 0.02 (FEZ1+Munc18), respectively.
Eight growth cones were taken for each set of analysis. Images of the entire
growth cones are shown in Fig. S4.
FEZ1 colocalizes with Stx, Munc18, and α-tubulin in neuronal growth
| www.pnas.org/cgi/doi/10.1073/pnas.1113819109Chua et al.
compared to unc-76 mutants, as would be expected if the motor
function itself was directly disrupted. Importantly, unc-76;unc-
116 double mutants exhibit the strongest transport defect with
significant amounts of GFP-UNC-64 being retained as large
accumulations in cell bodies in addition to the aforementioned
axonal aggregates (Fig. 4A, e). Indeed, the percentage of such
cells is highest in double mutants (47.06%, n = 119) compared
with unc-116 (33.93%, n = 56) or unc-76 (16.51%, n = 109)
mutants or wild-type background (3.70%, n = 108).
Finally, using electron microscopy, we indeed observed accu-
mulation of abnormal membranous structures in axonal pro-
cesses of ventral cord neurons in both unc mutants (Fig. 4C).
Whereas wild-type axons are normally devoid of vesicular clus-
ters, unc-76 and unc-116 mutant axons contain clusters of ve-
sicular structures including synaptic and dense core vesicles,
multivesicular bodies, and autophagosomes (Fig. 4C). These
clusters are more pronounced in unc-116 mutants compared with
unc-76 mutants. The appearance of autophagosomes surround-
ing aberrant vesicle clusters in the mutants might represent an
attempt to clear these aggregates from the axons.
Together, these results demonstrate that UNC-76 functions as
an adaptor that links UNC-64 to UNC-116 in anterograde axonal
trafficking. However, axons still form in both unc-76 and unc-116
mutants, with some Stx being present in the axonal membrane,
suggesting that the UNC-76/Kinesin-1 transport complex is not
the only cargo-motor complex capable of delivering Stx to the
axonal plasma membrane (6, 7).
Phosphorylation Regulates FEZ1-Mediated Interactions. In Drosoph-
to synaptotagmin and controls loading of kinesin with synaptic
vesicles (3). Using liquid chromatography-tandem mass spectrom-
etry (LC-MS/MS), we identified four phospho-serine sites (S58,
S134, S301, and S316) from GFP-FEZ1 immunoprecipitated from
in vivo. Importantly, S58 corresponds to S143 in Drosophila UNC-
76 and is flanked by highly conserved amino acids (Fig. S7B).
To determine whether FEZ1 phosphorylation affects its bind-
ing to Munc18, Stx, and KIF5C, we treated lysates of HEK 293
cells expressing various combinations of the four proteins with
alkaline phosphatase (AP) and then tested by immunoprecipita-
tion if any of the interaction was affected by the treatment.
Phosphatase treatment virtually abolished interaction of FEZ1
with Munc18 and KIF5C (Fig. 5 A and B). In contrast, binding to
ventral nerve cords of C. elegans. (A) Axonal distribution of GFP-tagged UNC-
18 and UNC-64 in ventral nerve cords (VNC) in wild-type and mutant C. ele-
gans. (a–c) Distribution of GFP-UNC-64 differs between wild-type (a), unc-76
(e911) mutants (b), and unc-116 (e2310) mutants (c). (c) Unc-116 mutants ex-
hibit greater axonal clustering of GFP-UNC-64 than unc-76 mutants (b). Ex-
pression of mCherry-UNC-76 rescues GFP-UNC-64 clustering in unc-76 mutants
(d). Unc-76;unc-116 double mutants exhibit both axonal clustering and sig-
nificantly more GFP-UNC-64 accumulation in cell bodies (e). Expression pat-
terns of GFP-UNC-18 in wild-type (f), unc-76 (g), and unc-116 (h) mutants show
no significant differences. (Scale bar, 10 μm.) Diagram in i shows the schematic
organization of C. elegans ventral nerve chord (VNC) as a continuous row of
neuronal cell bodies (somata) with axonal bundles running adjacent to the
ventral hypodermis. En passant synapses between motor neurons and muscle
arms are regularly spaced along the entire length of the VNC. (B) Quantifi-
cation of GFP-UNC-64 and GFP-UNC-18 clustering using intensity variations
along the nerve (line scan analyses). SDs of pixel intensities obtained from the
line scans were used to compute an index to compare the extent of clustering
(SI Text). Eight to nine worms were taken for each analysis. Error bars repre-
sent SEM. (C) Abnormal membranous structures and autophagosomes
(arrows) were found within neurites of the ventral cord neurons in unc-116
and unc-76 mutants but not in wild-type animals. (Scale bar, 200 nm.)
Mutation of FEZ1 (unc-76) affects trafficking of syntaxin (UNC-64) in
KIF5C, or Stx treated with or without AP were immunoprecipitated using tag-
specific antibody (anti-GFP) and immunoblotted using tag-specific antibodies
(anti-FLAG, anti-GFP, anti-Myc, or anti-V5). The amount of immunoprecipitated
protein was determined by densitometry, with the untreated sample serving as
obtained from three independent experiments. Error bars represent SDs. (A)
Treatment of cell lysates with AP significantly reduces Munc18 binding to FEZ1.
(B) Phosphorylation regulates assembly of the trimeric FEZ1-Munc18-Kinesin-1
dissociates the ternary complex of FEZ1, Munc18, and KIF5C (right chart). (C)
Dephosphorylation reduces the amount of KIF5C bound to FEZ1 but significant
amounts of Stx remain bound to FEZ1. (D) Significant amounts of FEZ1-Stx-
Munc18 complexes remain isolatable even after AP treatment. HEK 293 cell
lysates expressing tagged versions of Munc18, Stx, and FEZ1 treated with or
without AP were immunoprecipitated with tag-specific (anti-FLAG) antibody
and immunoblotted using tag-specific antibodies (anti-FLAG, anti-Myc, or anti-
GFP). Data were obtained from the average of two independent experiments.
Phosphorylation of FEZ1 regulates binding to its interaction partners.
Chua et al.PNAS
| April 10, 2012
| vol. 109
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Stx was only marginally affected, whereas kinesin binding was
abolished regardless of the presence of Stx or Munc18 (Fig. 5 B
and C). Intriguingly, Munc18 binding was preserved in the pres-
ence of Stx, again demonstrating that Munc18 can bind to the
trimeric complexes via its interaction with Stx (Fig. 5D).
To determine which of the phospho-serines is involved in
regulating FEZ1 binding to Munc18 and Kinesin-1, we gener-
ated a series of FEZ1 point mutations in which one or several
of the four identified serines were mutated to alanines. The
mutants were then tested in an Y2H assay against KIF5A,
KIF5C, and Munc18. Significantly, mutation of the conserved
S58 alone specifically abolished its interaction with Munc18
(Table S1). Furthermore, mutation of any other site also dis-
rupted interaction with Kinesin-1. This contrasts with previous
observations in Drosophila where binding of UNC-76 to Kinesin-
1 was not influenced by the phosphorylation status of S143 (3).
Finally, we tested whether S58 is critical for FEZ1/Kinesin-1
transport of Stx in neurons. In these experiments, we took ad-
vantage of the fact that human FEZ1 rescues the unc-76 un-
coordinated phenotype (30) and expressed the human protein in
unc-76 mutant strains of C. elegans. As expected, wild-type FEZ1
rescued UNC-64 transport defects observed in unc-76 mutants
(Fig. 6). In contrast, expression of FEZ1 S58A did not ameliorate
UNC-64 clustering in these mutants. In fact, quantitative image
analysis revealed that the clustering phenotype is even more pro-
nounced than observed in unc-76 mutants. These findings confirm
that phosphorylation of FEZ1 at S58 is essential for its function as
a cargo adaptor for Kinesin-1-dependent axonal transport of Stx.
The trafficking routes involved in biogenesis, maintenance, and
turnover of the axonal and presynaptic plasma membrane are
largely unclear (1, 31). In contrast to the relatively well-charac-
terized synaptic vesicle precursors, the nature and composition
of vesicles carrying proteins such as Stx, presynaptic potassium,
and calcium channels is unknown. It is also unclear whether all of
these proteins are transported along the axon by a single class of
vesicles derived from the Golgi complex as “constitutive” se-
cretory vesicles, or whether several vesicle populations coexist
that are differentially regulated. Stx is one of the most abundant
neuronal membrane proteins that is concentrated in synapses but
that is also widely distributed along the axonal plasma membrane
(32, 33). Stx has been detected both in purified synaptic vesicles
(34) (albeit is less abundant than in the plasma membrane) and
in Piccolo-Bassoon transport vesicles (PTVs) (35), as well as in
amyloid-precursor protein (APP)-containing transport vesicles
transported by Kinesin-1 (36), but it is unlikely that the bulk of
Stx is transported by any of these routes.
Our data show that transport of Stx along axons is mediated in
part by KIF5C via its specific adaptor FEZ1. Strong colocalization
of Stx, Munc18, and FEZ1 in growth cones from young neurons
and data from transgenic worms indicate that transport via FEZ1/
Kinesin-1 transport complexes play an important role in bringing
Stx and Munc18 to the tips of rapidly developing axons during
neuritogenesis. Thus, we identified a second transport complex
for Stx distinct from, but that may act in conjunction with, the
previously identified adaptor syntabulin that mediates Stx trans-
port via another member of the Kinesin-1 family (KIF5B) (6, 7).
It will be interesting to clarify whether FEZ1 and syntabulin can
functionally substitute for each other in the transport of Stx-
containing vesicles and whether their cargo spectrum is similar.
Importantly, Munc18 also resides in the same FEZ1/Kinesin-1
transport complex. This observation is significant given previous
reports that correct plasma membrane trafficking of Stx strongly
depends on the presence of Munc18 (8–13, 22). Noteworthy, Stx
is able to bind FEZ1 in the absence of Munc18. Thus, the motor
complex is, by itself, unable to correctly distinguish processed Stx
(bound by Munc18) from improperly processed (“free”) Stx. Our
findings, thus, support the current view that Munc18 serves to
protect Stx from spurious interactions during Golgi maturation
and provides additional evidence for the involvement of Munc18
in post-Golgi transport of Stx. In the latter role, it is conceivable
that the Munc18-FEZ1 interaction serves to signal to the motor
that a properly processed cargo has been loaded. The observa-
tion that the binding of Munc18 to FEZ1 is also phosphoryla-
tion-dependent offers a tantalizing possibility that such events
may mediate loading/unloading of Stx trafficking vesicles to
KIF5C as has been reported for other Kinesin adapters (4).
Phosphorylation of UNC-76 by UNC-51 affects the binding of
UNC-76 to synaptotagmin and is essential for its axonal transport
(3). Flies lacking UNC-51 or expressing a phosphorylation-de-
fective UNC-76 mutant exhibit axonal transport defects resulting
from loss of cargo binding to UNC-76. Like its Drosophila
counterpart, alanine substitution of FEZ1 at the conserved S58
created a phosphorylation-defective mutant that failed to trans-
port UNC-64 along neuronal axons. However, unlike UNC-76,
this defect cannot be directly attributed to the loss of cargo
binding because Stx remains attached to FEZ1. Instead, lack of
phosphorylation at S58 abrogates binding of Munc18 and Kine-
sin-1. Importantly, we observe that binding of FEZ1 to Kinesin-1
is strictly dependent on phosphorylation at multiple sites (in-
cluding S58) unlike UNC-76. Conceivably, loss of phosphory-
lation of FEZ1 detaches the adapter-cargo complexes from the
motor, leading to their accumulation within the axon.
Our findings can be integrated into a model where FEZ1
serves to connect Stx-containing transport vesicles to Kinesin-1
to deliver them to the presynapse (Fig. S8). Accordingly, FEZ1 is
phosphorylated in the cell body, which activates the protein and
promotes binding to both Kinesin-1 and Munc18. Based on our
data, Stx has two options for binding: one by directly interacting
with FEZ1 in a phosphorylation-independent manner, and the
other via binding to Munc18 in an inactive (closed) conforma-
tion. Conversely, Munc18 can also bind to FEZ1 via Stx in
a phosphorylation-independent manner. Although it is currently
not possible to discern between these alternatives, the fact that
Munc18 is needed for axonal transport of Stx favors the view that
Munc18 binding to FEZ1 constitutes the primary interaction.
After reaching presynaptic sites, activation of phosphatases is
expected to dissociate the cargo from the motor protein. Again,
it remains to be established whether the Stx cargo vesicles are
transport of Stx (UNC-64) in axons. Localization of GFP-
UNC-64 in VNC of unc-76(e911) mutants coexpressing
mCherry-FEZ1 (wild-type) or mCherry-FEZ1(S58A). Irregular
distribution of GFP-UNC-64 in unc-76 mutants was rescued
by expression of wild-type FEZ1, whereas expression of
FEZ1(S58A) aggravated the clustering phenotype of GFP-
UNC-64 in these worms. Quantifications of GFP-UNC-64
clustering for both transgenic worms are shown (Right).
Eight worms were taken for each analysis. Error bars rep-
resent SEM. (Scale bar, 10 μm.)
S58A is essential for phosphorylation-dependent
| www.pnas.org/cgi/doi/10.1073/pnas.1113819109 Chua et al.
also dissociated from FEZ1 or whether the phosphorylation-in- Download full-text
dependent binding of Stx may serve to retain some FEZ1 bound
to Stx after reaching the final destination in the synaptic/axonal
Perturbations of microtubule-based transport are implicated in
an increasing number of neurodegenerative diseases (NDs) (37).
FEZ1 not only transports Stx but also synaptic vesicles (3) and
mitochondria (38). Importantly, FEZ1 also interacts with proteins
and huntingtin (40). We hope that further characterization of
FEZ1’s role in axonal transport will provide additional insight into
how transport disorders contribute to the progression of NDs.
Materials and Methods
Plasmids and Antibodies. All plasmids used in this study were generated by
standard cloning. Descriptions of procedures and antibodies used are de-
tailed in SI Materials and Methods.
Automated Y2H Screening. Automated Y2H screening was carried out as
published previously with minor modifications (41). Details concerning the
screen are provided in SI Materials and Methods.
Coimmunoprecipitations. One day after transfection, cells were lysed with ice-
cold HNE buffer [50 mM Hepes (pH 7.2), 150 mM NaCl, 1% (vol/vol) Triton X-
100, 1 mM EDTA] containing Complete EDTA-free protease inhibitor mixture
(Roche). Cell lysates were cleared by centrifugation, and the resultant
supernatant was incubated with anti-FLAG, anti-V5, or anti-GFP antibodies
for 3 h. Thirty microliters of protein G-Sepharose were subsequently added to
the mixture and incubated continued for an additional hour. Immunopre-
cipitates were washed 4× with HNE buffer. Proteins were eluted with 2× LDS
buffer and analyzed by immunoblotting.
C. elegans Strains. Generation of constructs and worm culturing were per-
formed according to standard procedures. Details are provided in SI Materials
Immunocytochemistry. Primary hippocampal neurons were fixed with 3.6%
(wt/vol) paraformaldehyde at 2–3 d in vitro (DIV). Neurons were perme-
abilized with 0.3% Triton-X100 in PBS (pH 7.3) and blocked with 10% normal
goat serum diluted in PBS. Coverslips were incubated with primary anti-
bodies for 1 h. After washing, cells were incubated with Cy2- or Cy3-conju-
gated goat anti-rabbit and donkey anti-mouse antibodies, respectively
(Jackson ImmunoResearch). Images were acquired using a Leica SP2 confocal
ACKNOWLEDGMENTS. We thank Maria Druminski, Ina Maria Herfort, Kirstin
Rau, and Jan Timm for excellent technical assistance. This work received
funding from European Union Sixth and Seventh Framework Programme
Grants LSHM-CT-2005-019055 (“EUSynapse”) and HEALTH-F2-2009-241498
(European Study Programme in Neuroinformatics “EuroSPIN”) and from Na-
tional Genome Research Network (NGFN) Grants NGFNp NeuroNet-TP1/TP3,
01GS08170, and 01GS08171. M.G. was supported by a research fellowship
from The Alfred Benzon Foundation.
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Chua et al. PNAS
| April 10, 2012
| vol. 109
| no. 15