1 Department of Neuroscience (D13), 2-2 Yamadaoka, Suita, Osaka 565-0871,
Japan
2 Ophthalmology (E7) Osaka University Graduate School of Medicine, 2-2
Yamadaoka, Suita, Osaka 565-0871, Japan
3 Department of Pharmacology, Yamaguchi University School of Medicine, 1-1-1
Minamikogushi, Ube, Yamaguchi 755-8505, Japan
4 Department of Neuroplasticity, Research Center on Aging and Adaptation,
Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621,
Japan
5 Department of Neuroanatomy, Iwate Medical University School of Medicine, 19-1
Uchimaru, Morioka 020-8505, Japan
* Author for correspondence (e-mail: sobue{at}nbiochem.med.osaka-u.ac.jp)
Accepted 28 August 2002
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Summary |
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Key words: Postsynaptic density, PSD, Dendritic raft, Microvilli, Hippocampus, Myristoylation, Leucine-zipper
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Introduction |
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Rafts, which are enriched in glycosphingolipids and cholesterol, are
discrete membrane microdomains that form in the lipid bilayer
(Simons and Ikonen, 1997;
Resh, 1999
). Specific classes
of membrane proteins (e.g. GPI-linked proteins), signaling molecules (e.g. src
family kinases and G protein subunits), cytoskeletal proteins (e.g. ERM
proteins), scaffold proteins (e.g. GAP-43 and MARCKS), and endocytotic
machinery-related proteins (e.g. caveolins) are anchored to lipid rafts and
form insoluble complexes after lysis in cold detergents such as Triton X-100.
Several proteins in rafts contain combinations of N-terminal myristoylated and
adjacent palmitoylated sites. Some proteins are singly myristoylated and
possess an adjacent or distant polybasic cluster. Accumulating evidence
suggests that the most apparent roles of rafts are in membrane trafficking,
signal transduction and cytoskeletal connections
(Ikonen, 2001
). However, rafts
in neurons have not been well documented, with a few exceptions. Suzuki et al.
isolated dendritic rafts from the rat forebrain and provided evidence for the
localization of AMPA receptor subunits to the dendritic raft
(Suzuki et al., 2001
). Amyloid
ß peptides (Aß), ß-and
-secretase-cleaved products of
amyloid precursor protein (APP) and presenilins are potent pathogens for
Alzheimer's disease. Lee et al. detected Aß, APP and presenilin-1 in the
brain raft fraction and suggested the occurrence of APP processing during
membrane transport (Lee et al.,
1998
). Recently, parkin, a Parkinson's disease-related protein,
was also localized to the brain raft fraction
(Fallon et al., 2002
).
Here, using one of our PSD mAbs, we identified and performed the molecular
cloning of a 70 kDa protein that is concentrated in PSDs and is highly
homologous to the human FEZ1/LZTS1 gene product
(Ishii et al., 1999). We found
that this protein contains an N-terminal myristoylation consensus sequence and
a distant polybasic cluster in the N-terminal domain in addition to four
leucine-zipper motifs in the C-terminal domain. Furthermore, the expression of
this protein is exclusively dominant in the neurons of some cerebral areas,
including the cerebral cortex, hippocampus, olfactory bulb, striatum, and pons
and is further localized to the PSD and the dendritic rafts. Experiments in
cultured hippocampal neurons to test how this protein is targeted to the
membrane and synapse revealed that the N-terminal myristoylation and the
polybasic cluster of the N-terminal domain are required for the membrane
localization and that the C-terminal domain is involved in the synaptic
targeting. Because this PSD-enriched 70 kDa protein is likely to function
primarily in neurons, and especially in the postsynaptic sites, we refer to
this protein as PSD-Zip70 (Tachibana et
al., 1999
).
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Materials and Methods |
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cDNA cloning and plasmid construction
A cDNA library was constructed from the cerebrum of 7-week-old
Sprague-Dawley (SD) rats using a ZAP ExpressTM cDNA synthesis kit
(Stratagene). The cDNA library was immunoscreened with mAb204H, and two
positive clones were isolated. The plasmids thus obtained were prepared by the
in vivo excision method and sequenced. The wild-type PSD-Zip70 (PSD-Zip70WT)
and its N-terminus (PSD-Zip70N, amino-acid residues 1-255) and C-terminus
(PSD-Zip70C, amino-acid residues 246-601) were amplified by polymerase chain
reaction (PCR) using Pfu polymerase (Stratagene) and subcloned into myc-tagged
pcDNA3.1/Zeo(+) (modified from Invitrogen). The C-terminus of PSD-Zip70WT and
PSD-Zip70N was tagged, as was the N-terminus of PSD-Zip70C. A point mutation
of glycine to alanine (PSD-Zip70N/G2A), and of lysine and arginine to
asparagine (R26N, K27N, K32N, K33N; PSD-Zip70Nmut) and a deletion mutant
lacking amino-acid residues 21-40 (PSD-Zip70N21-40) were made by
site-directed mutagenesis. For the production of GST-fusion protein, the cDNAs
encoding PSD-Zip70WT, GST-Zip70C, and PSD-Zip70N were subcloned into pGEX6P1
(Amersham Pharmacia Biotech) by the same method. The sequences of all the
constructs were confirmed by DNA sequence analysis.
Analysis of myristoylation
Transiently transfected COS7 cells were preincubated for 30 minutes with
cerulenin (2 µg/ml; SIGMA) in DMEM containing fatty-acid-free bovine serum
albumin (10 mg/ml; SIGMA). Cells were then labeled with 500 µCi of
[3H]myristic acid (40-60 Ci/mmol; Amersham Pharmacia Biotech) for 5
hours in the same medium. [3H]Myristic acid, which is supplied in
ethanol solution, was concentrated under N2 so that the final
concentration of ethanol in the labeling medium was 1%.
For immunoprecipitation, [3H]-labeled cells were scraped and lysed in IP buffer (20 mM Tris, pH 7.4, 1 mM EDTA, 150 mM NaCl, 1% Triton X100, 200 µM phenylmethylsulfonyl fluoride [PMSF], 10 µg/ml pepstatin, 10 µg/ml leupeptin and 10 µg/ml aprotinin) with a 26-G needle. The samples were then spun at 17,000 g for 10 min at 4°C. The supernatants were precleaned with 30 µl of protein A-Sepharose suspension (1:1 in IP buffer) and then incubated with 2 µg of polyclonal anti-myc antibody pre-coupled with protein-A-Sepharose for 3 hours at 4°C. After incubation, the Sepharose gels were pelleted, washed three times with TNE buffer and boiled with SDS-PAGE sample buffer. In brief, one half of each sample was loaded on a gel, separated by SDS-PAGE and transferred to a nitrocellulose membrane. The radioactivity was analyzed using a FUJIFIX Bioimage Analyzer (BAS5000). For the quantification of precipitated PSD-Zip70, the other half of each sample was analyzed by Western blotting as described below. The anti-myc pAb (Santa Cruz) was used as the primary antibody.
Northern blotting
Northern blotting was performed as described previously
(Hayashi et al., 1999). Total
RNAs were extracted from the embryonic whole brain, neonatal and adult
cerebrum and cerebellum and other adult tissues of SD rats. 10 µg of total
RNA per lane were separated by electrophoresis, transferred to nylon membranes
and hybridized with specific probes. For the preparation of the probes, cDNA
fragments of PSD-Zip70 were labeled with [
-32P]dCTP by the
random priming method. The blots were subjected to autoradiography.
In situ hybridization
Fresh-frozen sections of whole brains from 7-week-old SD rats were prepared
for in situ hybridization. These sections were prepared and treated as
described previously (Sun et al.,
1998) and hybridized with antisense or sense probes. For the
preparation of the probes, PSD-Zip70 cDNAs (344-2,149 nt) were transcribed in
the presence of [
-35S]UTP using the Riboprobe system
(Promega). The hybridized signals were visualized with a BAS-5000 phosphor
imager (Fujifilm).
Subcellular fractionation, solubilization and western blotting
The subcellular and tissue distribution and developmental change of
PSD-Zip70 were examined by western blotting. The subcellular fractions were
prepared from the cerebrum of 7-week-old rats by a previously described method
(Wu et al., 1986). In brief,
the brain was homogenized and spun at 1475 g. The supernatant (S1)
was further spun at 17,300 g, and the pellet (P2) and supernatant
(S2) fractions were retained. The P2 fraction was further separated by sucrose
density gradient (0.32, 1.0 and 1.4 M sucrose) centrifugation at 82,500
g for 65 minutes. The synaptosome fraction was obtained at the 1.0
M-1.4 M interface. This fraction was lysed by hypo-osmotic shock with 1 mM
NaHCO3, treated with 0.5% Triton X-100, and centrifuged at 15,000
g for 60 minutes. The pellet was further separated by sucrose density
gradient (0.32, 1.0, 1.5, and 2.1 M sucrose) centrifugation at 201,800
g for 120 minutes. The PSD-I fraction was obtained between 1.5 M and
2.1 M sucrose. 20 µg of protein per lane were analyzed. To examine the
solubilization of PSD-Zip70, the synaptosome fraction was suspended with 10
volumes of 1 mM NaHCO3 and centrifuged at 17,000 g for 10
minutes at 4°C. After centrifugation, 1 mg protein of each pellet was
resuspended with a 26-G needle in 500 µl of extraction buffer (10 mM Hepes,
pH 7.4, 1 mM EGTA, 1 mM MgCl2 150 mM NaCl, 200 µM PMSF, 10
µg/ml pepstatin A, 10 µg/ml leupeptin and 10 µg/ml aprotinin)
containing various additional reagents as indicated in
Fig. 5, rotated for 30 minutes
and centrifuged at 100,000 g for 30 minutes at 4°C. The pellets
were resuspended in the original volume of extraction buffer. 10 µl of the
pellet (ppt) and supernatant (sup) fractions were analyzed by western
blotting. Samples were loaded on gels, separated by SDS-PAGE and transferred
to nitrocellulose membranes. mAb204H or pAbZip70 were used as the primary
antibodies and visualized using peroxidase-conjugated secondary antibodies
followed by ECL (Amersham Pharmacia). For the preabsorption of antibodies,
mAb204H and pAbZip70 diluted for western blotting were preabsorbed with an
excess amount of purified GST-PSD-Zip70.
|
Cells
Hippocampal neurons were prepared from rat embryonic brains at embryonic
day 18 (E18). The hippocampus was dispersed with 0.25% trypsin in HBSS
solution, and the cell suspension was plated onto 0.5 mg/ml
poly-L-lysine-coated glass coverslips at a density of 10,000-15,000
cells/cm2. Neurons were cultured in a Neurobasal medium (Life
Technologies) containing 2% B27 supplement (Life Technologies) and 0.5 mM
L-glutamine. Half of the medium was changed per week. COS7 and MDCK cells were
cultured in DMEM supplemented with 10% FCS and transfected with expression
vectors using Lipofectamine 2000 (Life Technologies). The cells were fixed at
16 hours (for immunocytochemistry) or 36 hours (for biochemical analysis)
after transfection.
Transfection using microinjection
Using a micromanipulator (Narishige), plasmid DNAs (5-200 ng/µl) were
microinjected through a glass capillary into the nuclei of hippocampal
neurons. After 24 hours, the neurons were fixed for immunocytochemistry as
described below.
Immunocytochemistry
After 6 or 30 days in culture, hippocampal neurons were fixed in 4%
paraformaldehyde and 4% sucrose in PBS for 1 hour at 4°C. In some cases,
the neurons were further treated with methanol at -20°C for 10 minutes.
Fixed neurons were permeabilized with the blocking solution containing Triton
X-100 (10% normal goat serum, 0.2% BSA, and 0.1% Triton X-100 in PBS) for 1
hour at 37°C and incubated with primary antibodies in the blocking
solution for 2 hours at 37°C. Anti-MAP2 (clone HM-2, SIGMA) and anti-Tau-1
(Boehringer Mannheim) mAbs were used for dendritic and axonal markers,
respectively. Presynaptic and postsynaptic sites were labeled with
anti-synaptotagmin mAb produced by our laboratory (mAbSV96) and anti-PSD-95
mAb (clone 6G6-1C9, Affinity Bioreagents), respectively. F-actin was labeled
with Alexa FluorTM 568-phalloidin. For PSD-Zip70 staining, pAbZip70 was
used. After incubation with the primary antibody, the neurons were labeled
with Alexa FluorTM 488 and/or 546 secondary antibodies (2 µg/ml,
Molecular probes) in the blocking solution. After washing with PBS, the
coverslips were mounted onto glass slides using a Prolong Antifade Kit
(Molecular Probe). Fluorescence images were acquired using a cooled CCD camera
(Roper Scientific) mounted on an Olympus IX70 microscope with Metamorph
imaging software (Universal Imaging Corporation).
Transfected MDCK cells were fixed in 4% paraformaldehyde in PBS for 10 minutes at 37°C. For in vivo extraction, cells were washed with PBS and treated with the extraction buffer (0.2% Triton X-100, 20 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 1 mM MgCl2, 1 mM EGTA, 200 µM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin and 10 µg/ml aprotinin) for 1 minute at 4°C. After extraction, cells were washed with the extraction buffer without Triton X-100 and then fixed. The anti-myc mAb and pAb (Santa Cruz) and an anti-ezrin mAb (CHEMICON) were used as the primary antibodies. To stain the mitochondria, MitoTracker (Molecular Probes) was added to the growth medium and the cells were further cultured for 30 minutes. After labeling, the cells were fixed and double stained as described above.
Immunohistochemistry
To prepare the sections, rat brains (7-week-old) were fixed by perfusion
with 4% paraformaldehyde in 0.1 M phosphate buffer and further fixed in the
same buffer for 4 hours at 4°C. 50 µm sections were prepared using a
vibratome. These sections were pre-treated with the blocking solution as
described above for 1 hour at room temperature and incubated with pAbZip70 or
control rabbit IgG (0.5 µg/ml) in the blocking solution for 36 hours at
4°C. A peroxidase-conjugated anti-rabbit IgG antibody was used as the
secondary antibody, and the staining was visualized using an ABC kit
(Vector).
Immunoelectron microscopy
Adult SD rats were perfused with a fixative containing 4% paraformaldehyde,
0.01 M sodium periodate and 0.1 M lysine-HCl in 0.1 M phosphate buffer. Small
pieces of forebrain tissues including the cerebral cortex and hippocampus were
dissected out and kept in the same fixative for 4 hours at 4°C. After
washing with 0.1 M phosphate buffer, specimens were infused with a mixture of
20% poly(vinylpyrrolidone) and processed for ultracryotomy on an Ultracuts
microtome (Reichert) equipped with FCS (Reichert) following the method of
Tokuyasu (30). Briefly, sections of frozen specimens were collected with drops
of 2.3 M sucrose, placed on a grid and thawed at room temperature. After
blocking with 10% normal goat serum, they were incubated in 0.1 M
Tris-buffered saline (TBS) containing pAbZip70 for 24-48 hrs at 4°C, then
washed with TBS and incubated with goat anti-rabbit IgG antibodies conjugated
with 5 or 10 nm gold particles (Amersham) for 2 hours at room temperature.
Thereafter, the sections were rinsed and embedded with a mixture of 1%
poly(vinyl alcohol) containing 0.1% uranyl acetate and observed under an
electron microscope (Hitachi H-7100) after drying.
Sucrose floatation gradient separation of a crude synaptosomal
fraction and isolation of dendritic rafts
All procedures were carried out at 4°C. 1 mg protein of the synaptosome
fraction obtained from the rat cerebrum was suspended with a 26-G needle in 1
ml of TNE (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 200 µM PMSF, 10
µg/ml pepstatin A, 10 µg/ml leupeptin and 10 µg/ml aprotinin)
containing 1% Triton X-100 and rotated for 30 minutes. The sample was adjusted
to 40% sucrose using 80% sucrose in TNE, placed in the bottom of a centrifuge
tube and overlaid with 3 ml of 30% sucrose, 3 ml of 25% sucrose and 5 ml of 5%
sucrose in TNE. The sample was then centrifuged at 201,000 g for 14
hours. After centrifugation, the upper 3 ml of the gradient solution was
discarded and 10 fractions were collected from the top. The pellet fraction
(ppt) was resuspended in 50 µl of TNE, and 500 µl of each collected
fraction was precipitated with tricarboxylic acid (TCA), resuspended in 50
µl of TNE and separated by SDS-PAGE followed by western blotting as
described above. The methods for preparing the synaptic plasma membrane (SPM),
dendritic raft and PSD fractions have been described previously
(Suzuki et al., 2001). 5 µg
of each sample per lane were analyzed by western blotting. Anti-PSD-95 mAb,
pAbZip70, anti-synaptophysin mAb (PROGEN), anti-fyn pAb (Upstate
biotechnology), anti-
-internexin pAb (CHEMICON) and anti-NR1 pAb
(Upstate biotechnology) were used as the primary antibodies. The methods for
electron microscopy and the lipid assay for the SPM, dendritic rafts and PSD
were described previously (Suzuki et al.,
2001
). The PSD preparation purified by this method was referred to
`PSD-II'. For electron microscopy, the dendritic raft preparation was fixed
with 2% glutaraldehyde in HEPES/KOH buffer (5 mM, pH 7.4), spun briefly,
further fixed with 1% osmium tetroxide, dehydrated through a graded ethanol
series and embedded in Epon. An ultrathin section was cut and stained with
uranyl acetate and lead citrate. For negative staining, dendritic raft
suspension was dropped on a Formvar-coated grid and stained with 4% uranyl
acetate. Specimens were examined with an electron microscope (JE-M-1200 EX;
JEOL). For the lipid assay, lipids were extracted from the SPM, dendritic raft
and PSD-II preparations and separated by thin layer chromatographies (TLC).
The separated lipids were re-extracted and quantified using an enzymatic
colorimetric assay kit for cholesterol and sphingomyelin.
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Results and Discussion |
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A rat cerebrum cDNA library (3x105 clones) was screened
with mAb204H, and two positive clones were isolated. These clones had
identical sequences in the open reading frame (GenBank Accession Number
AB075607), which encoded a 601 amino-acid protein with the calculated
Mr of 67,562 (Fig.
1B). An N-terminal myristoylation consensus sequence (MGxxxS/T)
and a polybasic cluster (RKxxxxKK) were found in the N-terminus, and four
leucine-zipper motifs were present in the C-terminal
region*
(Fig. 1B). A database search
revealed that the predicted amino-acid sequence of PSD-Zip70 was 89% identical
to that of the human FEZ1/LZTS1 gene product
(Ishii et al., 1999), which
contains multiple leucine-zipper motifs; therefore, PSD-Zip70 is a rat
homologue of the human FEZ1/LZTS1 gene product. Recent allelotyping
studies have shown that the allelic loss of chromosome region 8p21-22 is
closely associated with various human tumors
(Jenkins et al., 1998
). The
FEZ1/LZTS1 gene was identified by Ishii et al. in a genetic analysis
of tumor suppressor genes at 8p22; they found this gene to be altered in many
tumors including esophageal, prostate, and breast cancers
(Ishii et al., 1999
). The
PSD-Zip70 protein recognized by mAb204H and pAbZip70 was a closely related 70
kDa doublet in the rat cerebral subcellular fractions
(Fig. 1A). When an expression
plasmid carrying wild-type PSD-Zip70 was transfected into COS7 cells, the
expressed protein also appeared as a 70 kDa doublet (data not shown). As
described above, this protein contains a consensus sequence for N-terminal
myristoylation. Indeed, the N terminus of PSD-Zip70 (PSD-Zip70N) was
myristoylated when it was expressed in COS7 cells
(Fig. 1C), but its mutant
(PSD-Zip70N/G2A) was not, indicating that the N-terminal myristoylation of
PSD-Zip70 in vivo is specific. In this case, myristoylated PSD-Zip70WT and
non-myristoylated PSD-Zip70N/G2A both appeared as the doublet
(Fig. 1C). Although PSD-Zip70N
also contains a candidate sequence for palmitoylation
(15SKHCRA20), it was not palmitoylated in COS7 cells
(data not shown). These results suggest that the doublet of PSD-Zip70 may be
due to post-translational modifications other than acylation. Consistent with
our results, Ishii et al. demonstrated the serine phosphorylation of the
FEZ1/LZTS1 gene product by cAMP-dependent protein kinase, which
resulted in differently migrating protein bands on SDS-PAGE
(Ishii et al., 2001
).
Unique expression of PSD-Zip70 in the brain
Northern blotting revealed that the approximately 5.0 kb PSD-Zip70 mRNA was
already expressed in whole embryonic brains at 15 days (E15) and that its
expression gradually increased in the cerebrum thereafter. By contrast, the
PSD-Zip70 mRNA in the cerebellum increased abruptly between 1 and 3 weeks
after birth and then disappeared (Fig.
2A). Western blotting using pAbZip70 showed only a trace amount of
PSD-Zip70 protein in embryonic brains at E15-18, whereas the protein
expression in the cerebrum increased between 1 and 3 weeks after birth and
reached a plateau at 7-12 weeks (Fig.
2B). The protein in the cerebellum was present at lower levels
than in the cerebrum during all developmental stages. Thus, PSD-Zip70 showed
distinctly different expression patterns at the mRNA and protein levels during
brain development. This discrepant expression of PSD-Zip70 in the brain may be
due to extremely different turnover rates of the PSD-Zip70 mRNA and protein or
to the critical regulation at the translational level during development.
Further studies are required to elucidate the precise mechanism governing
these discrepant expression patterns.
|
We next examined the expression of PSD-Zip70 at the mRNA and protein levels in adult rat tissues. Northern and western blot analyses revealed the presence of PSD-Zip70 mRNA and protein only in the cerebrum and not in the other tissues examined (Fig. 2C,D). A long exposure of northern and western blots, however, showed trace amounts of the PSD-Zip70 mRNA and protein in the cerebellum, lung, kidney and stomach (data not shown).
In situ hybridization of adult rat brain showed a unique distribution of the PSD-Zip70 mRNA, in which intense signals were found in the cerebral cortex, hippocampus, olfactory bulb, striatum and pons (Fig. 3A). In the hippocampus, the transcripts were intensely labeled in the CA2 region compared with the CA1 and CA3 regions. Faint signals were detected in other cerebral regions, such as the thalamus and brain stem. However, no signals were observed in the cerebellum. In control sections using a sense probe, no signals were detected (Fig. 3B). Immunostaining for pAbZip70 showed intense labeling in the cerebral cortex, hippocampal CA2 region, subiculum and other cerebral regions. In the cerebral cortex and hippocampal CA2 region, immunolabeling was localized to the soma and dendritic processes of the principal neurons but not to glial cells (Fig. 3C-E). These immunoreactivities were specific for PSD-Zip70, because no immunolabeling was detected using a control IgG as the primary antibody (Fig. 3F). These results indicate that PSD-Zip70 is specifically expressed in the neurons of some cerebral areas.
|
Postsynaptic localization of PSD-Zip70
Immunocytochemistry of cultured hippocampal neurons after 6 days in vitro
(DIV) revealed patchy immunofluorescence for PSD-Zip70 in the dendrites and
the cell bodies (Fig. 4A1,B1).
Double immunofluorescence for PSD-Zip70 and MAP2, a dendritic marker,
completely overlapped in the dendrites and the cell bodies
(Fig. 4A1-A3). However,
PSD-Zip70 and tau protein, an axonal marker, showed distinctly different
distributions (Fig. 4B1-B3).
These results indicated a preferential localization of PSD-Zip70 to the
dendrite. In cultured mature neurons (30 DIV), numerous
PSD-Zip70-positive neurons were seen among some populations of morphologically
excitatory neurons but not in all excitatory neurons. This finding may reflect
the dominant expression of PSD-Zip70 in the hippocampal CA2 region, as shown
in Fig. 3A,D. In addition to
the patchy fluorescence in the dendrites, intense immunofluorescent clusters
of PSD-Zip70 were distributed along the dendrites
(Fig. 4C1,D1 and E1). To
further characterize the localization of PSD-Zip70, double staining was
performed using pAbZip70 and antibodies against pre- and postsynaptic markers
or phalloidin for F-actin. Clusters of PSD-Zip70 were colocalized with PSD-95
clusters (Fig. 4C1-C3 and
C1'-C3') and with many F-actin clusters
(Fig. 4D1-D3 and
D1'-D3'). High-resolution images showed that the areas
of PSD-Zip70 cluster within the dendritic spines were much wider than those of
PSD-95 clusters (Fig.
4C1'-C3') and that the PSD-Zip70 clusters also
partially overlapped with synaptotagmin clusters
(Fig. 4E1-E3 and
E1'-E3'), suggesting the synaptic localization of
PSD-Zip70.
|
To further define the synaptic localization of PSD-Zip70, we performed postembedding immunoelectron microscopy. Immunogold labeling for PSD-Zip70 was predominantly observed in axospinous and axodendritic asymmetric synapses of pyramidal neurons in the hippocampal CA2 region and cerebral cortex. Most of the immunogold label appeared over the perisynaptic regions of the PSD and the postsynaptic membrane in excitatory asymmetric synapses of pyramidal neurons in the hippocampus and cerebral cortex (Fig. 4F-I), indicating the postsynaptic localization of PSD-Zip70.
Solubility of PSD-Zip70 from the synaptic membrane fraction
We examined the solubility of PSD-Zip70
(Fig. 5). Non-ionic detergents
(Triton or Lubrol WX), CHAPS or high salt alone did not significantly
solubilize the PSD-Zip70 from the synaptosome membrane. A combination of
non-ionic detergent or CHAPS and high salt could solubilize 10-30% of the
total PSD-Zip70, indicating that high salt is partially effective for the
solubilization of this protein from the detergent-resistant synaptosome
membrane. A high concentration of Tris-HCl, which can partly solubilize the
membrane cytoskeleton, including spectrin
(Tanaka et al., 1991), had no
effect on the solubility of PSD-Zip70. Alkaline conditions, which solubilize
the peripheral membrane proteins, did not solubilize PSD-Zip70. Deoxycholate
(1%, pH 9.0), which was previously used to extract the PSD proteins
(Husi et al., 2000
), and urea
could release nearly a half of the total PSD-Zip70. Consistent with the
perisynaptic localization of PSD-Zip70 in the PSD and the postsynaptic
membrane, N-lauroyl sarcosinate (NLS) completely solubilized the protein.
These results suggest that most of the PSD-Zip70 tightly associates with the
detergent-insoluble compartments of the postsynaptic membrane, such as the
PSD, rafts, and detergent-insoluble cytoskeleton via lipid-lipid and/or
lipid-protein interactions.
Localization of PSD-Zip70 to the PSD and the dendritic rafts
To investigate whether PSD-Zip70 was associated with the membrane rafts, we
performed a sucrose floatation gradient separation of the Triton-X-100-treated
synaptosome fraction modified from a previously reported method
(Röper et al., 2000). Fyn
and PSD-95 are reported to be raft-associated proteins
(Wolven et al., 1997
;
Perez and Bredt, 1998
). As
shown in Fig. 6A, PSD-Zip70 and
PSD-95 were recovered in both the detergent-insoluble low-density fraction
(the raft fraction, fractions 2-4) and the pellet fraction, which probably
included the PSD. Fyn was mainly found in the raft fraction. By contrast,
synaptophysin was detected in the detergent-soluble fraction (fractions 7-10).
To confirm the neuronal distribution of PSD-Zip70, we prepared the dendritic
rafts by the method of Suzuki et al.
(Suzuki et al., 2001
). The
dendritic raft fraction thus obtained was homogenous in its morphology
according to both negative staining and thin-sectioned electron micrographs
(Fig. 6B). Both types of
imaging showed membrane sacs of 200-1000 nm in diameter, to which fuzzy
structures that probably consist of proteins were attached. One of the
criteria for the raft structure is a highly ordered concentration of
cholesterol and sphingomyelin in the raft membrane
(Simons and Ikonen, 1997
). In
agreement with this criterion, the levels of cholesterol and sphingomyelin in
the dendritic raft fraction were much higher than those in the SPM and PSD
fractions (Table 1). As shown
in Fig. 6C, PSD-Zip70 and
PSD-95 were found in both the PSD-II and the dendritic raft fractions, and Fyn
was mainly in the dendritic raft fraction. Consistent with our previous report
(Suzuki et al., 2001
), the NR1
subunit of the NMDA receptors and
-internexin were only detected in the
PSD fraction. Thus, two different fractionations indicated that PSD-Zip70 was
localized to both the PSD and the dendritic rafts.
|
|
Targeting of PSD-Zip70 into the microvilli and the plasma membrane of
non-neuronal cells
As shown in Fig. 6,
PSD-Zip70 was localized to the membrane raft microdomain in the brain. We
further examined if PSD-Zip70 is also targeted to the microvilli-like
structure of the plasma membrane, which is a membrane lipid raft-rich
structure in MDCK cells (Röper et
al., 2000), and determined the critical domains of PSD-Zip70 for
its localization.
The PSD-Zip70 protein was not detected in MDCK cells by immunocytochemistry
or western blotting (data not shown). However, when myc-tagged PSD-Zip70WT or
PSD-Zip70N was expressed in MDCK cells cultured under serum-supplemented
conditions, the immunolabeling with the anti-myc antibody was highly
concentrated in structures at the apical plasma membrane and also showed a
submembranous distribution (Fig.
7A1,B1). Double labeling for PSD-Zip70WT or PSD-Zip70N (with the
anti-myc antibody) and F-actin (with phalloidin;
Fig. 7A1-A3 and B1-B3) or
ezrin, a marker for microvilli-like structures (with an anti-ezrin antibody;
Fig. 7E1-E3 and
E1'-E3'), revealed speckled immunolabeling for
PSD-Zip70WT or PSD-Zip70N overlapping with the positive staining for F-actin
or ezrin in the apical structures. After in vivo extraction with Triton X-100,
PSD-Zip70N was retained in the microvilli, but its membrane localization was
abolished (data not shown). It has been demonstrated that such
detergent-insoluble microvilli belong to a subclass of membrane rafts
(Röper et al., 2000).
Ishii et al. demonstrated the colocalization of cytoplasmic microtubules and
the exogenous FEZ1/LZTS1 gene product in HEK 293 cells and some
cancer cells and reported the FEZ1/LZTS1-dependent inhibition of cell
growth mediated through a suppression of microtubule assembly and interaction
with p34cdc2 (Ishii et al.,
2001
). However, we found that myc-tagged PSD-Zip70WT and
PSD-Zip70N in MDCK cells were predominantly targeted to the microvilli and the
plasma membrane but not to the cytoplasmic microtubules. One possibility for
this discrepancy may be due to a different cell line used; however, the exact
reason is not yet known.
|
Many proteins have covalently attached fatty acids (myristate and
palmitate) at their N-terminus (Resh,
1999). This is seen in src family tyrosine kinases, G protein
subunits, endothelial nitric oxide synthase and others. Several proteins are
singly myristoylated and possess an adjacent or distant polybasic cluster.
Other proteins contain two or more covalently linked palmitates at their
N-terminus. In all cases, multiple N-terminal acylation sites or an acylation
site juxtaposed to a polybasic cluster are required for the membrane anchorage
of these modified proteins. The presence of these modified proteins and
additional protein-protein interactions are further required to form the raft
structure. Because PSD-Zip70 is myristoylated on its N-terminus
(Fig. 1D) and also contains the
distant polybasic cluster (Fig.
1B), we examined whether PSD-Zip70's localization to the
microvilli may be mediated through N-terminal myristoylation and/or the
polybasic cluster. PSD-Zip70N/G2A exclusively accumulated in the nucleus and
the perinuclear region (Fig.
7C1-C3). By contrast, PSD-Zip70C was diffusely distributed
throughout the cytoplasm but was not localized to the microvilli and the
plasma membrane. Deletion of amino-acid residues 21-40 containing the
polybasic cluster (PSD-Zip70N/
21-40) resulted in the loss of the
protein's localization to the microvilli and plasma membrane; instead, this
mutant protein translocated to the mitochondria (data not shown). A mutation
of the polybasic cluster (PSD-Zip70N/mut) resulted in a protein that showed
the same distribution as PSD-Zip70N/
21-40
(Fig. 7D1-D3). Staining the
mitochondria with MitoTracker clearly showed that the polybasic cluster mutant
was localized to the mitochondria (data not shown). These results indicate
that both N-terminal myristoylation and the polybasic cluster are required for
the targeting of PSD-Zip70 to the microvilli and the plasma membrane in
epithelial cells.
Synaptic targeting of PSD-Zip70
We also analyzed the synaptic targeting of PSD-Zip70 in cultured
hippocampal neurons. The synaptic localization of the exogenous proteins was
monitored by observing the colocalization with PSD-95. When myc-tagged
PSD-Zip70WT was expressed, it was localized to the dendrites and the dendritic
spines. The fluorescence intensity of the PSD-Zip70WT in the dendritic spines
was much higher than that in the dendrites
(Fig. 8A1-A3 and
A1'-A3'). PSD-Zip70C was also localized to the
dendrites and dendritic spines, but the spine localization of PSD-Zip70C was
slightly lower than that of PSD-Zip70WT
(Fig. 8C1-8C3 and
8C1'-8C3'). By contrast, PSD-Zip70N was ubiquitously
distributed from the dendrites to the dendritic spines. The fluorescence
intensities of PSD-Zip70N in the dendritic spines and the dendrites were
identical, suggesting the submembranous localization of PSD-Zip70N
(Fig. 8B1-B3 and
B1'-B3'). The non-myristoylated form of the
full-length protein, PSD-Zip70WT/G2A, was also localized to postsynaptic sites
similar to PSD-Zip70C (Fig. 8D1-D3 and
D1'-D3'). By contrast, PSD-Zip70N/G2A lost the
submembranous localization but accumulated in the nuclei and the perinuclear
regions (Fig. 8E1-E3). These
results indicate that, consistent with the results using MDCK cells, the
N-terminal myristoylation of PSD-Zip70 is required for its plasma membrane
localization in hippocampal neurons and the C-terminal domain is critically
involved in synaptic targeting. We have identified a 200 kDa protein that is
also concentrated in PSDs as a candidate binding partner for the C-terminal
domain of PSD-Zip70, which mainly contains leucine-zipper motifs (data not
shown). Therefore, these motifs may be critically involved in the synaptic
targeting of PSD-Zip70 via this protein. We are currently investigating the
characterization of this 200 kDa protein and its role in the PSD.
|
Here, we have demonstrated that the PSD-Zip70 protein is exclusively and
highly expressed in the neurons of restricted cerebral areas and is mainly
localized to the PSD and the dendritic rafts. These results suggest that the
main function of PSD-Zip70 is likely to be carried out at its postsynaptic
site. The N-terminal myristoylation of PSD-Zip70 is required for its membrane
localization, and the C-terminal domain, which contains leucine-zipper motifs,
is critically involved in its targeting to the synapse, which is mediated
through protein-protein interaction. It has been well documented that the
leucine-zipper motif is important for the dimerization of several
transcription factors, such as c-fos and c-jun. Furthermore, this motif is
also present in a variety of cytoplasmic and transmembrane proteins, including
cytoskeletal proteins (Lupas et al., 1996). Leucine-zippermotif-containing
proteins in the postsynaptic region of the neuromuscular junction have been
reported and include rapsin, dystrophin and dystrobrevin. Rapsin, a
neuromuscular 43 kDa myristoylated protein, is involved in acetylcholine
receptor (AchR) clustering. The other two neuromuscular proteins, dystrophin
and dystrobrevin, bind to each other to form heterodimers via their
leucine-zipper motifs (Sadoulet-Puccio et
al., 1997). A few leucine-zipper proteins in the central nervous
system have also been reported. One of them is PSD-Zip45, which is involved in
the clustering of mGluRs mediated through self-multimerization via the extreme
C-terminal leucine-zipper of PSD-Zip45
(Tadokoro et al., 1999
).
Together, previous reports on the neuromuscular junction and our previous and
present studies suggest that the leucine-zipper motif might be a critical
motif for protein-protein interactions contributing to the postsynaptic
cytoarchitecture and function.
The currently accepted concept is that both the PSD and the rafts are
cytoarchitectural centers for receptor-linked signal transduction, membrane
trafficking and cytoskeletal connections
(Sheng and Lee, 2000;
Kennedy, 2000
;
Scannevin and Huganir, 2000
;
Xiao et al., 2000
;
Sheng and Sala, 2001
;
Simons and Ikonen, 1997
;
Resh, 1999
;
Ikonen, 2001
). However, the
relationship between the rafts and the PSD in neurons is largely unknown, with
a few exceptions. Recently, several studies have indicated the importance of
acylation for postsynaptic functions. El-Husseini Ael-D et al. reported that
the palmitoylated PSD protein, PSD-95, regulates synaptic plasticity through
the activity-dependent regulation of palmitate cycling
(El-Husseini Ael-D et al.,
2002
). They suggest that in synapses, protein acylation is not
only involved in simple membrane anchoring but also in the regulation of
several cell processes, including endocytosis, that are reported to be raft
functions. One of us previously demonstrated the localization of AMPA
receptors to the dendritic rafts (Suzuki
et al., 2001
). It is well documented that AMPA receptors are
recruited to postsynaptic sites in a neuron-stimulation-dependent fashion
(Carroll et al., 1999
;
Shi et al., 1999
;
Hayashi et al., 2000
).
Furthermore, EphrinB ligands recruit GRIP family members, which are
AMPA-receptor-binding proteins, into raft membrane microdomains
(Brückner et al., 1999
).
As demonstrated here (Fig. 6)
and in previously reported studies (Wolven
et al., 1997
; Perez and Bredt,
1998
), PSD-95 and Fyn are also localized to both the PSD and the
dendritic rafts. Thus, some important postsynaptic molecules are present in
both structures.
As shown in Fig. 4,
PSD-Zip70 was localized to the edge of the PSD and/or the perisynaptic region.
The significance of this localization is not clear. However, several studies
report that some neurotransmitter receptors, such as the 7 nicotinic
acetylcholine receptor subunits (
7 nAChRs) and type I mGluRs, also
localize to the perisynaptic region of the postsynapse
(Fabian-Fine et al., 2001
).
Specifically,
7 nAChRs are localized to membrane lipid rafts, and rafts
are necessary for their maintenance in ciliary neurons
(Bruses et al., 2001
). These
reports and our data suggest that a close relationship exists between the
dendritic rafts and postsynaptic structures, including the PSD. Taken
together, the findings support the idea that the dendritic rafts may be active
centers of recruitment for some proteins to postsynaptic sites, especially to
the PSD, in addition to the generally accepted roles of rafts. The unique
expression and localization of PSD-Zip70 indicate that it may be involved in
the dynamic properties of postsynaptic structure and function.
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Acknowledgments |
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Footnotes |
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