doi: 10.1242/10.1242/jcs.00127
The postsynaptic density and dendritic raft localization of PSD-Zip70, which contains an N-myristoylation sequence and leucine-zipper motifs
Daijiro Konno1,
Ji-Ae Ko1,3,
Shinichi Usui1,2,
Kei Hori1,
Hisato Maruoka1,
Makoto Inui3,
Takashi Fujikado2,
Yasuo Tano2,
Tatsuo Suzuki4,
Koujiro Tohyama5 and
Kenji Sobue1,*
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
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Fig. 5. Solubilization of PSD-Zip70 from the synaptosome membrane fraction. The
synaptosome membrane fraction was treated with extraction buffer with or
without (cont) various detergents and salts as indicated on the left side of
the panels. After centrifugation, the pellet (ppt) and the supernatant (sup)
fractions were analyzed by western blotting using pAbZip70. NLS indicates
N-lauroyl sarcosinate.
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Fig. 1. Subcellular distribution, predicted amino-acid sequence and N-terminal
myristoylation of PSD-Zip70. (A) The subcellular distribution of PSD-Zip70 in
the rat cerebrum using mAb204H (204H, upper panel). Each fraction was prepared
as described in Materials and Methods. WB, Syn and mito indicate the whole
brain lysate, synaptosome and mitochondria fraction, respectively. pAbZip70
recognized a 70 kDa protein in the PSD-I fraction (pAb70, lower right panel).
Preabsorption (pre) means that mAb204H and pAbZip70 were preabsorbed with an
excess amount of GST-PSD-Zip70 (lower two panels). PSD-95 is shown as a
control of the subcellular fractionation (middle panel). (B) The predicted
amino-acid sequences of rat PSD-Zip70 (upper letters) and the human
FEZ1/LZTS1 gene product (lower letters). The N-terminal
myristoylation consensus sequence is boxed. The four putative leucine-zipper
motifs of PSD-Zip70 and the FEZ1/LZTS1 gene product are marked in
gray. The thin line and hatch marks in the FEZ1/LZTS1 sequence
indicate identical amino acids within PSD-Zip70 and the polybasic region,
respectively. (C) N-terminal myristoylation of PSD-Zip70 in COS7 cells. Left
and right panels show the autoradiographs of incorporated [3H]
myristic acid and western blotting, using anti-myc pAb, respectively.
PSD-Zip70N/G2A is mutated at the myristoylation site. (D) Design of the
PSD-Zip70 variants used for transfection.
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Fig. 2. Expression of PSD-Zip70 mRNA and protein during brain development and in
tissue distribution. (A,C) Northern blotting using specific
32P-labeled probes for PSD-Zip70 mRNA. 20 µg of total RNAs per
each lane were analyzed. The two lower panels indicate methylene blue staining
of 28S ribosomal RNAs, which was used to quantify the amounts of total RNA
loaded. The positions of the 28S and 18S ribosomal RNAs are indicated at the
right. (B,D) Western blotting using pAbZip70. 20 µg of the indicated
protein samples per lane were analyzed.
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Fig. 3. Distribution of PSD-Zip70 in the brain. In situ hybridization (A,B) and
immunohistochemistry (C-F) were performed to determine the distribution of
PSD-Zip70 mRNA and protein in the brain. Sagittal sections from the adult rat
brain were hybridized with specific 35S-labeled cRNA antisense (A)
or sense (B) probes for PSD-Zip70. Ob, olfactory bulb; Cr, cerebral cortex;
Hi, hippocampus; St, striatum; Po, pons; Cl, cerebellum. Immunohistochemistry
of sagittal sections from the adult rat brain was performed using pAbZip70.
The cerebral cortex (C), hippocampal CA2 region (D) and subiculum (E) are
shown. The cerebral cortex in a control section using normal rabbit IgG is
shown in F.
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Fig. 4. Synaptic localization of PSD-Zip70. (A-E) Hippocampal neurons cultured in
vitro for 6 days (DIV6, A1-A3 and B1-B3) or 30 days (DIV30, C1-C3, D1-D3 and
E1-E3) were double-labeled using pAbZip70 (left panels) and anti-MAP2 (A2),
anti-tau (B2) and anti-PSD-95 (C2 and C2') antibodies, phalloidin (D2
and D2') or anti-synaptotagmin antibody (E2 and E2'). Right panels
show the merged images of the left and middle panels. C1'-C3',
D1'-D3', and E1'-E3' show higher magnifications of the
boxed regions in C1-C3, D1-D3 and E1-E3, respectively. Bar, 10 µm. (F-I)
Immunoelectron microscopy of the adult rat brain was performed using pAbZip70.
The cerebral cortex (F-H, 5 nm gold particles) and the CA2 region of
hippocampus (I, 10 nm gold particles) are shown. Bar, 100 nm.
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Fig. 6. The PSD and raft localization of PSD-Zip70 in the synaptosome fraction. (A)
Sucrose floatation gradient separation of the 1% Triton-X-100-treated
synaptosome fraction was performed at 4°C. Gradient fractions were
analyzed by western blotting using pAbZip70, anti-PSD-95, anti-Fyn, and
anti-synaptophysin antibodies. Fraction 1 is from the top of gradient, and ppt
indicates the pellet fraction. Fractions 2-4 are the raft fraction, and
fractions 7-10 mainly contain Triton-soluble proteins. (B) Electron microscope
observation of the dendritic raft fraction prepared from the rat forebrain.
Photographs show negative staining (upper panel) and thin-section (lower
panel) of the dendritic raft fraction. Bar, 200 nm. (C) Localization of
PSD-Zip70 in the PSD-II and raft preparations. The synaptic plasma membrane
(SPM), dendritic raft (raft) and PSD-II fractions were prepared using the
method of Suzuki et al. (Suzuki et al.,
2001 ). These preparations were analyzed by western blotting using
pAbZip70, anti-PSD-95, anti-Fyn, anti- -internexin and anti-NR1
antibodies.
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Fig. 7. Localization of myc-tagged PSD-Zip70 and its deletion mutants in MDCK
cells. Madin-Darby canine kidney (MDCK) cells were transfected with myc-tagged
PSD-Zip70 and its deletion and point mutant expression vectors. Expressed
PSD-Zip70WT (A1-A3), PSD-Zip70N (B1-B3, E1-E3, and E1'-E3'),
PSD-Zip70N/G2A (C1-C3) and its mutation in the polybasic region, PSD-Zip70Nmut
(D1-D3) were visualized with an anti-myc antibody (all left panels) and
double-labeled with phalloidin (A2, B2, C2 and D2) and the anti-ezrin antibody
(E2 and E2'). E1'-E3' show higher magnifications of the
boxed regions in E1-E3. Right panels show the merged images of the left and
middle panels. Bar, 5 µm.
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Fig. 8. Synaptic targeting of PSD-Zip70 in cultured hippocampal neurons. Myc-tagged
PSD-Zip70WT (A1-A3), PSD-Zip70N (B1-B3), PSD-Zip70C (C1-C3), PSD-Zip70WT/G2A
(D1-D3) and PSD-Zip70N/G2A (E1-E3) were expressed in hippocampal neurons using
the microinjection method. Expressed PSD-Zip70 variants were visualized using
an anti-myc antibody (all the left panels) and double-labeled for PSD-95 (all
the middle panels). All the right panels show the merged images of the left
and middle panels. A1'-A3', B1'-B3',
C1'-C3', and D1'-D3' show higher magnifications of the
boxed regions in A1-A3, B1-B3, C1-C3 and D1-D3 respectively. Bar, 10
µm.
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© The Company of Biologists Ltd 2002
