Xie et al., 2009 TARGETING ACETYLCHOLINESTERASE (ACHE)

27 Pages·2009·0.28 MB·English

JBC Papers in Press. Published on February 10, 2010 as Manuscript M109.038711
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M109.038711
Xie et al., 2009
TARGETING ACETYLCHOLINESTERASE (ACHE) TO MEMBRANE RAFTS:
A FUNCTION MEDIATED BY THE PROLINE RICH MEMBRANE ANCHOR (PRIMA)
IN NEURONS*
Heidi Q. Xie1, Dong Liang2, K. Wing Leung1, Vicky P. Chen1, Kevin Y. Zhu1, Wallace K.B.
Chan1, Roy C.Y. Choi1, Jean Massoulié2 and Karl W.K. Tsim1
From 1Department of Biology and Center for Chinese Medicine, The Hong Kong University of
Science and Technology, Clear Water Bay Road, Hong Kong, China and 2Laboratoire de
Neurobiologie, CNRS UMR 8544, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France
Running head: PRiMA directs G AChE to membrane rafts in neurons
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Address correspondence to Dr. Karl W.K. Tsim, Department of Biology, The Hong Kong University
of Science and Technology, Clear Water Bay Road, Hong Kong SAR, China
Tel: [phone]; Fax: [phone]; E-mail: [email protected]

In the mammalian brain, cholesterol-binding motif, called CRAC
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acetylcholinesterase (AChE) is anchored in (cholesterol recognition/interaction w
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cell membranes by a transmembrane protein consensus), located at the junction of d
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PRiMA (Proline-Rich Membrane Anchor). transmembrane and cytoplasmic domains of fro
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We present evidence that at least part of the both PRiMA I and II isoforms. The ttp
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PRiMA-linked AChE is integrated in cytoplasmic domain of PRiMA, which differs w
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membrane microdomains called rafts. A between PRiMA I and PRiMA II, appeared .jb
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significant proportion of PRiMA-linked to play some role in stabilizing the raft brg/
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AChE tetramers from rat brain was localization of G4 AChE, since the Triton gue
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recovered in raft fractions: this proportion X-100 resistant fraction was smaller with the t o
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was markedly higher at low than at high shorter PRiMA II isoform than that with the pril 3
concentration of cold Triton X-100. The longer PRiMA I isoform. , 2
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detergent-resistant fraction increased during
brain development. In NG108-15 Acetylcholinesterase (AChE; EC 3.1.1.7)
neuroblastoma cells transfected with cDNAs controls synaptic and neurohumoral cholinergic
encoding AChE and PRiMA, PRiMA-linked activity by hydrolyzing acetylcholine. The
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G AChE was found in membrane rafts, and function of AChE relies on precise regulations
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showed the same sensitivity to cold Triton on its expression and localization. Alternative
X-100 extraction as in the brain. The splicing in the 3’ region of the primary transcript
association of PRiMA-linked AChE with rafts generates subunits of AChE that contain the
was weaker than that of same catalytic domain but distinct C-terminal
glycosylphosphatidylinositol (GPI)-anchored peptides, which determine their
G AChE or G Q -H -linked AChE. It was post-translational maturation and oligomeric
2 4 N C
found to depend on the presence of a assembly (1, 2). In mammals, the AChE variant
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Copyright 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
Xie et al., 2009
produces a soluble monomer that is reported to domain of PRiMA I (40 residues) contains
be up regulated in the brain during stress (3, 4), several serines that are predicted to be
the AChE variant produces a constitutive phosphorylation sites (5, 13). The
H
glycosylphosphatidylinositol (GPI)-anchored cytoplasmic domain of PRiMA II has only 11
dimer that is mainly expressed in blood cells, residues and does not contain these serines. Both
and the AChE variant, characterized by a PRiMA I and PRiMA II can anchor AChE
T T
40-residue C-terminal “t” peptide, forms a tetramers in cell membranes (13, 14). The
number of oligomeric assemblies (5). AChE expression of PRiMA I is up regulated during
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subunits are predominant in all cholinergic development, and is the major form being
tissues and represent the physiologically active expressed in the brain. In contrast, PRiMA II is
variant; these subunits form tetramers associated only detected as a minor component at the adult
with the collagen ColQ and with the stage in the brain (12, 13). This regulation
transmembrane protein PRiMA (Proline-Rich during brain maturation suggests that the
Membrane Anchor), which allow their difference between the two splice variants could
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functional localization. ColQ-associated AChE have functional implications. The structure of w
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is attached to the basal lamina at vertebrate PRiMA, and in particular its possible fatty d
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neuromuscular junctions, and PRiMA-linked acylation, suggest that it may interact with the fro
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AChET tetramers (G4) is anchored in cell cholesterol-sphingolipid rich membrane ttp
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membranes. The association of AChET subunits microdomains or rafts (15, 16), which have been w
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with ColQ and PRiMA proteins is mostly based shown to be involved in synaptic signalling and .jb
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on a tight interaction between the four plasticity (17, 18). brg/
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C-terminal “t” peptides of AChET subunits and a Here, we demonstrate that part of the gue
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"Proline-Rich Attachment Domain" (PRAD) PRiMA-linked G AChE is integrated in t o
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that exists in both ColQ and PRiMA (6-8). In membrane rafts in the rat brain. The fraction of pril 3
mammals, PRiMA-linked G AChE exists in the membrane-bound AChE recovered in raft , 2
4 0
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brain and muscle, and is the predominant AChE fractions was lower in the presence of a high
species in the adult brain (9-12). than a low detergent concentration (0.05% and
PRiMA is a type I transmembrane protein of 0.5% Triton X-100 in the cold). The proportion
~20 kDa, consisting of a secretory signal peptide, of high detergent resistant AChE in rafts
an N-terminal extracellular domain containing increased during brain development, suggesting
the PRAD and an N-glycosylation site, a that it may fulfil a possible physiological
transmembrane domain and a C-terminal function. In cultured neuroblastoma cells, we
cytoplasmic domain. Two splice variants, demonstrated a critical role of PRiMA in
PRiMA I and II, differ only by their C-terminal directing the association of AChE with
domains: the first seven cytoplasmic residues are membrane rafts, and its heterogeneity in
common and include a cysteine that may detergent-resistance. PRiMA contains a
represent a palmitoylation site. The cytoplasmic cholesterol-binding motif, or CRAC (cholesterol
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recognition/interaction consensus), L/V-(X) - between the signal peptide and the N-terminus
(1-5)
Y-(X) -R/K), where (-X-) represents of the mature protein. AChE was also tagged
(1-5) (1-5) T
between one and five residues of any amino acid similarly at its C-terminus. NG108-15 cells were
(19, 20). We found that this motif is absolutely transfected using jetPEITM reagent
required for raft association. The longer (Polyplus-transfection, Illkirch, France)
intracellular domain of PRiMA I seems to according to the manufacturer’s instructions.
participate in the stability of this association, The transfection efficiency was consistently
since the proportion of raft-associated AChE at 50-60 %. The cells were collected for analysis
high detergent concentration was higher with 48 hours after the transfection.
PRiMA I than with PRiMA II.
Preparation of membrane raft fractions- Brains
EXPERIMENTAL PROCEDURES were obtained from 7-day-old (P7), 14-day-old
(P14) and 100-day-old (P100) male
Cell culture- Neuroblastoma X glioma Sprague-Dawley rats. The preparation of raft
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NG108-15 hybrid cells were maintained in membrane fractions was carried out at 4 oC, with w
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Dulbecco’s Modified Eagle’s Medium (DMEM) different concentrations of detergent, as d
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supplemented with 5% fetal bovine serum (FBS), described previously with modifications (24, 25). fro
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100 units/ml penicillin and 100 μg/ml In brief, fresh tissues were homogenized (1 g/10 ttp
streptomycin and incubated at 37 oC in a ml) in buffer A (50 mM Tris-HCl, pH 8.0, 150 ://ww
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water-saturated 5% CO incubator. All reagents mM NaCl, 5 mM benzamidine HCl, 10 μg/ml .jb
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for cell cultures were from Invitrogen (Carlsbad, aprotinin, 10 μg/ml leupeptin) for 10 sec 6 times brg/
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CA). at 9,500 rev/min (IKA, Staufen, Germany). The gu
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cells were also suspended in buffer A. The t o
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DNA construction and transfection- All tissues or cell suspensions were then sonicated at pril 3
constructs were expressed in the pEF-Bos vector low intensity 3 times for 0.5 sec periods, with 30 , 2
0
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(21), except rat alkaline phosphatase (ALP) sec intervals to avoid heating. The preparation
tagged with a C-terminal t peptide (ALP-t), was centrifuged at 500 xg for 5 min to remove
which was in pcDNA3 vector (22). Mutagenesis cell debris and nuclei, and the post-nuclear
was performed as previously described (23). The extracts of tissues or cells were centrifuged at
cDNA encoding full length mouse PRiMA 35,000 rpm for 30 min in a Sorvall TST 60.4
isoform I was tagged with a HA epitope (YPY rotor. The resulting pellet was washed and
DVP DYA) inserted before the stop codon at the resuspended by sonication in 600 µl of buffer A
C-terminus. The cDNAs encoding full length containing 5% glycerol. This was used as the
mouse PRiMA isoform II, and mouse PRiMA total membrane fraction. For analysis of
isoform I with C-terminal cysteine or serines membrane rafts, a 500 μl sample was incubated
mutations, and with CRAC motif mutations with Triton X-100 at the indicated
were tagged with the same HA epitope inserted concentrations on ice for one hour, and applied
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to a discontinuous flotation gradient: it was 1:2,000; Santa Cruz Biotechnology, Santa Cruz,
mixed with an equal volume of 80% sucrose in CA), and anti-HA antibody (1:2,000; Sigma) as
buffer A, placed at the bottom of a 4 ml primary antibodies; secondary anti-IgG
ultracentrifugation tube and overlaid with 2.4 ml antibodies against different species were
of buffer A containing 30% sucrose and with 0.6 conjugated with horseradish peroxidase. (HRP)
ml of buffer A containing 5% sucrose. The The immune complexes were visualized using
resulting discontinuous gradient was spun at the enhanced chemiluminescence (ECL) method
50,000 rpm in a Sorvall TST 60.4 rotor for 18 (Supersignal West Pico kit, Thermo Fisher
hours at 4 °C. Twenty fractions of ~190 μl were Scientific, Waltham, MA). The exposure time
collected from the top and used for various was 1-10 min.
assays.
Sucrose density gradients- For sedimentation
Cholesterol depletion assay- Three hours after analyses, 100 – 300 μl samples were mixed with
transfer of the cells to serum-free medium, the E. coli β-galactosidase and ALP (Sigma), as
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cultures were treated with 10 mM cyclodextrin, internal sedimentation markers, layered onto w
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MβCD (Sigma, St Louis, MO), for 1 hour at 5-20% sucrose gradients (containing 50 mM d
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37 °C and analyzed as described above. The Tris-HCl pH 7.5; 20 mM MgCl , in the presence fro
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effectiveness of MβCD was determined by a of 0.2% Brij-97 or 0.2% Triton X-100), and ttp
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cholesterol assay kit (Thermo Electron Co. centrifuged in a Beckman SW41 rotor at 38,000 w
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Australia) and mass spectrometry rpm, for 16 hours at 10 oC. Fractions of .jb
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(Supplementary information). approximately 200 μl were collected and brg/
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assayed for AChE, β-galactosidase and ALP gu
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Western blots- Aliquots in 300 μl of fractions activities, as described previously (7). The t o
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from discontinuous flotation gradients were fraction of AChE activity corresponding to the pril 3
incubated with 20% trichloroacetic acid (final PRiMA-linked or Q -H -linked G forms was , 2
N C 4 0
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concentration) on ice, and a protein pellet was determined from the sedimentation profiles.
obtained by high speed centrifugation at 4 °C for
15 min. The pellet was washed with ice-cold Enzyme assay and statistical tests- AChE
acetone twice and air dried, then dissolved at enzymatic activity was assayed according to a
100 oC for 5 min in a denaturing buffer modified method of Ellman et al. (1961) (11,
containing 1% SDS and 1% dithiothreitol, and 26). The cell lysates were incubated with 0.1
analyzed by electrophoresis in 8% or 12% mM tetraisopropylpyrophosphoramide (iso
SDS-polyacrylamide gels. For Western blot -OMPA, Sigma) for 10 min to inhibit the BChE
analysis, we used anti-PSD 95 antibody (1:2,000; activity; 5-20 μl samples were then added to the
Millipore, Billerica, MA), anti-flotillin-2 reaction mixture containing 0.625 mM
antibody (1:5,000; BD Biosciences, San Jose, acetylthiocholine iodide (ATCH, Sigma) and 0.5
CA), anti-tranferrin receptor antibody (H-300; mM 5,5’-dithiobis-2-nitrobenzoic acid (DTNB,
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Sigma) in 80 mM Na HPO (pH 7.4). The To prepare raft–enriched fractions, the
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increase in absorbance at 410 nm was recorded, membrane pellets were treated with 0.5% Triton
and the specific enzyme activity was expressed X-100 in the cold and subjected to flotation in
as absorbance units/min/μg of protein. For the discontinuous sucrose gradients. About 20% of
measurement of ALP activity, 50 μl samples the total membrane-bound G AChE activity in
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from the gradient fractions were mixed with 50 the cerebrum was recovered in the raft-enriched
μl of 5 mM p-nitrophenylphosphate (pNPP, fractions at 5% - 30% sucrose interface. These
Sigma) in a buffer containing 0.1 M glycine, 1 fractions contained 60-70% of total
mM MgCl and 1 mM ZnCl (pH 10.4). After membrane-bound ALP, which served as a
2 2
incubation at 37 °C for 1-5 hours, 50 μl of 2 M membrane raft marker (28) (Fig. 1B). In
NaOH was added to stop the reaction, and the addition, Western blots analyses of these
absorbance was measured at 405 nm. The raft-enriched fractions showed the presence of
statistical significance of differences between the raft-associated proteins flotillin-2 (29) and
experimental data was evaluated by the PSD-95 (30). The transferrin receptor (TfR),
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PRIMER program, version 1 (Primer of which is not associated with rafts (31), was not w
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Biostatistics, S. A. Glantz, McGraw-Hill, Inc. detected in raft-enriched fractions, but only at d
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1988). the bottom of the gradient, in fractions fro
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containing proteins that had been solubilized by ttp
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RESULTS the detergent. The distribution of all these w
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marker proteins indicated that the method used .jb
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Membrane rafts isolated from adult rat brain for the isolation of membrane rafts was reliable. brg/
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contain PRiMA-linked G4 AChE. Total To analyze the molecular forms of AChE gue
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membrane preparations were obtained from that were associated with rafts, a sample of the t o
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fresh cerebrums of adult rats, as described in the raft fractions was solubilized with 1% Triton pril 3
experimental section. Sedimentation analysis X-100 and subjected to sucrose density , 2
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after solubilization with 0.5% Triton X-100 sedimentation, as illustrated in Fig. 1C: it
showed that they contained mostly AChE contained only the PRiMA-linked G form.
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tetramers (G ), with a small proportion of dimers
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(G ) and/or monomers (G ) (Fig. 1A). In order The association of PRiMA-linked AChE with
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to establish the association of PRiMA with G membrane rafts is developmentally regulated.
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AChE in the cerebrum, the protein extract was The association of PRiMA-linked G AChE with
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immunoprecipitated by anti-PRiMA antibody, as membrane rafts in the brain may be relevant to
previously reported (11, 15, 27). As shown in its physiological function. To address the
Fig. 1A, the antibody treatment depleted the significance of this association, we analysed this
peak of G AChE, but not the G or G forms. raft association during maturation of the brain.
4 1 2
This result confirmed that G AChE in the Because proteins differ in the detergent
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cerebrum is mostly associated with PRiMA. sensitivity of their attachment to rafts (32, 33),
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we used low (0.05%) and high (0.5%) examined the role of PRiMA in recruiting AChE
concentrations of cold Triton X-100 to evaluate into rafts. Mouse neuroblastoma X rat glioma
the association of AChE activity with rafts in the NG108-15 cells spontaneously express very
cerebrums of P7, P14 and P100 rats. The little PRiMA-linked AChE (34). They were
membrane-bound amphiphilic G AChE was the transfected with various cDNAs encoding
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major form of AChE at the three chosen stages HA-tagged PRiMA constructs together with
(Fig. 2A). In all cases, a higher proportion of HA-tagged AChE cDNA. Transient
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membrane-bound AChE was found to be transfection with AChE cDNA markedly
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associated with the rafts isolated with low Triton increased the level of AChE monomers, but only
X-100 than with high Triton X-100 (Fig. 2B), slightly that of membrane-bound tetramers,
which was opposite to the case of ALP (Fig. 2B indicating that the expression of PRiMA limited
and C). The fraction of AChE activity recovered the formation of PRiMA-linked G AChE in this
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in low detergent resistant rafts remained system (Fig. 3A). This was confirmed by the
essentially constant (50 to 60% of total fact that co-expression of AChE with the mouse
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membrane G AChE activity), while the fraction PRiMA I isoform did produce a high level of w
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in high detergent resistant rafts increased during membrane-bound G tetramers (Fig. 3A). d
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postnatal development, from ~10% at P7 to Because the recruitment of AChE in the raft fro
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more than 15% at P14 and P100 (Fig. 2C). fraction of cerebrum membranes was more ttp
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The fact that most of AChE was weakly obvious at a low detergent concentration, we w
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associated with rafts at low detergent was analysed its distribution in NG108-15 cell .jb
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demonstrated by subjecting a raft-enriched membrane fractions under similar conditions. brg/
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fraction (from 0.05% Triton X-100) to a second Considering the lower total protein gu
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flotation in the presence of 0.5% Triton X-100 concentration of the cell preparations, we used t o
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(Fig. 2D). The resulting high detergent resistant 0.02% instead of 0.05% cold Triton X-100. pril 3
raft fraction only contained 10 ± 0.5% (Mean ± When AChE subunits, tagged with HA, , 2
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SEM, n=3) of the AChE activity associated with were expressed alone in cultured NG108-15
the original low detergent resistant raft fraction. cells, AChE enzymatic activity and protein were
This result suggests the presence of two only present in the soluble fractions, i.e. at the
populations of G AChE in the rafts. The AChE bottom of the gradients (Fig. 3B left panel).
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fraction remaining associated with rafts at high When they were co-expressed with HA-tagged
detergent concentration could represent the PRiMA I, a significant fraction of both proteins
functional enzyme in the adult brain. was detected in the rafts (Fig. 3B right panels).
However, the soluble fractions appeared to
PRiMA directs G AChE to membrane rafts via contain a higher proportion of AChE protein
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its cholesterol-binding motif CRAC. Since the than the raft fractions, as shown in the Western
membrane rafts from the cerebrum only contain blots, but a lower proportion of AChE activity.
PRiMA-linked amphiphilic G AChE form, we This discrepancy between AChE protein and
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enzymatic activity suggests that the non-raft membrane proteins, named CRAC, located at
soluble fractions contained inactive AChE the membrane interface, is thought to interact
protein, probably not associated with PRiMA. with cholesterol; its sequence is defined as
The existence of inactive AChE has been L/V-(X) -Y-(X) -R/K-, where (-X-)
(1-5) (1-5) (1-5)
reported previously (34, 35, 36). As shown in represents between one and five residues of any
the brain, the raft-associated AChE in NG108-15 amino acid (19, 20). The 109-120 peptidic
cells expressing AChE and PRiMA was sequence overlapping the junction between the
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exclusively amphiphillic G AChE (Fig. 3C). transmembrane and the cytoplasmic domains,
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To verify that PRiMA-linked AChE was which is common to both PRiMA I and PRiMA
associated with cholesterol-sphingolipid II (VLV IIC YKA IKRK) (13, 15), corresponds
membrane rafts, we treated the cultured to a CRAC motif and has the appropriate
NG108-15 cells expressing AChE and PRiMA location. This motif is conserved among human,
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with 10 mM MβCD to deplete cholesterol. After mouse, rat and chick PRiMA sequences (Fig.
one hour, the cells started to become spherical. 5A). Therefore, the CRAC motif of PRiMA may
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This observation indicates that they could not be responsible for the association with w
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tolerate any longer treatments. However, they cholesterol and targeting PRiMA-linked AChE d
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were not detached from the plates, and MTT to the rafts. fro
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assay showed that the cell viability was not To assess the role of the CRAC motif, we ttp
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significantly changed after the treatment (data constructed a mutant called PRiMA IΔCRAC in w
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not shown). The efficiency of the cholesterol which key residues were modified, as underlined .jb
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depletion by MβCD was shown by the fact that (GIA IIC LKA ISDM). The amounts of brg/
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the cholesterol content of the total membrane PRiMA-linked G4 AChE in the transfected gue
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fraction, determined by a cholesterol detection NG108-15 cells expressing the wild type and t o
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kit and mass spectrometry, was decreased by mutated PRiMA were similar (Fig. 5B). This is pril 3
about 70 ± 2.5 % (mean ± SEM, n=3). in agreement with the fact that the mutation does , 2
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Cholesterol depletion reduced the ALP content not affect the PRAD motif, which is responsible
of membrane raft fractions by about half, and the for the assembly with AChE subunits. However,
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proportion of AChE activity in raft-enriched the proportion of membrane G AChE activity
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fractions even more markedly (Fig. 4 lower associated with the raft fraction, isolated with
panel). This was confirmed by Western blots of 0.02% Triton X-100, was only about 20% with
AChE and PRiMA proteins (Fig. 4 upper PRiMA I as compared to 50% with wild
T ΔCRAC
panel). These results indicated that the type PRiMA I; the amount of raft-associated
association of PRiMA-linked AChE with ALP, used as a control, was the same in both
membrane rafts depends on cholesterol. cultures (Fig. 5C). Thus, the CRAC motif of
There are several hypotheses on the PRiMA plays an important role in the
partitioning of proteins between raft and non-raft recruitment of AChE in the rafts, in cultured
domains (19, 37). A peptidic motif of integral neurons.
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complex was associated with rafts at low
PRiMA is responsible for the lower resistance to detergent concentration, while only ~10% was in
detergent for the membrane raft-associated G the raft fraction at high detergent concentration
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AChE. Because GPI-linked proteins are known (Fig. 6B), as previously observed for
to be associated with raft microdomains, we membranes from rat cerebrum (Fig. 2C).
wished to compare PRiMA-linked G AChE Therefore, the association of PRiMA with
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with GPI-linked AChE (G ) forms. In mammals, membrane rafts is much less resistant to
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AChE subunits possess a C-terminal GPI detergent than that of GPI-anchored AChE.
H
addition signal, they are mostly expressed in To confirm that anchoring through PRiMA
blood cells and produce GPI-anchored dimers, is responsible for the reduced association of
exposed at the cell surface (1). AChE tetramers AChE with rafts at high detergent concentration,
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could also be assembled and anchored in cell we analyzed a complex made from a mutant of
membranes through a GPI glycolipid when human ALP-t, in which the C-terminal
co-expressed with a fusion protein, Q -H , GPI-addition signal was replaced by a “t”
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composed of the N-terminus of Torpedo ColQ peptide derived from AChE (39). The w
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containing the PRAD, followed by the co-expression of ALP-t and PRiMA I in d
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C-terminal GPI-addition signal of Torpedo transfected NG108-15 cells formed amphiphilic, fro
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AChEH (8, 38). In cultured NG108-15 cells, membrane-anchored tetramers (Fig. 7A). Thus, ttp
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AChEH, as well as AChET + QN-HC and AChET the ALP-t protein could associate with PRiMA I, w
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+ PRiMA were over expressed (Fig. 6A), and like AChE . We analyzed the distribution of .jb
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their distribution in membrane fractions with ALP-t in raft and non-raft fractions, when brg/
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PRiMA-linked AChE were compared. expressed with or without PRiMA. When ALP-t gue
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The production of membrane-bound AChE was expressed alone, the proportion of t o
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in transfected cells was demonstrated by membrane-bound ALP activity in the raft pril 3
sedimentation in sucrose density gradients. As fraction was less than 20% at low detergent , 2
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expected, G AChE was produced by cells concentration (Fig. 7B): this proportion was
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expressing AChE with either PRiMA or Q -H , similar to that of control without any
T N C
while G AChE was produced by cells transfection and corresponded to the endogenous
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expressing AChE (Fig. 6A). The proportion of GPI-anchored ALP. The exogenous ALP
H
AChE activity recovered in the raft fractions of activity derived from ALP-t cDNA was
transfected NG108-15 cells was determined recovered at the bottom of the gradient (data not
under low (0.02%) and high detergent (0.2%) shown). When ALP-t was co-expressed with

conditions. The GPI-anchored molecules were PRiMA I, the proportion of membrane-bound
found to be associated with the membrane raft ALP activity recovered in the raft fraction
fraction at similar levels under low and high reached ~35% at low detergent concentration,
detergent conditions. In contrast, more than 50% but ~20% at high detergent concentration (Fig.
of the membrane-bound AChE -PRiMA 7B). Thus, the ALP-t-PRiMA complex is
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Xie et al., 2009
efficiently targeted to the raft microdomains, and 14). Both PRiMA variants could anchor AChE
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its association with rafts is sensitive to detergent, tetramers in cell membranes, but might differ in
like the AChE-PRiMA complex. The relatively their localization and interactions, and in
high proportion of ALP activity in the raft under particular in their recruitment into the membrane
high Triton X-100 condition, as compared to rafts.
that of PRiMA-linked AChE, may be accounted To analyze the possible role of
by the presence of endogenous GPI-anchored post-translational modifications on the insertion
ALP (Fig. 7B). of PRiMA-linked AChE complexes in the rafts,
To eliminate the influence of the we mutated the corresponding sites in the
endogenous GPI-anchored ALP, the ALP-t and intracellular C-terminus, as shown in Fig. 8A.
PRiMA co-transfected cells were pre-treated We compared the recruitment of AChE in rafts,
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with 0.4 U/ml phosphatidylinositol when co-expressed with PRiMA I, the mutants
phospholipase C (PI-PLC) at 30 oC for 1 hour PRiMA I-C114S and PRiMA I-SA under low
before the raft extraction (38). By eliminating and high detergent conditions (Fig. 8C). Similar
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the GPI-anchored ALP (background), the as already observed for the wild type PRiMA I, w
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proportions of PRiMA-linked ALP activity the proportion of membrane-bound AChE- d
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being recruited to the raft were around 35% and PRiMA complexes associated with rafts for all fro
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10% under low and high detergent condition, those mutants on the potential functional ttp
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respectively (Fig. 7B). These results support the residues of the C-terminus was ~40% at low w
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notion that the low affinity of the raft association detergent concentration (0.02% Triton X-100) .jb
c
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of PRiMA-linked G4 AChE is due to PRiMA, and ~10% at a higher concentration (0.5% brg/
y
but not AChET. Triton X-100) (Fig. 8C). The distribution of gue
s
endogenous ALP in raft and non-raft fraction t o
n
A
Role of the C-terminus of PRiMA in its was analyzed for comparison: it was similar pril 3
association with membrane rafts. We examined among all the transfected cells but varied under , 2
0
1
9
the possible role of the intracellular cytoplasmic different detergent conditions, i.e. ~20% of total
domain of PRiMA I in raft association was activity input with low detergent and ~60% with
analyzed. The cytoplasmic domain of PRiMA I high detergent, similar with the result shown in
contains a putative palmitoylation site (C114), Fig. 2C (lower panel).
close to the transmembrane domain, and In addition, we also compare the difference
potential serine phosphorylation sites (S131, between the shorter C-terminus in PRiMA II and
S138-140 and S142) (Fig. 8A). The the longer one in PRiMA I for raft association
corresponding post-translational modifications (Fig. 8B). We found that both PRiMA I and
may be involved in intracellular protein PRiMA II could recruit AChE complexes into
interactions and signaling (15). The minor splice rafts to the same degree under low detergent
variant PRiMA II also possesses C114, but not conditions. Under high detergent conditions,
the potential phosphorylation sites (Fig. 8B)(13, however, the recruitment of the complexes was
9
Xie et al., 2009
significantly lower with PRiMA II (~5%) than membrane rafts, the situation is in fact quite
with PRiMA I (~10%), suggesting that the complex (33, 40, 41), because of the
intracellular domain of PRiMA I might heterogeneity of low-density detergent-resistant
participate in the stabilization of AChE in rafts membrane domains (41, 42). These domains
(Fig. 8C). This might involve an interaction with differ in their lipid and protein composition, as
some scaffold proteins, associated with the rafts. well as in the detergent solubility of individual
Since PRiMA II and PRiMA I possess the same raft proteins (32, 41). The association of
CRAC motif, except for the final lysine residue PRiMA-linked G AChE with rafts is clearly
4
(Fig. 8B), the role of the C-terminus in heterogeneous in its sensitivity to the cold
recruiting AChE to the raft needs further detergent, both in brain membranes and in
investigation in the absence of a CRAC motif. transfected cells. Under low detergent condition
Therefore, we compared a PRiMA II mutant in (0.05% Triton X-100 for tissue and 0.02%
which the CRAC motif was suppressed (PRiMA Triton X-100 for cultured cells), around 50% of
II ) with PRiMA I . PRiMA II membrane-bound G AChE was found to be
ΔCRAC ΔCRAC ΔCRAC 4 D
o
recruited significantly less AChE than PRiMA associated with membrane rafts (Fig. 2C, 3B). A w
n
lo
a
I to the rafts, in either high or low detergent large part of this raft-associated AChE d
ΔCRAC e
d
conditions (Fig. 8C). This result strengthens the population, anchored through PRiMA I or fro
m
h
notion that the C-terminus of PRiMA plays a PRiMA II, could be dissociated by 0.5% cold ttp
://w
role in raft targeting. Triton X-100, perhaps reflecting a high mobility w
w
between raft and non-raft domains. A fraction of .jb
c
.o
DISCUSSION PRiMA-linked AChE remained to be associated brg/
y
with rafts at high detergent concentration, gu
e
s
Here, we demonstrate a partial association perhaps because of interactions with intracellular t o
n
A
of PRiMA-linked G4 AChE with membrane rafts raft-associated proteins (Figs. 2D, 8C). pril 3
in the rat cerebrum. This association was The proportion of membrane-bound AChE , 2
0
1
9
considerably reduced when the rafts were associated with rafts under high detergent
isolated in the presence of cold 0.5% Triton conditions increased from 10% at P7 up to 15%
X-100, rather than cold 0.05% Triton X-100, at P14 (Fig. 2C), suggesting a maturation
and therefore appeared weaker than that of process during the brain development and
GPI-anchored proteins, including the AChE G therefore a physiological role of this raft
2
form possessing a GPI anchor or a G AChE localization. Thus, two compartments of
4
form associated with a PRAD-containing raft-associated PRiMA-linked AChE may be
GPI-anchored construct (Q -H ), indicating that physiologically distinct.
N C
PRiMA determines the detergent sensitivity of It has long been known that the interaction
raft-associated AChE in the brain. between nicotinic acetylcholine receptors and
Although the resistance to extraction in cold scaffold proteins in muscle membranes reduces
Triton X-100 was initially considered to define their mobility and detergent extractability (43,
10