P25a
⁄
TPPP expression increases plasma membrane
presentation of the dopamine transporter and enhances
cellular sensitivity to dopamine toxicity
Anja W. Fjorback
1,2,
*, Sabrina Sundbye
2
, Justus C. Da
¨
chsel
2,
, Steffen Sinning
1
, Ove Wiborg
1
and Poul H. Jensen
2
1 Centre for Psychiatric Research, Aarhus University Hospital, Denmark
2 Department of Medical Biochemistry, Aarhus University, Denmark
Keywords
dopamine transporter; p25a; Parkinsons
disease; toxicity; TPPP; tubulin
polymerization promoting protein
Correspondence
P.H. Jensen, Department of Medical
Biochemistry, Aarhus University, Ole
Worms Alle 1170, DK-8000 Aarhus C,
Denmark
Fax: +45 86131160

and the dopamine transporter in HEK-293-MSR cells sensitized them to
the toxicity of extracellular dopamine. Neuronal expression of p25a thus
holds the potential to sensitize the cells toward dopamine and toxins
carried by the dopamine transporter.
Structured digital abstract
l
MINT-8055798: DAT (uniprotkb:Q01959) and p25 alpha (uniprotkb:O94811) colocalize
(
MI:0403)byfluorescence microscopy (MI:0416)
l
MINT-8054201: p25 alpha (uniprotkb:B1Q0K1), bip (refseq:GI:194033595), Synaptophysin
(uniprotkb:
Q62277), Alpha-synuclein (uniprotkb:Q3I5G7) and DAT (uniprotkb:C6KE31)
colocalize (
MI:0403)bycosedimentation in solution (MI:0028)
l
MINT-8055878: Synaptophysin (uniprotkb:Q62277), bip (refseq:GI:194033595) and p25-alpha
(uniprotkb:
O94811) colocalize (MI:0403)bycosedimentation through density gradient
(
MI:0029)
Abbreviations
a-syn, a-synuclein; DA, dopamine; DAT, dopamine transporter; NET, norepinephrine transporter; PBSCM, phosphate-buffered saline
supplemented with Ca and Mg; PD, Parkinson’s disease; SERT, serotonin transporter; VMAT-2, vesicle monoamine transporter-2.
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 493
Introduction
Parkinson’s disease (PD) is a progressive neurodegen-
erative disorder that is characterized by motor dys-
functions, including resting tremor, postural
imbalance, slowness of movement and muscle rigidity.

balance between the functional levels of plasma mem-
brane DAT and vesicular VMAT-2.
The protein a-synuclein (a-syn) is known to play a
central role in PD because mutations in its gene cause
familial PD, and it is a major component of Lewy
bodies in the sporadic forms of the disease [9]. All
though little is known about events that triggers the
death of dopaminergic neurons, it has been shown that
a-syn affects cellular DA homeostasis as cellular a-syn
decreases DAT-mediated DA uptake via mechanisms
inhibited by PD-causing mutations [10,11]. Because of
the a-syn-mediated effect on DA uptake, abnormal
function or aggregation of a-syn in dopaminergic neu-
rons may affect the DA balance, and thereby increase
oxidative stress in the cell.
The brain-specific protein p25a is normally only
expressed in oligodendrocytes, where it is thought to
affect myelin metabolism [12], possibly via modulation
of microtubule dynamics [13]. However, p25a is abnor-
mally expressed in neurons in a range of neurodegener-
ative diseases, where it is found in the cytoplasm and
nucleus and is often associated with inclusions contain-
ing aggregated a-syn [12,14–16]. It has been shown
that p25a directly stimulates the aggregation of a-syn,
and thereby induces cytotoxicity [16,17]. We hypothe-
sized that expression of p25a in dopaminergic neurons,
in addition to its putative effects on a-syn aggregation,
may increase the sensitivity to dopaminergic stress,
thus contributing to cell vulnerability. We demonstrate
that p25a increases DAT-mediated DA uptake in tran-

tive smear at  150 kDa that was also faintly visible for
DAT-expressing cells. The immunofluorescence analysis
demonstrated strong DAT immunoreactivity on the
plasma membrane of the DAT-expressing cells, with
essentially no signal for the mock-transfected control
p25a regulates dopamine transporter function A. W. Fjorback et al.
494 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
(Fig. 1B). Hence, the C-20 antibody binds specifically
and selectively to DAT in our cellular system, in agree-
ment with previous reports [18].
Figure 1C shows that expression of DAT caused a
cocaine-inhibitable and saturable uptake of [
3
H]-DA in
HEK-293-MSR cells, and this was enhanced by
co-expression with p25a. As a control, we also
A
C
D
E
B
Fig. 1. p25a increases dopamine uptake by increasing the surface
expression of the dopamine transporter. (A) HEK-293-MSR cells
were transiently transfected with DAT expression vector or empty
control vector for 48 h, and extracted for immunoblot analysis. Pro-
tein aliquots (20 lg) were resolved by 8–16% SDS ⁄ PAGE followed
by electroblotting. The filter was first probed with the C-20 goat
antibody, after which the membrane was stripped and reprobed
with an anti-actin antibody. Top panel, anti-DAT immunoreactivity;
molecular size markers indicated on the left. Lower panel, actin

MSR cells transiently transfected with DAT and p25a, a-syn or the
empty vector were either extracted directly in lysis buffer or sub-
jected to cell surface biotinylation with EZ-Link Sulfo-NHS-SS-Biotin
followed by extraction. This cross-linker allows cleavage between
biotin and the target by reducing the disulfide bridge. The biotinyla-
ted proteins were captured by incubating with NeutraAvidin beads.
The total and surface-bound protein fractions were analyzed by
reducing SDS ⁄ PAGE followed by Western blotting with antibodies
toward DAT, p25a, a-syn and b-actin (as loading control). Total cel-
lular DAT is present as two bands representing glycosylated and
non-glycosylated DAT, and was slightly lower in the double trans-
fected cells compared to those expressing DAT alone. By contrast,
surface-bound DAT was increased in p25a-expressing cells and
decreased in a-syn-expressing cells compared to control-transfected
cells. (E) Quantification of the surface DAT bands in (B) normalized
against b-actin as analyzed by densitometry and shown as a per-
centage of the control. The columns represent means ± 1 SEM of
three experiments. Comparison of the three conditions by one-way
ANOVA was significant (P < 0.05). Individual comparison of p25a or
a-syn to control by Student’s t test was also significant (*P < 0.05).
A. W. Fjorback et al. p25a regulates dopamine transporter function
FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 495
co-expressed DAT with a-syn, which is known to
decrease DAT activity [10], and noted a decrease in
DAT-mediated uptake. Kinetic analysis showed that
p25a increased the V
max
without affecting the K
m
(Table 1). This effect was not caused by increased DAT

p25aDN, which lacked N-terminal residues 3–43, and
p25aDC, which lacked C-terminal residues 156–219
(Fig. 2A). The truncated p25a proteins were compared
with full-length p25a with respect to their ability to
induce increased DA uptake when co-expressed with
DAT. Figure 2B demonstrates that the full-length and
two truncated p25a proteins all induce a similar
increase in DA uptake when expressed in the HEK-
293-MSR cells expressing DAT alone. This suggests
that the central folded core domain is responsible for
the stimulatory effect on DAT.
DAT belongs to the family of neurotransmitter
transporters that includes the norepinephrine and sero-
tonin transporters (NET and SERT), which share a
range of structural characteristics. The expression of
a-syn has been shown to decrease the membrane
expression of all three transporters [10,23,24]. Table 1
shows that p25a selectively stimulates uptake via DAT
Table 1. Determination of V
max
and K
m
for DAT, NET and SERT in
the presence and absence of p25a. HEK-293-MSR cells were tran-
siently transfected with DAT, NET or SERT and with p25a or mock
vector. The cells were subsequently used for analysis of DA and
serotonin uptake, respectively.
3
[H]-DA was used to determine V
max

SERT ⁄ pcDNA3 0.648 ± 0.04 3.2 ± 1.3
SERT ⁄ p25a 0.588 ± 0.06 3.81 ± 0.96
NET ⁄ pcDNA3 0.157 ± 0.01 1.25 ± 0.14
NET ⁄ p25a 0.135 ± 0.007 1.85 ± 0.46
DAT ⁄ pcDNA3 0.591 ± 0.013 2.49 ± 0.80
DAT ⁄ p25a 0.792 ± 0.016 2.47 ± 0.63
A
B
Fig. 2. Effect of p25a deletions on dopamine uptake. (A) p25a con-
tains a folded central core (gray box) and unfolded termini. Expres-
sion vectors for deletion mutants p25aDN, lacking residues 3–43,
and p25aDC, lacking residues 156–219, were constructed. (B) [
3
H]-
DA (2.5 l
M) uptake in HEK-293-MSR cells transfected with DAT
and control vector or DAT in combination with wild-type p25a,
p25aDM or p25aDC vector. Bars represent mean ± 1 SEM of three
independent experiments performed in triplicate, and are normal-
ized against a control expressing DAT alone. Comparison of the
four conditions by one-way ANOVA indicates significant differences
(P < 0.05). *P < 0.05 as compared to control by Student’s t test.
p25a regulates dopamine transporter function A. W. Fjorback et al.
496 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
but not via SERT and NET. Hence, the central folded
core of p25a selectively increases the surface expression
of DAT.
p25a and DAT are associated with light brain
vesicles
Immunoprecipitations and subcellular fractionations

biology. For comparison, the presence of a-syn was
also investigated because this protein is known to be
both cytosolic and vesicle-associated. a-syn showed a
less distinct localization, being present in most frac-
tions in significant concentrations. For instance, when
the synaptosomal fraction P2 is subjected to hypotonic
lysis, a-syn appear in the cytosolic fraction LS2 and
the vesicular fraction LP2 in almost equal amounts.
By contrast, p25a appears almost exclusively in the
light vesicular fraction LP2, with no protein in the
cytosolic fraction LS2. This indicates some specificity
in vesicle binding when compared to the membrane-
associated proteins DAT and synaptophysin.
Having established for the first time that p25a is a
vesicle-associated protein in subcellular brain fractions,
we investigated whether purified recombinant p25a can
bind directly to brain vesicles. A vesicle-binding experi-
ment comprising a vesicle-flotation assay was used,
as described previously for a-syn [25], wherein brain
homogenate was supplemented with 55% sucrose,
overlaid with a sucrose density gradient, and subjected
to ultracentrifugation. Lipid-containing vesicles have a
low density and float up into the gradient, whereas
A
B
Fig. 3. Co-fractionation of DAT and p25a in subcellular fractions of
porcine striatal tissue and localization in cells. (A) Isolated porcine
striatal tissue (nucleus caudatus) was fractionated as described in
Experimental procedures. The fractions were crude pellet (P1),
microsomal fraction (P3), cytosolic fraction (S3), lysed dense synap-

not shown). The full-length p25a protein was subse-
quently biotinylated to allow it to be distinguished
from endogenous porcine brain p25a. Incubation of
brain vesicles with biotinylated p25a prior to vesicle
flotation resulted in the recovery of biotinylated p25a
in light fractions 2–6. As a control, a sample was
supplemented with 1% Triton X-100 and 1% SDS to
A
B
Fig. 4. p25a binds to porcine brain vesicles. (A) The wild-type and deletion constructs of p25a p25aDN, p25aDC and the p25a core, corre-
sponding to residues 44–156, were cloned into pET11d vector, expressed in E. coli and purified. The purified proteins were subjected to
reducing SDS ⁄ PAGE and Coomassie blue staining. Molecular size markers are shown on the left. (B) The association of p25a with brain ves-
icles was demonstrated by subjecting a porcine brain homogenate to a vesicle flotation assay. The assay is based on the light vesicles float-
ing in the density gradient and the cytosolic proteins remaining in the bottom fractions. The homogenate was supplemented with sucrose to
55%, overlaid with a sucrose density gradient of 48–20%, and subjected to ultracentrifugation, after which nine fractions were isolated from
the top of the gradient. The isolated fractions were subjected to reducing SDS ⁄ PAGE and immunoblotting for detection of endogenous p25a
(E-p25a), BIP and synaptophysin. BIP and synaptophysin immunoreactivity were detected on the filter where biotinylated p25a (B-p25a) had
been resolved. To ensure that the flotation was due to binding to light vesicles, 1% Triton X-100 and 1% SDS were added to dissolve the
membranes, as demonstrated for BIP, synaptophysin and biotinylated p25a (B-p25a + TX). As a positive control for the immunoblot, purified
recombinant p25a is shown on the left (R-p25a). To measure direct binding of recombinant full-length p25a to the brain vesicles, purified
protein was biotinylated (B-p25a) and incubated with the brain homogenate prior to vesicle flotation, followed by visualization with horse-
radish peroxidase-conjugated streptavidin. Similarly, the truncated peptides p25aDN and p25aDC were incubated with the homogenate and
treated like B-p25a, except they were detected with using p25a-1 antibody. Representative data from one of three independent experiments
are presented.
p25a regulates dopamine transporter function A. W. Fjorback et al.
498 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
dissolve the vesicles prior to analysis. This caused the
biotinylated p25a tracer to remain in the bottom frac-
tions, thus demonstrating that the flotation was due to
binding to vesicles. Because the vesicle-binding profile

compatible with an association with vesicles and the
plasma membrane. p25a showed cytosolic staining, was
localized in the cytosol with a less granular appearance
than DAT. Focusing on the finer neurites revealed that
p25a and DAT co-localize in some instances, but it
should be remembered that both proteins are highly
over-expressed, and this may saturate specific inter-
actions and reduce the signal-to-noise ratio (Fig. 3B).
Therefore, we conclude that p25a is a brain vesicle-
binding protein that may associate with DAT-contain-
ing brain vesicles.
p25a increases dopamine toxicity
Oxidative stress caused by cytoplasmic DA may con-
tribute to PD-associated cell death. To investigate a
putative role for aberrant neuronal p25a expression in
this scenario, we incubated HEK-293-MSR cells with
0.5 mm DA for 24 h, and quantified their viability
using the MTT assay. Expression of DAT, p25a and
a-syn alone, or p25 or a-syn together with DAT
respectively, did not affect survival (data not shown),
but the presence of 0.5 mm DA for 24 h caused a 20%
reduction in the survival of DAT-expressing cells but
had no effect on p25a- and a-syn-expressing cells
(Fig. 5). Co-expression of DAT and p25a enhanced
the DA toxicity to 50%, in agreement with an
increased DA uptake (Fig. 5). By contrast, co-expres-
sion of DAT with a-syn was used as a control for
protein over expression. a-syn eliminated the toxicity
produced by addition of DA (Fig. 5).
Discussion

cal role of these systems in degeneration of dopaminer-
gic neurons has been shown through both genetic and
chemical evidence. DAT is an import molecule for
environmental toxins causing PD [27], and there is an
increased sensitivity toward DAT-dependent toxins in
VMAT-2 heterozygous mice [28]. The instability of
DA at the neutral pH of the cytosol causes the forma-
tion of toxic aminochromes, and these species are not
formed when DA is protonated at the acidic pH of the
storage vesicles. Factors that increase the surface
expression of DAT holds the potential to increase
cytosolic DA by disturbing the balance between DAT
and VMAT-2 and causing chronic oxidative stress due
to an increased concentration of toxic DA metabolites
[26]. Abnormal expression of p25a in nerve cells has
been shown in the Parkinson’s disorders PD, multiple
systems atrophy and Lewy body dementia [16,29,30],
in which it is principally associated with a-syn-contain-
ing inclusions but also with other a-syn-negative cyto-
plasmic and nuclear inclusions [14].
The p25a protein is normally expressed in myelinat-
ing oligodendrocytes [12,31]. p25a is a microtubule-
associated protein that stimulates the aggregation of
tubulin in vitro [13], but cellular experiments have also
shown an effect on the actin system via (LIMK1) a
cytoplasmic serine/threonine kinase [32]. The impor-
tance of vesicular transport in regulating DAT sorting
to the plasma membrane, combined with ectopic
expression of the microtubule regulator p25a in degen-
erating dopaminergic neurons, suggested that p25a

the two antigens. We performed a vesicle binding experi-
ment to confirm that the association of p25a with brain
vesicles could be mediated by direct binding, and also to
investigate structural requirements for an association.
The assay is based on subjecting a brain homogenate to
density gradient centrifugation, whereby the lower den-
sity of vesicles makes them float in the gradient whereas
soluble proteins remain at the bottom. Biotinylated full-
length p25a did bind to vesicles, and addition of deter-
gents to solubilize the vesicles abolished the flotation of
biotinylated p25a. Non-biotinylated N- and C-termi-
nally truncated p25a proteins showed a vesicular bind-
ing pattern similar to that of endogenous p25a,
suggesting that the folded core domain possesses the
structure necessary for the vesicle binding function.
However, direct evidence could not be obtained because
the recombinant p25a core protein was insoluble at neu-
tral pH. The similarity between the core sequences of
the a, b and c p25 gene products suggests that the vesi-
cle-binding function may be a common property for this
protein family [35]. It should be remembered that p25a
is preferentially expressed in oligodendrocytes, and is
present in both the cell body and myelin sheets [12,31],
and its putative functions related to microtubules and
vesicle transport warrant further investigations. The
specificity of vesicle association in combination with
the direct binding of p25a to vesicles suggest the pres-
ence of vesicular p25a receptors, and this is now under
investigation.
The functional role of the vesicle interaction was

pcDNA3 vectors expressing DAT and NET were a kind
gift from Dr Susan Amara (Center for Neuroscience, Uni-
versity of Pittsburgh, PA, USA). SERT was cloned into the
pcDNA3 vector as previously described [37]. cDNA encod-
ing a-syn or p25a was cloned into the pcDNA3 vector as
described previously [16,38].
Prokaryotic expression vectors containing inserts for
human p25a, N-terminally truncated by amino acid resi-
dues 3–43 (p25aDN) and C-terminally truncated by residues
156-219 (p25aDC) were used, and were purified as described
previously [17,19,22,39]. The central folded core of p25a,
corresponding to amino acid residues 44–156, was amplified
and tagged with six histidine residues by PCR using for-
ward primers 5¢-CACCATCACGGAGCATCCCCTGAG-
3¢,5¢-TCGCATCACCATCACCATCACGGAGCA-3¢ and
5¢-CACCCATGGGATCGCATCACCAT-3¢, and reverse
primer 5¢-CACGGATCCCTACGTCACCCCTGA-3¢. After
digestion with NcoI and BamHI restriction enzymes, the
insert was ligated into pET11d vector (Novagen, Rodovre,
Denmark). Correct insertion was verified by DNA sequenc-
ing (Eurofins-MWG, Martinsried, Germany). The protein
was expressed in Escherichia coli BL21 (DE3) cells (Strata-
gene, La Jolla, CA, USA), and extracted by sonication on
ice in 50 mm NaH
2
PO
4
, pH 7.0. Cell debris was removed
by centrifugation at 1000 g and the supernatant was fil-
trated. The hexahistidine-tagged p25a core protein was

fied Eagle’s medium supplemented with 10% fetal calf
serum, 2 mm glutamine, 100 lgÆmL
)1
streptomycin and
100 UÆmL
)1
penicillin at 37 °C and 5% CO
2
. The cells were
transfected 48–72 h prior to the experiments with appropri-
ate amounts of plasmid and Genejuice (Novagen) mixed
with medium according to the manufacturer’s recommenda-
tions. SH-SY5Y cells were grown in Dulbecco’s modified
Eagle’s medium supplemented with 5% FCS and 100 lg/mL
pen ⁄ strep, and zeocin (25 lgÆmL
)1
) was added to the med-
ium for selection of stably transfected cell lines. The cells
were not differentiated prior to experimentation.
Immunofluorescence microscopy
For cellular localization studies, HEK and SH-SY5Y cells
were transiently transfected with human DAT and p25a.At
36 h after transfection, the cells were fixed in 4% parafor-
maldehyde for 10 min, and subsequently permeabilized for
30 min (50 mm glycine, 0.1% Triton-X-100, 3 mm CaCl
2
,
2mm MgCl
2
). After blocking for 30 min with 3% BSA in

ton X-100, 0.1% SDS and protease inhibitor cocktail
(Complete EDTA-free tablets, Roche, Denmark)] for
30 min at 4 °C under gentle shaking. The cell lysate was
cleared by centrifugation at 12 000 g at 4 °C for 15 min,
and the remaining supernatant was mixed with SDS sample
buffer (250 mm Tris ⁄ HCl, pH 6.8, 5% SDS, 0.25% brom-
ophenol blue, 25% glycerol) and left at 37 °C for 30 min.
Fractions of the cell samples were analyzed by 8–16%
SDS ⁄ PAGE and transferred to nitrocellulose membrane.
The membrane was blocked for 2 h in 5% dry milk in
TBST buffer (pH 8.0, 50 mm Tris ⁄ HCl, 150 mm NaCl,
0.5% Tween-20), and then probed overnight with primary
antibody (1 : 1000, except ASY1 at 1 : 500), followed by
incubation with horseradish peroxidase-conjugated second-
ary antibody: anti-goat antibody (1 : 2500) or anti-rabbit
antibody (1 : 1000). Proteins were visualized using an ECL
Advance Western blotting detection kit (GE Healthcare,
Denmark) and developed on a Kodak Image station 440
(Denmark).
Uptake activity
Dopamine or serotonin uptake assays were performed 48 h
after transfection of the cells. The medium was removed and
the cells were washed with NaCl ⁄ P
i
(137 mm NaCl, 2.7 mm
KCl, 4.3 mm Na
2
HPO
4
, 1.4 mm KH

of dopamine and serotonin ranged from 0–10 lm. For single
V
max
determinations, only one concentration (5 lm)of[
3
H]-
dopamine diluted 20 times with unlabeled dopamine was
used. Washing twice with PBSCM terminated the uptake. All
washing steps were performed using an automated plate
washer. Following uptake, cells were solubilized in scintilla-
tion solution (MicroScient-20, Packard Bell, Denmark), and
plates were counted in a Packard Top counter. Values are the
mean of six replicates. The specific uptake was determined by
subtracting the uptake counts in the absence of inhibitor
from the uptake counts in the presence of cocaine or S-cita-
lopram, respectively. We also confirmed that no uptake
occurred in mock-transfected HEK cells. Assuming Michal-
is–Menten kinetics, the data were plotted and analyzed by a
non-linear-squares curve fit (GraphPadPrism, Denmark).
The amount of cellular protein per well for each condition
was determined as the mean values for the contents of three
individual wells extracted in 0.1 m NaOH and measured
using the bicinchoninic acid method.
DA toxicity assay
Dose-dependent DA toxicity was evaluated in the
0.1–2.5 mm DA concentration range, and a concentration
of 0.5 mm was used for further survival studies. HEK-
MSR-293 cells were transfected in a solution with a fixed
concentration of vector DNA (empty vector or the DAT,
AS or p25a plasmids), and 5000 cells ⁄ well were plated in

retained for determination of total protein. The remainder
of the supernatant was incubated with NeutraAvidin
(Pierce, Rockford, IL, USA) beads to precipitate the bioti-
nylated proteins. The beads were washed four times in
PBSCM before elution with 50 lLof20mm dithioerythrei-
tol-containing SDS sample buffer, and incubated for 30 min
at 37 °C. The dithioerythreitol reduces the disulfide bridge
in the cross-linker between biotin and the target proteins,
and thus allows their analysis by SDS ⁄ PAGE.
Subcellular fractionation of porcine striatal brain
tissue
Porcine brain cut in half in the saggital plane was obtained
fresh from a local abattoir and immediately cooled on ice.
Within 2 h, the nucleus caudatus, i.e. the part of the
p25a regulates dopamine transporter function A. W. Fjorback et al.
502 FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS
striatum facing the lateral ventricle, was dissected and fro-
zen in liquid nitrogen followed by storage at ) 80 °C. A
method originally described for rat brain tissue was used
for subcellular fractionation [40]. All procedures were per-
formed on ice or at 4 °C. Frozen tissue (2 g) was thawed in
10 mL ice-cold homogenization buffer [320 mm sucrose,
4mm HEPES ⁄ NaOH, pH 7.4, 2 mm EDTA, Complete
proteinase inhibitor (Roche)]. The brain was homogenized
using a glass-Teflon Dounce homogenizer, and the homoge-
nate was centrifuged for 10 min at 1000 g. The resulting
pellet (P1) was frozen. The supernatant (S1) was collected
and centrifuged for 15 min at 12 000 g, yielding S2 and P2.
P2 was washed by resuspension in 8.5 mL of homogeniza-
tion buffer, and re-centrifuged for 15 min at 10 200 g,

lation reaction was quenched using 1 m Tris, pH 8.2, and
excess NHS-PEO4-Biotin was removed by fast desalting on
a PC3.2 ⁄ 10 column (Amersham, Denmark).
Vesicle isolation and binding were performed as
described previously [25]. Briefly, 1 g porcine brain was
homogenized in 2.5 mL of 5 mm dithiothreitol, 2 mm
EDTA, 9% sucrose, 25 m m MES, pH 7.0, in the presence
of protease inhibitors (Complete EDTA-free tablets;
Roche). Nuclei and debris were removed by centrifugation
at 500 g for 5 min at 4 °C, and a crude vesicle fraction was
isolated by ultracentrifugation of the supernatant at
100 000 g for 1 h at 4 °C. After ultracentrifugation, the
pellet was resuspended in 400 lL homogenization buffer,
and 100 lL of resuspended vesicles were incubated with
1 lm recombinant p25a protein (biotinylated p25a or unla-
beled truncated p25DN or p25DC) for 2 h at 4 °C. The
truncated peptides migrate faster than endogenous p25a,
and thus need no biotinylation. For the negative control,
1% Triton X-100 and 1% SDS were added to the sample
to solubilize the vesicles. The solution was brought to 55%
sucrose in a volume of 0.47 mL, and overlaid with 4 mL
48–20% sucrose gradient. The samples were then subjected
to ultracentrifugation at 100 000 g for 16 h at 4 °C. The
gradient was divided into nine fractions starting from the
top. The protein content of each fraction was precipitated
with 20% trichloroacetic acid and subjected to SDS ⁄ PAGE
followed by Western blotting. Endogenous p25a as well as
recombinant p25DN and p25DC were visualized using
anti-p25a1 IgG (rabbit) (1 : 1000) followed by horseradish
peroxidase-conjugated anti-rabbit IgG (1 : 1000) (DAKO).

FEBS Journal 278 (2011) 493–505 ª 2010 The Authors Journal compilation ª 2010 FEBS 503
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