Download Protein 4.2 interaction with hereditary spherocytosis mutants of the

Biochem. J. (2011) 433, 313–322 (Printed in Great Britain)
313
doi:10.1042/BJ20101375
Protein 4.2 interaction with hereditary spherocytosis mutants of the
cytoplasmic domain of human anion exchanger 1
Susan P. BUSTOS* and Reinhart A. F. REITHMEIER†1
*Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8, and †Department of Medicine, University of Toronto, Toronto, ON, Canada, M5S 1A8
AE1 (anion exchanger 1) and protein 4.2 associate in a protein
complex bridging the erythrocyte membrane and cytoskeleton;
disruption of the complex results in unstable erythrocytes and HS
(hereditary spherocytosis). Three HS mutations (E40K, G130R
and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur
with deficiencies of protein 4.2. The interaction of wild-type
AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1),
kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian
ovalocytosis AE1) with protein 4.2 was examined in transfected
HEK (human embryonic kidney)-293 cells. The HS mutants had
wild-type expression levels and plasma-membrane localization.
Protein 4.2 expression was not dependent on AE1. Protein 4.2
was localized throughout the cytoplasm and co-localized at the
plasma membrane with the HS mutants mdAE1 and kAE1, but
at the ER (endoplasmic reticulum) with AE1SAO. Pull-down
assays revealed diminished levels of protein 4.2 associated with
the HS mutants relative to AE1. The mdAE1 did not bind protein
4.2, whereas kAE1 and AE1SAO bound wild-type amounts of
protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had
decreased plasma-membrane localization compared with wildtype protein 4.2, and co-expression with AE1 enhanced its plasmamembrane localization. Subcellular fractionation showed the
majority of wild-type and G2A/C173A protein 4.2 was associated
with the cytoskeleton of HEK-293 cells. The present study shows
that cytoplasmic HS mutants cause impaired binding of protein 4.2
to AE1, leaving protein 4.2 susceptible to loss during erythrocyte
development.
INTRODUCTION
The locations of the three HS mutations are indicated and do not
cluster together. We have previously shown that these mutations
have little or no effect on the oligomerization, stability or folded
structure of the isolated domain, other than a slight destabilization
caused by the P327R mutation [7].
Protein 4.2 is a 72 kDa peripheral membrane protein [8] that
binds to both AE1 and ankyrin and is believed to strengthen
the membrane−cytoskeletal link [9,10]. The 72 kDa isoform of
protein 4.2 is referred to as Type II and is the smaller and more
predominant of two protein 4.2 splice variants. Type I is the less
common larger isoform and has an additional 30 amino acids near
the N-terminus [11]. Amino acid numbering of protein 4.2 in the
present paper will be according to the Type II isoform. Protein 4.2
is a member of the TG (transglutaminase) family of enzymes, but
has no catalytic activity [12]. It is myristoylated at Gly2 [13] and
palmitoylated at Cys173 [14], which may allow association with
cellular membranes. Protein 4.2 has been shown to associate
with the plasma membrane of Sf9 insect cells [15] and Xenopus
oocytes [16] in the absence of AE1. In Sf9 cells, myristoylation
of Gly2 was required for plasma-membrane localization.
In Figure 1 (right-hand panel), a homology model we
constructed of protein 4.2 using human TG2 [17] as a template,
is placed next to the crystal structure of cdAE1 to show their
relative sizes. The N-terminal region of protein 4.2, encompassing
residues 1−238, has been shown to represent the cdAE1-binding
domain [14]. Residues 157−181 were found to be critical for the
interaction and predicted formation of a β-hairpin in the centre
of protein 4.2 with Cys173 located at the bend. Another study
HS (hereditary spherocytosis) is an inherited human haemolytic
anaemia characterized by erythrocytes that become unstable due
to weakened interactions between the erythrocyte membrane and
cytoskeleton. The AE1 (anion exchanger 1, Band 3) membrane
glycoprotein and the cytoskeletal proteins ankyrin, spectrin and
protein 4.2 are involved in this interaction, and 20 % of HS cases
are caused by mutations in AE1 [1]. AE1 is the major membrane
glycoprotein of the erythrocyte and exists as dimers and tetramers.
Each monomer comprises two distinct domains: the C-terminal
52 kDa membrane domain (mdAE1) spans the bilayer up to
12 times and carries out the electroneutral exchange of chloride
for bicarbonate [2]; the N-terminal 43 kDa cytoplasmic domain
(cdAE1) binds glycolytic enzymes, haemoglobin and cytoskeletal
proteins, including ankyrin and protein 4.2.
Three mis-sense mutations in the cdAE1, E40K (Band 3
Montefiore), G130R (Band 3 Fukuoka) and P327R (Band 3
Tuscaloosa), cause HS while maintaining a normal amount of AE1
in the mature erythrocyte [3−5]. However, a marked deficiency
of protein 4.2 is seen, suggesting a destabilization of the protein
4.2−AE1 linkage. The homozygous E40K and G130R mutations
result in 88 % and 55 % decreases in protein 4.2 respectively,
whereas the heterozygous P327R mutation results in a 29 %
decrease in protein 4.2. This deficiency may be due to degradation
of unstable protein 4.2 or mis-sorting of protein 4.2 during
enucleation of reticulocytes. Figure 1 (left-hand panel) shows the
crystal structure of cdAE1 found to exist as a symmetric dimer [6].
Key words: anion exchanger 1 (AE1), erythrocyte cytoskeleton,
hereditary spherocytosis (HS), protein 4.2, protein interaction,
erythrocyte membrane.
Abbreviations used: AE1, anion exchanger 1; AE1SAO, Southeast Asian ovalocytosis AE1; cdAE1, cytoplasmic domain of AE1; CNX, calnexin; Cy3,
indocarbocyanine; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplamic reticulum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HA,
haemagglutinin; HEK, human embryonic kidney; HS, hereditary spherocytosis; kAE1, kidney isoform of AE1; mdEA1, membrane domain of AE1; Ni-NTA,
Ni2+ -nitrilotriacetate; PNA, peanut agglutinin; TG, transglutaminase; TM, transmembrane.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
314
Figure 1
S. P. Bustos and R. A. F. Reithmeier
Crystal structure of cdAE1 and homology model of protein 4.2
The symmetric dimer of cdAE1 (left-hand panel) has been crystallized and its structure determined by X-ray diffraction (PDB code 1HYN) [6]. The two subunits are coloured in grey and green.
Residues Gly130 and Pro327 , the locations of the HS mutations G130R and P327R, are indicated. Residues 1−54 (red dotted line) were not resolved in the structure. The E40K HS mutation is located
here and the approximate location of residue Glu40 is indicated on the structure. Residues 1−65 (red) are missing in kAE1. The structure of cdAE1 is shown alongside the homology model of protein
4.2 (right-hand panel). The protein 4.2 structure was created with SWISS-MODEL [45]. The human TG2 structure (PDB code 1KV3) [17] was used as the template instead of sea bream TG, as was
used previously [16], since it is a human homologue, is found in erythrocytes, and the mouse TG2 interacts with mouse cdAE1 and protein 4.2 [46]. Our protein 4.2 model is oriented such that the
palmitoyl group at Cys173 points upwards towards the conventional placement of the plasma membrane. This orientation matches that of the published crystal structure of cdAE1. The regions in blue
and green of the protein 4.2 model represent the 23 kDa N-terminal cdAE1-binding region. Residues 33−45 are coloured in light green. Residues 157−181 are coloured in dark green, and Cys173
is indicated. Structures of cdAE1 and protein 4.2 are on the same scale. aa, amino acids.
found that amino acids 33−45 of protein 4.2 contained a cdAE1binding site [18]. Basic residues Arg34 and Arg35 were essential
for the interaction and may bind to an acidic region, such as the
extreme acidic N-terminus of cdAE1.
Since the E40K, G130R and P327R AE1 mutations all affect
the levels of protein 4.2 in erythrocytes, we believe that these
sites form part of a large binding surface for protein 4.2. We
hypothesize that each of these positively charged HS mutations
change the surface of cdAE1, thereby weakening protein 4.2
binding. To test this hypothesis, the interaction of protein 4.2 with
wild-type AE1 and the three cytoplasmic HS mutants (AE1HS)
was studied in transfected HEK (human embryonic kidney)-293
cells. The mdAE1, which is missing the cytoplasmic domain, is
unable to bind protein 4.2 [8] and was included in the present
studies as a negative control.
A truncated kidney isoform of AE1 (kAE1) is missing the first
65 amino acids [19], which are shown in red in Figure 1 (left-hand
panel) and include a central β-strand. This isoform exchanges Cl −
for HCO3 − at the basolateral membrane of α-intercalated cells in
the collecting ducts of the distal nephron [20]. The cytoplasmic
domain of kAE1 (cdkAE1) is less stable than erythroid cdAE1
and exists in a more open structure [21]. In addition it does not
bind ankyrin or glycolytic enzymes [22,23], most probably due
to absence of the acidic N-terminal tail. We included kAE1 in
the present studies to see what effect the missing tail and altered
structure would have on protein 4.2 binding. Also, protein 4.2 is
expressed in kidney cells [24], where it may perform a similar
function as in erythrocytes.
AE1SAO (Southeast Asian ovalocytosis AE1) is a condition
caused by a nine amino acid deletion at the beginning of the first
TM (transmembrane) domain [25]. The deletion results in rigid,
oval-shaped erythrocytes [26] caused by an increased cytoskeletal
attachment [27−29]. We wondered whether this translated into an
enhanced interaction with protein 4.2, so we included AE1SAO in
c The Authors Journal compilation c 2011 Biochemical Society
the present studies. This mutant traffics to the plasma membrane in
erythrocyte precursors [30], but is retained in the ER (endoplasmic
reticulum) in HEK-293 cells [31], allowing us to determine
whether protein 4.2 interaction with AE1 can occur at the ER.
The localization and interaction of protein 4.2 with these AE1
variants was examined in HEK-293 cells, as well as the role of
fatty acid modifications on protein 4.2 localization. Protein 4.2
had a broad distribution in transfected cells, was predominantly
associated with the cytoskeletal fraction and co-localized with
AE1 at the plasma membrane. The AE1HS mutants, but neither
kAE1 nor AE1SAO, had impaired protein 4.2 binding; this
weakened interaction may account for the loss of protein 4.2
during erythrocyte development.
EXPERIMENTAL
Materials
The following is a list of materials used: pcDNA3 vector
(Invitrogen); mutagenic primers (ACGT Corporation);
QuikChange® site-directed mutagenesis kit (Stratagene); HEK293 cells (A.T.C.C.); DMEM (Dulbecco’s modified Eagle’s
medium), calf serum, penicillin and streptomycin (Gibco BRL);
LipofectamineTM 2000 (Invitrogen); C12 E8 (Nikko Chemicals);
Protein G−Sepharose (Amersham Biosciences); Poly-L-lysine
(Sigma−Aldrich); Ni-NTA (Ni2+ -nitrilotriacetate)–agarose
resin (Qiagen); mouse anti-AE1 (BRIC 6) antibody, which
recognizes an extracellular epitope at the C-terminus of AE1
(Bristol Institute for Transfusion Sciences, Bristol, U.K.); mouse
anti-AE1 antibody, which recognizes an intracellular epitope of
AE1 (a gift from Dr Michael L. Jennings, University of Arkansas
for Medical Sciences, Little Rock, AR, U.S.A.); rabbit anti-Cterminal AE1 antibody raised against the last 16 amino acids of
AE1 (SynPep Corporation); mouse anti-HA (haemagglutinin)
Interaction of protein 4.2 with HS mutants of AE1
antibody (Covance); rat anti-HA antibody (Roche); rabbit
anti-CNX (calnexin) antibody (Stressgen Biotech); lectin PNA
(peanut agglutinin) Alexa Fluor® 488 conjugate (Molecular
Probes); mouse anti-actin antibody (Chemicon); mouse
anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
antibody (Millipore); goat horseradish-peroxidase-conjugated
anti-rabbit IgG and anti-mouse IgG (New England Biolabs);
Cy3 (indocarbocyanine)-conjugated goat anti-mouse, anti-rat
and anti-rabbit antibodies (Jackson ImmunoResearch); Alexa
Fluor® 488-conjugated goat anti-mouse and anti-rat antibodies
(Molecular Probes); and a chemiluminescence kit (Roche).
Site-directed mutagenesis
The coding sequence for wild-type human AE1 was inserted into
the XhoI and BamHI sites of the pcDNA3 vector. The construction
of AE1SAO and kAE1 have been described previously in
[32] and [33] respectively. The mdAE1 was constructed using
PCR on the full-length AE1 protein with a methionine residue
engineered at the start of the DNA sequence coding for amino
acids Asp369 −Val911 , followed by subcloning into the pcDNA3
vector. Asp369 is the first perfectly conserved residue in the
human AE1 family (AE1, AE2 and AE3) defining the beginning
of the membrane domain. The AE1HS mutants were created
using the QuikChange® mutagenesis kit using complementary
mutagenic primers, with wild-type AE1 as template. The coding
sequence for wild-type human protein 4.2, Type II, was subcloned
from the pGEM-7Zf(+/ − ) vector into the EcoRI site of the
pcDNA3 vector. The C-terminal HA tag on protein 4.2 was
created by PCR, inserting the tag after Ala691 . The G2A/C173A
fatty acylation mutant of protein 4.2 was created using the
QuikChange® mutagenesis kit using complementary mutagenic
primers, with wild-type HA-tagged protein 4.2 as a template. HAtagged protein 4.2 and the HA-tagged G2A/C173A mutant were
used in all experiments, but are referred to as protein 4.2 and
G2A/C173A in the text respectively. Sequencing of constructs
was performed by ACGT Corporation.
Transient transfection and expression of AE1 and protein 4.2 in
HEK-293 cells
HEK-293 cells were grown in DMEM supplemented with 10 %
(v/v) calf serum, 0.5 % penicillin and 0.5 % streptomycin under
5 % CO2 at 37 ◦ C as described previously [34]. Cells were
transfected using the LipofectamineTM method [35], with 2 μg
of plasmid DNA per well of a six-well plate.
SDS/PAGE and immunoblotting
Proteins were resolved by SDS/PAGE (8 % or 10 % gels) [36] and
transferred to nitrocellulose membrane [37]. AE1 was detected
using a mouse monoclonal anti-AE1 antibody [38]. HA-tagged
protein 4.2 was detected using a mouse monoclonal anti-HA
antibody. Actin was detected using a mouse anti-actin antibody,
and GAPDH was detected using a mouse anti-GAPDH antibody.
Goat horseradish-peroxidase-conjugated anti-mouse IgG was
then added, followed by detection by chemiluminescence and
film exposure or by a VersaDoc Imaging System Model 5000.
Band intensities of immunoblots in the linear range of intensity
were determined using ImageJ 1.41o software.
Immunofluorescence and confocal microscopy
HEK-293 cells transfected with pcDNA3 plasmids were grown on
glass coverslips. In some cases, coverslips were coated with PolyL-lysine, but this made no difference to the growth or adherence
315
of the cells. Cells were fixed with 3.8 % (w/v) paraformaldehyde
for 15 min and washed once with 100 mM glycine. Cells were
either non-permeabilized or permeabilized and incubated with
antibodies. Non-permeabilized cells were blocked with 0.2 %
BSA for 30 min, followed by incubation with 1:100 diluted mouse
anti-AE1 (BRIC 6) antibody or 1:100 diluted PNA Alexa Fluor®
488-conjugated antibody in 0.2 % BSA for 30 min. These cells
were then permeabilized with 0.2 % Triton X-100 for 5 min and
blocked with 0.2 % BSA for 30 min. Next, 1:250 diluted rat antiHA antibody was added in 0.2 % BSA for 30 min. Cells that were
permeabilized at the beginning were permeabilized with 0.2 %
Triton X-100 for 5 min and blocked with 0.2 % BSA for 30 min.
These cells were then incubated with 1:500 diluted mouse antiAE1 antibody, 1:100 diluted mouse anti-AE1 (BRIC 6) antibody,
1:250 diluted rat anti-HA antibody or 1:250 diluted rabbit antiCNX antibody in 0.2 % BSA for 30 min. Following several
washes, samples were incubated with a 1:1000 dilution of Alexa
Fluor® 488-conjugated goat anti-mouse antibody, Alexa Fluor®
488-conjugated goat anti-rat antibody, Cy3-conjugated goat antimouse antibody, Cy3-conjugated donkey anti-rat antibody or Cy3conjugated donkey anti-rabbit antibody for 30 min. A Zeiss laser
confocal microscope LSM 510 was used to observe the samples.
Ni-NTA pull-down
HEK-293 cells transiently transfected with His6 -tagged AE1 and
HA-tagged protein 4.2 constructs, were harvested with lysis buffer
(1 % C12 E8 , 300 mM NaCl and 10 mM imidazole with protease
inhibitors in PBS). Cell lysates were centrifuged at 14 000 g for
30 min at 4 ◦ C to remove insoluble material. The supernatants
were added to 50 μl of a 50 % slurry of Ni-NTA–agarose in
binding buffer (0.1 % C12 E8 , 300 mM NaCl and 10 mM imidazole
with protease inhibitors in PBS) and incubated for 2 h at 4 ◦ C.
Resin was washed with 0.3 ml of wash buffer (0.2 % C12 E8 ,
300 mM NaCl and 30 mM imidazole with protease inhibitors in
PBS) three times. Bound proteins were eluted with elution buffer
(0.5 % C12 E8 , 300 mM NaCl and 500 mM imidazole in PBS) and
solubilized in 2 × SDS sample buffer. Samples were analysed
by SDS/PAGE (8 % gels) and immunoblotting was performed
as described above. Band intensities were determined using
ImageJ 1.41o software. The amount of protein 4.2 associated
with the amount of AE1 eluted from the resin was calculated
from immunoblots from eight separate transfection experiments
and normalized to the total amount of protein 4.2 expressed in the
cells. Each value was reported as relative to that of AE1, which
was set to 100 %, to give a value of protein 4.2 relative binding.
Results for protein 4.2 binding are given as means +
− S.D. Mean
values were considered to be significantly different (P < 0.05)
when the Student’s t test was used.
Co-immunoprecipitation
HEK-293 cells transiently transfected with AE1 and HA-tagged
protein 4.2 constructs were harvested with lysis buffer (1 % C12 E8
with protease inhibitors in PBS). Cell lysates were centrifuged at
14 000 g for 30 min at 4 ◦ C to remove insoluble material. AE1
was immunoprecipitated from supernatants with 4 μl of rabbit
anti-C-terminal AE1 antibody followed by 100 μl of Protein
G−Sepharose. Proteins were eluted with 25 μl of 0.1 M glycine
(pH 2.5) on ice for 20 min. Then, 2 μl of 1 M Tris (pH 9.0) was
added to the effluent and proteins were solubilized in 2 × SDS
sample buffer. Samples were analysed by SDS/PAGE (8 % gels)
and immunoblotting was performed as described above. Band
intensities were determined using ImageJ 1.41o software from
various blot exposures, ensuring that the band intensities were
c The Authors Journal compilation c 2011 Biochemical Society
316
S. P. Bustos and R. A. F. Reithmeier
in the linear range. Linear dilutions of protein were included on
the blots to ensure linearity. The amount of protein 4.2 associated
with the amount of AE1 eluted from the resin was calculated
from immunoblots from eight separate transfection experiments
and normalized to the total amount of protein 4.2 expressed in the
cells. Each value was reported as relative to that of AE1, which
was set to 100 %, to give a value of protein 4.2 relative binding.
Results for protein 4.2 binding are given as means +
− S.D. Mean
values were considered to be significantly different (P < 0.05)
when the Student’s t test was used.
Subcellular fractionation of HEK-293 cells expressing wild-type or
G2A/C173A protein 4.2
HEK-293 cells transiently transfected with HA-tagged wildtype protein 4.2 or the G2A/C173A protein 4.2 mutant were
suspended in PBS with protease inhibitors, followed by sonication
at 50 % duty for 20 pulses on ice to lyse cells. Cell lysates were
centrifuged at 1500 g for 10 min at 4 ◦ C to remove cell debris
and unbroken cells. The supernatant, containing the membrane,
cytoskeletal and cytoplasmic fractions (labelled the total fraction)
was collected and centrifuged at 50 000 rev./min for 1 h at 4 ◦ C.
The resulting supernatant contained the soluble cytosolic fraction
and was labelled S1. The pellet was washed with 1 ml of PBS with
protease inhibitors and centrifuged at 50 000 rev./min for 1 h at
4 ◦ C and the wash fraction was labelled Sw. The pellet, containing
the membrane and cytoskeletal fractions, was resuspended in
lysis buffer (1 % C12 E8 with protease inhibitors in PBS) and
was labelled P1. Samples were slowly rotated for 1 h at 4 ◦ C to
solubilize the membrane proteins in the detergent. Samples were
then centrifuged at 50 000 rev./min for 1 h at 4 ◦ C. The resulting
supernatant contained detergent-soluble membrane protein and
was labelled S2. The pellet contained the cytoskeletal fraction
and was labelled P2. Samples were solubilized in 2 × SDS sample
buffer and analysed by SDS/PAGE (10 % gels). Immunoblotting
was performed as described above and band intensities were
determined using ImageJ 1.41o software. The total amount of
wild-type and G2A/C173A protein 4.2 was set to 100 %, with
the cellular fractions reported as relative percentages of the total.
Results for the amount of protein 4.2 in each fraction are given
as means +
− S.D. Detection of GAPDH was used as a marker for
soluble cytosolic proteins. Actin was used as marker for soluble
(G-actin) and cytoskeletal (F-actin) proteins.
Figure 2
Expression of AE1 proteins and protein 4.2 in HEK-293 cells
Immunoblot analysis was performed on whole-cell detergent extracts from HEK-293 cells
expressing AE1 proteins and protein 4.2. A mouse monoclonal anti-AE1 antibody was used
to detect AE1 proteins (A) and a mouse monoclonal anti-HA antibody was used to detect
HA-tagged protein 4.2 (B). Incubation with horseradish-peroxidase-conjugated anti-mouse IgG
and detection by chemiluminescence was then performed (see the Materials and methods
section). The molecular mass in kDa is indicated on the left-hand side.
RESULTS
the non-transfected control cells (results not shown). In multiple
transfection experiments, there was no statistically significant
difference in the amount of protein 4.2 expressed in the absence or
presence of AE1, AE1HS mutants, mdAE1, kAE1 or AE1SAO.
These results show that the AE1HS mutants had stabilities similar
to those of AE1, and that co-expression with AE1 was not
necessary for the expression of protein 4.2 in transfected HEK293 cells.
Expression of protein 4.2 and AE1 proteins in transfected HEK-293
cells
Co-localization of protein 4.2 and AE1 in HEK-293 cells
Protein 4.2 was expressed alone or co-expressed with AE1, the
HS mutants (E40K, G130R and P327R), as well as mdAE1,
kAE1 and AE1SAO in transiently transfected HEK-293 cells.
Figure 2 shows a representative immunoblot of total cell extracts.
AE1 and AE1HS proteins were detected as approx. 95 kDa bands
(Figure 2A, lanes 2, 4, 5 and 6). No immunoreactive band was seen
in the empty-vector-transfected control lane (Figure 2A, lane 1).
All three AE1HS proteins had similar expression levels compared
with the wild-type protein. The immunodetection of mdAE1
(Figure 2A, lane 3) was variable and often lower than AE1 owing
to the presence of multiple bands. Expression (results not shown)
of kAE1 was comparable with AE1, whereas AE1SAO was lower,
as reported previously [31]. HA-tagged protein 4.2 expressed
in HEK-293 cells in the absence or presence of AE1 proteins
was readily detected as an approx. 72 kDa immunoreactive band
(Figure 2B, lanes 1−6). No HA-immunoreactive band was seen in
Localization of wild-type and mutant AE1 proteins expressed
in HEK-293 cells was determined by immunofluorescence
and confocal microscopy (Supplementary Figure S1 at
http://www.BiochemJ.org/bj/433/bj4330313add.htm). AE1 was
readily detected at the cell surface, indicating that it had trafficked
to the plasma membrane, as seen in previous studies [39]. The
three AE1HS mutants, kAE1 and mdAE1 were also detected at the
cell surface, at similar intensities, indicating their ability to traffic
to the plasma membrane. In contrast, AE1SAO was not detected at
the cell surface, but only at the ER, indicating its inability to traffic
from the ER to the plasma membrane in transfected HEK-293
cells, as was reported previously [31].
HA-tagged protein 4.2 was expressed alone or with AE1,
AE1HS, mdAE1, kAE1 and AE1SAO proteins and visualized
by immunofluorescence and confocal microscopy to determine
its intracellular localization in HEK-293 cells. The first row of
c The Authors Journal compilation c 2011 Biochemical Society
Interaction of protein 4.2 with HS mutants of AE1
317
Figure 3 shows immunofluorescence of protein 4.2 expressed
alone in permeabilized cells and CNX, an ER-resident protein.
Protein 4.2 (green) displayed a wide distribution throughout
the cell and also co-localized with CNX (blue) as seen by the
cyan colour in the merged image, suggesting that some protein
4.2 localizes at the ER. In the first column of the remaining
rows, protein 4.2 displayed a similar wide distribution when
co-expressed with all AE1 proteins, similar to when expressed
alone. The second column shows the immunofluorescence of AE1
proteins in permeabilized cells. The proteins were predominantly
localized at the cell surface, with the exception of AE1SAO, which
was intracellular, similar to when the proteins were expressed in
the absence of protein 4.2 (Supplementary Figure S1). In the
merged images in the third column, the yellow colour indicates
that a fraction of protein 4.2 co-localized to the plasma membrane
with AE1, AE1HS, mdAE1 and kAE1 proteins. Its co-localization
with mdAE1 is interesting, since mdAE1 lacks the cytoplasmic
domain which is needed for protein 4.2 interaction [8]. Protein 4.2
also co-localized with AE1SAO, indicating its localization at the
ER. The green staining in the merged images indicates localization
of protein 4.2 in other cellular compartments. Thus protein 4.2
has a wide intracellular distribution in transfected HEK-293 cells,
localizing to the ER and other intracellular compartments, as well
as to the plasma membrane.
Interaction of protein 4.2 with AE1 proteins in HEK-293 cells
To examine the interaction of protein 4.2 with the AE1 proteins
we conducted Ni-NTA pull-down assays on HEK-293 cells
transiently expressing His6 -tagged AE1 proteins and HA-tagged
protein 4.2. Figure 4 shows representative immunoblots of protein
4.2 associated with AE1 (top panel), AE1 pulled down (middle
panel) and total protein 4.2 in the HEK-293 lysate (bottom panel).
The amount of protein 4.2 bound to AE1 proteins was calculated
as described in the Materials and methods section. The control
experiment with protein 4.2 expressed in the absence of AE1
(lane 1) showed background amounts of bound protein 4.2. The
AE1HS mutants consistently bound less protein 4.2 at levels of
54 +
− 14 % (E40K), 43 +
− 14 % (G130R) and 65 +
− 29 % (P327R),
n = 8, relative to wild-type AE1.
As expected, the mdAE1 was unable to pull-down protein
4.2 above background levels (lane 6). The kAE1 and AE1SAO
proteins bound similar amounts of protein 4.2 at levels of
110 +
− 33 %, n = 8, relative to wild-type AE1
− 21 % and 99 +
respectively. These small differences were not statistically
significant, indicating that the loss of the first 65 amino acids
for kAE1 or deletion of nine amino acids at the first TM domain
in AE1SAO does not diminish protein 4.2 binding.
To confirm the protein 4.2 binding impairment by the AE1HS
mutants seen in the Ni-NTA pull-down assays, we conducted coimmunoprecipitation experiments on HEK-293 cells transiently
expressing AE1HS proteins and protein 4.2. Figure 5(A) shows
representative immunoblots of protein 4.2 associated with AE1
(top panel), AE1 bound to the resin (middle panel) and total
protein 4.2 in the HEK-293 lysate (bottom panel). The amount
of protein 4.2 bound to AE1HS proteins relative to wild-type
Figure 3 Immunofluorescence images of protein 4.2 and AE1 proteins in
HEK-293 cells
Permeabilized cells expressing HA-tagged protein 4.2 alone or co-expressed with wild-type or
mutant AE1 proteins were incubated with rat anti-HA and rabbit anti-CNX or mouse monoclonal
anti-AE1 antibodies. Incubation of samples with fluorescently labelled secondary antibodies and
confocal microscopy was performed (see the Materials and methods section). In the merged
image of the top row, cyan indicates co-localization of protein 4.2 (green) and CNX (blue). In
the remaining merged images, yellow indicates co-localization of protein 4.2 (green) and AE1
(red). perm., permeabilized.
c The Authors Journal compilation c 2011 Biochemical Society
318
Figure 4
S. P. Bustos and R. A. F. Reithmeier
Ni-NTA pull-down of wild-type and mutant His6 -tagged AE1 with protein 4.2 in HEK-293 cells
His6 -tagged AE1 in whole-cell extracts was prepared with the detergent C12 E8 and pulled-down using Ni-NTA agarose. Total and bound fractions were resolved by SDS/PAGE, and immunoblots
were probed with mouse anti-AE1 antibody to detect AE1 and mouse anti-HA antibody to detect HA-tagged protein 4.2 (see the Materials and methods section). Immunoblot band intensities were
measured from eight independent experiments. The amount of protein 4.2 bound to the P327R HS mutant appears high in this blot. However, after correcting for bound AE1 and total protein 4.2, the
amount of protein 4.2 bound to P327R HS mutant was less than that bound to AE1. The molecular mass in kDa is indicated on the left-hand side.
AE1 was calculated as described in the Materials and methods
section. The control experiment with protein 4.2 expressed in the
absence of AE1 (Figure 5A, lane 1) showed no bound protein
4.2. The AE1HS proteins (Figure 5A, lanes 3−5) consistently
co-purified less protein 4.2 relative to AE1 (Figure 5A, lane 2). In
Figure 5(B) we show the results for AE1 (Figure 5B, lane 1)
and mdAE1 (Figure 5B, lane 2) where the amount of
immunoreactive mdAE1 expressed was comparable with AE1.
As seen in lane 2 (Figure 5B), there was no bound protein
4.2. In agreement with the Ni-NTA pull-down results, the three
AE1HS mutants consistently bound less protein 4.2 at levels of
75 +
− 16 % (E40K), 68 +
− 16 % (G130R) and 79 +
− 17 % (P327R),
n = 8, relative to wild-type AE1. Clearly, protein 4.2 could still
interact with all three AE1HS mutants; however, at a statistically
significant lower level compared with wild-type AE1. Thus the
HS mutations in the cdAE1 result in impaired binding of protein
4.2 in HEK-293 cells.
Co-localization of wild-type and G2A/C173A protein 4.2 and AE1 in
HEK-293 cells
The ability of protein 4.2 to co-localize with mdAE1 at the plasma
membrane of HEK cells despite a lack of interaction was puzzling.
The requirement of protein 4.2 Gly2 myristoylation for plasmamembrane localization in Sf9 cells led us to believe its acylation
state may allow for similar localization in HEK-293 cells in the
absence of cdAE1. To confirm this, we constructed a double
mutant removing sites of myristoylation and palmitoylation
(G2A/C173A) and observed its co-localization with AE1 proteins.
Figure 6 shows the immunofluorescence of HA-tagged protein
4.2 and the G2A/C173A mutant expressed with AE1, mdAE1
and AE1SAO. In the first row, wild-type protein 4.2 is widely
distributed throughout the cell and co-localizes with AE1 at
the plasma membrane, as seen by the yellow colour in the
merged image. In the second row, the G2A/C173A mutant has
c The Authors Journal compilation c 2011 Biochemical Society
a similar cellular distribution and co-localizes to the plasma
membrane with AE1. In the third row, the co-localization of
protein 4.2 and mdAE1 is again seen by the yellow colour at
the plasma membrane. However, in the fourth row, when the
G2A/C173A mutant is expressed with mdAE1, there is very
little yellow staining at the plasma membrane, indicating poor
plasma-membrane localization. This suggests that the fatty acid
moieties promote protein 4.2 targeting to the plasma membrane.
This also suggests that the presence of wild-type AE1 allows the
G2A/C173A mutant to localize to the plasma membrane, as seen
in the second row, consistent with an interaction between these
proteins. The fifth row shows, as before, that wild-type protein
4.2 also co-localizes with AE1SAO at the ER. In the sixth row,
the G2A/C173A mutant is also seen to co-localize with AE1SAO
at the ER. Co-localization of protein 4.2 and the G2A/C173A
mutant with AE1SAO is not complete, as seen by distinct green
and red staining in the merged image. This indicates that both
wild-type and G2A/C173A protein 4.2 are associated with other
intracellular compartments in addition to the ER.
Co-localization of wild-type and G2A/C173A protein 4.2 and
cell-surface glycans in the absence or presence of AE1 in HEK-293
cells
In order to further support the idea that the G2A/C173A
mutant displays impaired plasma-membrane targeting, HAtagged protein 4.2 and the G2A/C173A mutant were expressed
either alone or with AE1, and their localization to the
plasma membrane using PNA binding was determined. PNA
is a lectin that binds to the disaccharide galactose-β1,3-Nacetylgalactosamine found in oligosaccharides of glycoproteins
[40] serving as a marker of the outer surface of the plasma
membrane. In the first row of Figure 7, protein 4.2 expressed
alone is widely distributed within the cell (green) and the red
Interaction of protein 4.2 with HS mutants of AE1
Figure 5
319
Co-immunoprecipitation of wild-type and mutant AE1 with protein 4.2 in HEK-293 cells
(A) AE1 in whole-cell extracts prepared with the detergent C12 E8 was immunoprecipitated with rabbit anti-C-terminal AE1 antibody followed by Protein G−Sepharose binding. Total and bound fractions
were resolved by SDS/PAGE and immunoblots probed with mouse anti-AE1 antibody to detect AE1 and mouse anti-HA antibody to detect any co-purifying HA-tagged protein 4.2 (see the Materials
and methods section). Immunoblot band intensities were measured from eight independent experiments. The mdAE1 lanes were excluded from these blots due to variable and low expression levels,
and the remaining lanes on the same blots were spliced together for clarity (splice points are denoted by vertical black lines). (B) Immunoblots following co-immunoprecipitation of AE1 and mdAE1
and associated protein 4.2 from an experiment with comparable amounts of bound AE1 and mdAE1 proteins. The molecular mass in kDa is indicated on the left-hand side.
stain, representing bound PNA, marks the outside surface of the
cell. Even though PNA is on the outside of the bilayer and protein
4.2 on the inner side, we see an overlap of fluorescence seen
by the yellow colour in the merged image since the confocal
microscope cannot resolve the thickness of the membrane. A
distinctive green/yellow/red pattern (going from the inside of the
cell outward) is seen in the merged image, indicating that protein
4.2 associates with the plasma membrane in the absence of AE1.
The same pattern is seen in the second row when protein 4.2 is
expressed with AE1. In the third row when the acylation mutant
is expressed alone, there is no yellow colour in the merged image,
indicating its inability to associate with the plasma membrane. In
the fourth row when the acylation mutant is co-expressed with
AE1, the yellow colour is seen in the merged image, indicating
that AE1 can localize the acylation mutant of protein 4.2 to the
plasma membrane.
Subcellular fractionation of HEK-293 cells expressing wild-type or
G2A/C173A protein 4.2
In order to confirm that impaired plasma-membrane localization
was not indirectly caused by impaired cytoskeletal attachment
by the double mutant, we performed subcellular fractionation
of HEK-293 cells expressing HA-tagged wild-type and
G2A/C173A mutant protein 4.2 (Supplementary Figure S2
at http://www.BiochemJ.org/bj/433/bj4330313add.htm). After
centrifugation at 50 000 rev./min the majority of protein 4.2
(73 +
− 3 %, n = 4) was associated with the membrane/cytoskeleton
fraction compared with the cytosolic fraction. Similar results
(71 +
− 6 %, n = 4) were seen with the acylation mutant. Detergent
extraction of the membrane/cytoskeletal fraction revealed that
most (87 %) of the membrane-associated protein 4.2 was in
the cytoskeletal fraction, with a small amount in the membrane
fraction. Similar results (92 %) were seen with the acylation
mutant, although the amount solubilized by detergent was
decreased relative to wild-type protein 4.2 (8 % compared with
13 %). These observations support a previous report [15] where
both myristoylated and non-myristoylated (G2A) protein 4.2
expressed in Sf9 cells were associated with the particulate
fraction. These results indicate that fatty acylation does not affect
protein 4.2 cytoskeleton association.
The majority of the actin (75 %) was seen in the
cytosolic fraction compared with the remainder in the
membrane/cytoskeletal fraction (Supplementary Figure S2), in
agreement with a previous study in HEK-293 cells [41]. Virtually
all of the GAPDH, a soluble protein, was detected in the cytosol,
with a negligible amount detected in the membrane/cytoskeleton
fraction. These results support our assignment of cellular
compartments to the various fractions, allowing us to localize
the majority of wild-type and G2A/C173A protein 4.2 to the
cytoskeletal compartment with confidence.
DISCUSSION
In the present study, we were able to express full-length HAtagged protein 4.2 with AE1 proteins in HEK-293 cells. The
AE1HS proteins expressed to similar levels as AE1 and could
traffic to the plasma membrane of the cells. These findings are
consistent with the observation that the levels of these HS mutants
in patient’s erythrocytes are normal [3−5]. This is also consistent
with our observation that the mutations do not have a major
effect on the structure or stability of the isolated cytoplasmic
domain [7]. We also demonstrated that protein 4.2 was expressed
to similar levels in HEK-293 cells in the absence or presence
of AE1 proteins. Thus protein 4.2 association with AE1 is not
required for its expression in HEK-293 cells.
When co-expressed in HEK-293 cells, protein 4.2 and AE1
co-localized at the plasma membrane. Similar observations were
made with the AE1HS mutants and kAE1, but also with mdAE1,
which does not interact with protein 4.2. In contrast, protein 4.2
co-localized at the ER with AE1SAO. Furthermore, protein
4.2 could target to the plasma membrane in the absence of
AE1, as seen by its co-localization with PNA. This result agrees
with previous reports of protein 4.2 localization at the plasma
membrane of Xenopus oocytes [16] and Sf9 cells [15] in the
absence of AE1. We also found protein 4.2 widely distributed
among intracellular compartments, including the ER, as seen by
its co-localization with CNX. These results show that protein 4.2
c The Authors Journal compilation c 2011 Biochemical Society
320
S. P. Bustos and R. A. F. Reithmeier
Figure 7 Immunofluorescence images of cell-surface glycans using PNA
and wild-type and G2A/C173A protein 4.2 expressed in the absence or
presence of AE1 in HEK-293 cells
Non-permeabilized HEK-293 cells expressing wild-type or mutant protein 4.2 proteins in the
absence or presence of AE1 were incubated with PNA Alexa Fluor® 488 conjugate to detect
the outer membrane. Cells were then permeabilized and incubated with rat anti-HA antibody
to detect HA-tagged protein 4.2 or G2A/C173A. Incubation of samples with fluorescently
labelled anti-rat secondary antibody and confocal microscopy was performed as described in
the Materials and methods section. In the merged image, yellow indicates the co-localization
of protein 4.2 (intracellular) and PNA (extracellular) at the level of the plasma membrane. GC,
G2A/C173A mutant; perm., permeabilized; non-perm., non-permeabilized.
Figure 6 Immunofluorescence images of wild-type and G2A/C173A protein
4.2 and AE1 proteins in HEK-293 cells
Non-permeabilized cells expressing wild-type or mutant protein 4.2 and wild-type or mdAE1
proteins (first four rows) were incubated with the mouse monoclonal anti-AE1 antibody (BRIC
6) against an external epitope. Cells were then permeabilized and incubated with a rat anti-HA
antibody. In the last two rows, permeabilized cells expressing wild-type or mutant protein 4.2 and
AE1SAO were incubated with mouse anti-AE1 and rat anti-HA antibodies. Incubation of samples
with fluorescently labelled secondary antibodies and confocal microscopy was performed (see the
Materials and methods section). In the merged images, yellow indicates co-localization of protein
4.2 and AE1. GC, G2A/C173A mutant; perm., permeabilized; non-perm., non-permeabilized.
associates with various membranes within the cell in the
absence of AE1, including the plasma membrane, the ER
membrane and other intracellular membranes in HEK-293 cells.
c The Authors Journal compilation c 2011 Biochemical Society
Pull-down experiments revealed that protein 4.2 can associate
with AE1, AE1HS, kAE1 and AE1SAO, but not mdAE1, in
whole-cell detergent extracts. Protein 4.2 binding to AE1HS
mutants was diminished relative to AE1. The binding impairment
is likely to be due to a change in the protein 4.2-binding surface on
cdAE1, since purified cdAE1 proteins with these mutations have
been shown not to cause any gross structural change in the domain
[7]. Introduction of a positive charge by the E40K, G130R or
P327R mutants may serve to disrupt the acidic protein 4.2-binding
surface on cdAE1. As shown in the model of cdAE1 (Figure 1),
these mutations are not localized to a ‘hot spot’, but are widely
distributed on one surface of the protein. Residues 40 and 130 on
one monomer occur on the same side of the domain as residue
327 from the other monomer, perhaps requiring dimer formation
to create the binding surface for protein 4.2. The cdAE1-binding
regions shown in the model of protein 4.2 are predicted to form a
binding surface that could be large enough to interact with all three
AE1HS mutation sites. It is not surprising, then, that alterations of
residues at these distant sites in cdAE1 affect protein 4.2 binding.
Interaction of protein 4.2 with HS mutants of AE1
The binding of protein 4.2 to kAE1 as measured in Ni-NTA pulldowns was not significantly different from that of AE1. This result
may seem puzzling when we consider that the kidney isoform is
missing the first 65 residues of the N-terminus, whereas the E40K
HS mutant which has a single point mutation in this region shows
impaired binding to protein 4.2. However, this mutation replaces
an acidic with a basic residue. This introduction of a positive
charge into a potential binding spot may compromise protein 4.2
binding in a way that absence of the region does not. As was
mentioned above, it is believed that the Arg−Arg motif of protein
4.2 (residues 34 and 35) is necessary for the cdAE1 interaction and
that this motif has an electrostatic interaction with an acidic region
of cdAE1, possibly at the N-terminus. If this acidic region were to
become more basic, the electrostatic repulsion could result in the
binding impairment we saw with E40K. The cytoplasmic domain
of kAE1 has been shown to be a more open structure than AE1
[21], and it is not known how this would affect protein binding.
It is possible that this open structure compensates for the missing
central β-strand. Wild-type binding levels of AE1SAO to protein
4.2 were not surprising as the cytoplasmic domain of this mutant
is unchanged, deficiencies of protein 4.2 in AE1SAO erythrocytes
have not been reported, and AE1SAO exhibits increased binding
to the cytoskeleton in erythrocytes [28].
The co-localization of protein 4.2 with mdAE1 at the plasma
membrane, despite a lack of interaction, turned our attention
to the possible role of fatty acid modifications in plasmamembrane localization. We found that G2A/C173A protein 4.2
had diminished plasma-membrane localization when expressed
alone or with mdAE1. This suggests that protein 4.2 is able
to associate with the plasma membrane via its lipid anchors,
as suggested in previous reports [15,42]. This is at least the
case in HEK-293 cells where AE1 is not needed for protein
4.2 stabilization or plasma-membrane localization. Subcellular
fractionation studies showed that the majority of wild-type
and G2A/C173A protein 4.2 expressed in HEK-293 cells was
associated with the cytoskeleton. This demonstrates that the
G2A/C173A mutation does not affect cytoskeletal binding. A
portion (<30 %) of wild-type and G2A/C173A protein 4.2
was seen in the cytosolic fraction, which agrees with the
immunofluorescence localization results where the protein was
broadly localized throughout the cell.
The present study shows that AE1 is not required for the
expression and plasma-membrane localization of protein 4.2
in HEK-293 cells. Also, the three AE1HS mutants, E40K,
G130R and P327R, have impaired binding to protein 4.2, which
contributes to the molecular basis of HS in patients with these
mutations. How this impaired binding translates into protein 4.2
deficiency in erythrocytes is yet to be determined. Protein
4.2 and AE1 have been found to appear simultaneously during
erythropoiesis and interact with each other as soon as they are
expressed [43]. Indeed, protein 4.2 may associate with AE1 at
the ER during their biosynthesis and traffic together to the cell
surface. The fate of protein 4.2 that is unable to fully associate
with AE1HS mutants during differentiation is not known. Protein
association with the cytoskeleton can determine protein sorting
during enucleation of the differentiating erythrocyte [44], where
an increased cytoskeletal association causes retention in the
reticulocyte. It is possible that the portion of protein 4.2 unable to
bind to AE1HS never makes it to the membrane, or does not
localize to a region of the membrane where protein 4.2 can
bind to the cytoskeleton. This would prevent its retention in
the reticulocyte during enucleation and explain its deficiency
in erythrocytes of patients with these three cytoplasmic AE1HS
mutations. Studies of protein 4.2 sorting during enucleation of
erythrocyte precursors from healthy individuals and HS patients,
321
or relevant HS mouse models, would help to determine the
mechanism of protein 4.2 loss in HS.
AUTHOR CONTRIBUTION
All experiments were carried out by Susan Bustos, who wrote the manuscript and prepared
the Figures. Reinhart Reithmeier developed and supervised the research project, and
edited the manuscript.
ACKNOWLEDGEMENTS
We thank Jing Li (University of Toronto, Toronto, ON, Canada) for her assistance in carrying
out replicates of some experiments and Dr Michael Jennings (University of Arkansas for
Medical Sciences, Little Rock, AR, U.S.A.) for the mouse monoclonal anti-AE1 antibody.
FUNDING
This work was supported by the Canadian Institutes of Health Research
[FRN15266/MOP102493]; and a Graduate Student Fellowship (to S.P.B.) from the
Canadian Blood Services.
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doi:10.1042/BJ20101375
SUPPLEMENTARY ONLINE DATA
Protein 4.2 interaction with hereditary spherocytosis mutants of the
cytoplasmic domain of human anion exchanger 1
Susan P. BUSTOS* and Reinhart A. F. REITHMEIER†1
*Department of Biochemistry, University of Toronto, Toronto, ON, Canada, M5S 1A8, and †Department of Medicine, University of Toronto, Toronto, ON, Canada, M5S 1A8
See the page that follow for Supplementary Figures S1 and S2.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2011 Biochemical Society
S. P. Bustos and R. A. F. Reithmeier
Figure S2 Subcellular fractionation of HEK-293 cells expressing wild-type
or G2A/C173A protein 4.2
Following subcellular fractionation of HEK-293 cells expressing HA-tagged wild-type and
G2A/C173A mutant protein 4.2, fractions were subjected to SDS/PAGE and transferred to
nitrocellulose. Blots were probed with mouse anti-HA antibody, mouse anti-actin antibody
and mouse anti-GAPDH antibody to detect protein 4.2, actin and GAPDH respectively (see
the Materials and methods in the main text). Immunoblot band intensities were measured
from four independent experiments. The molecular mass in kDa is indicated on the left-hand
side. S1, cytosolic fraction; Sw, wash fraction; P1, membrane/cytoskeletal fraction; S2,
detergent-solubilized membrane fraction; P2, cytoskeletal fraction; WT, wild-type.
Received 25 August 2010/28 October 2010; accepted 1 November 2010
Published as BJ Immediate Publication 1 November 2010, doi:10.1042/BJ20101375
Figure S1 Immunofluorescence images of wild-type and mutant AE1 in
HEK-293 cells
Non-permeabilized HEK-293 cells expressing wild-type or mutant AE1 proteins were incubated
with mouse anti-AE1 andtibody (BRIC 6) against an external epitope, followed by incubation with Alexa Fluor® 488-conjugated goat anti-mouse antibody to detect cell-surface
AE1 (green). Cells were then washed, permeabilized and incubated with the same primary
antibody followed by incubation with Cy3-conjugated goat anti-mouse antibody to detect total
AE1 (red). Confocal microscopy was then performed. An enlarged region of the cell is shown
in the inset for each image. In the merged image, yellow indicates the co-localization of cell
surface and total AE1. non-perm., non-permeabilized; perm., permeabilized.
c The Authors Journal compilation c 2011 Biochemical Society