From the Department of Cell and Animal Biology, Institute of Life
Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel and
Department of Pharmaceutical Sciences, School of
Pharmacy, University of Southern California,
Los Angeles, California 90033
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ABSTRACT |
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We provide morphological, biochemical, and
functional evidence suggesting that the AP-1 clathrin adaptor complex
of the trans-Golgi network interacts with the polymeric
immunoglobulin receptor in transfected Madin-Darby canine kidney cells.
Our results indicate that immunofluorescently labeled Polarized epithelial cells possess two surface domains as follows:
the apical plasma membrane that faces the external environment and the
basolateral plasma membrane that is in contact with internal milieu.
The apical and basolateral plasma membranes have very different protein
and lipid compositions. It has been proposed that sorting and targeting
events of membrane proteins and lipids largely contribute to the
polarized phenotype of the cell (1-3). Although in recent years we
have learned a great deal about relatively small stretches of amino
acids encoding sorting signals that mediate polarized trafficking of
membrane proteins (4-7), almost nothing is known about the sorting
machinery that decodes these signals and confers polarized trafficking
to a specific surface domain (1, 2, 6-8). Polarized trafficking steps
are modulated by signal transduction processes (4, 7, 9, 10), whose activity is thought to be superimposed on the activity of the sorting
machinery, but the mechanisms through which these processes facilitate
protein incorporation into a particular pathway are largely unknown.
Presently, we know four distinct features shown to determine sorting of
apical proteins to the apical domain as follows: first is the
glycosylphosphatidylinositol anchor of membrane proteins (11, 12); the
second is the mannose-rich core of N-glycans present in the
luminal portion of proteins (13); the third is O-glycosylated stalk domain of transmembrane protein (14);
and the fourth is a proteinaceous signal encoded by either the
transmembrane domain or the ectodomain (15). Many studies agree that
basolateral sorting of plasma membrane proteins is mediated by the
presence of relatively short but specific cytoplasmic sorting motifs
(reviewed in Refs. 4 and 5). Extensive mutagenesis studies have
uncovered two general types of basolateral sorting motifs. First there
are basolateral sorting signals for localization to clathrin-coated membranes that rely either on a critical tyrosine residue, such as
those found in the low density lipoprotein receptor (low density lipoprotein receptor proximal determinant, (16)), the vesicular stomatitis virus G protein (17), or on a di-leucine motif (18). The
second are basolateral sorting signals unrelated to localization to
clathrin-coated membranes. These signals can rely either on a Tyr
motif, e.g. the distal determinant in the low density
lipoprotein receptor, or rely on non-aromatic residues such as found
for the polymeric immunoglobulin receptor
(pIgR1 (4, 19)).
Interestingly, either the same or a closely related basolateral sorting
signal can mediate basolateral sorting from the TGN and recycling from
endosomes, after endocytosis from the basolateral plasma membrane (20,
21).
We use the pIgR expressed in Madin-Darby canine kidney (MDCK) cells as
a model system for studying the mechanisms that regulate polarized
trafficking of membrane proteins in epithelial cells. Previous studies
have intensively analyzed the intracellular trafficking of the pIgR in
MDCK cells. According to the simplest model, the pIgR is synthesized in
the endoplasmic reticulum (ER) as a single spanning type I membrane
protein. It is then targeted from the ER to the Golgi apparatus and
from the trans-Golgi network (TGN) pIgR molecules are
directed to the basolateral surface. However, the exact pathway that
newly synthesized membrane proteins take to the basolateral cell
surface is currently not understood. Although in the simplest model,
basolateral proteins are selectively sorted (packed) into basolateral
transport vesicles that are subsequently directly delivered to the
basolateral surface, recent studies have provided evidence that
targeting to the cell surface may be indirect, i.e.
involving passage through an endosomal compartment prior to
cell-surface delivery (22, 23). Whether a similar indirect route exists
in MDCK cells is currently unknown nor is the mechanism of sorting to
these endosomes known.
Upon arrival at the basolateral cell surface, polymeric Igs
(e.g. dimeric IgA; dIgA) specifically bind to the pIgR. The
pIgR·dIgA complexes are endocytosed via clathrin-coated pits,
delivered to basolateral endosomes, and then delivered to the opposite
pole of the cell via vesicular intermediates in a process termed
transcytosis. Transcytotic pIgR·dIgA complexes are not directly
routed from basolateral endosomes to the apical surface. Rather,
pIgR·Ig complexes are first targeted to, and subsequently accumulate
at, a pericentriolar subapical endosomal compartment that is active in
recycling of apical and perhaps basolateral membrane-bound ligands (24,
25). This compartment has been termed the "apical recycling
endosome." The final step of transcytosis involves pIgR targeting
from the apical recycling endosome to the apical surface. There, the
pIgR is cleaved by an endogenous protease, and the extracellular
ligand-binding domain (i.e. the secretory component, SC) is
released together with dIgA to external (mucosal) secretions, such as
milk, saliva, bile, tears, and intestinal fluids, where they form the
first immunological response against infections. The pIgR is also
transcytosed constitutively (i.e. in the absence of the
ligand), and this process is regulated by phosphorylation of
cytoplasmic Ser-664 (26).
Mutagenesis studies revealed that the cytoplasmic domain of the pIgR
contains several discrete sorting signals that mediate as follows: its
targeting from the TGN to the basolateral surface; endocytosis;
avoidance of lysosomes; and transcytosis (4, 6). Basolateral sorting
from the TGN is mediated by an autonomous and dominant 17-residue
membrane-proximal basolateral sorting signal (27), whose activity
depends mainly on three amino acids (His-656, Arg-657, and Val-660)
contained within the 17-residue signal (19). Structural studies of
oligopeptides corresponding to the 17-residue basolateral sorting
signal provided evidence for a Coat proteins such as clathrin and clathrin adaptor proteins (AP), AP-1
and AP-2, are thought to be involved in membrane protein sorting into
coated membrane domains and promote the budding of transport vesicles
from the trans-Golgi network (TGN) and from the plasma
membrane, respectively (for recent reviews see Refs. 28 and 29). The
AP-1 and AP-2 adaptor complexes are composed of two large subunits ( Basolateral targeting of the pIgR from the TGN is significantly
inhibited by BFA (40-42), suggesting that BFA-sensitive coat proteins
regulate pIgR exocytosis from the TGN to the basolateral surface. The
cytoplasmic tail of pIgR contains a phosphorylated Ser, Ser-726, that
functions in rapid internalization of the pIgR in the basolateral
plasma membrane via clathrin-coated pits (43). This Ser-based motif
resides in a putative CKII/PKA phosphorylation site upstream to a
di-leucine motif with yet undefined function (see Fig.
1A and Ref. 43). In this
respect this motif is interesting as it resembles to the putative
CKII/PKA motif present in the cytoplasmic tail of
cation-dependent mannose 6-phosphate receptor (CD-MPR),
shown to serve as an AP-1-binding site (44, 45). These observations led
us to propose that AP-1 adaptors bind to the Ser-726 motif of pIgR and
that these interactions may facilitate certain steps in basolateral
exocytosis of the receptor. The goal of the work reported herein was to
identify these interactions and to examine their role in pIgR
trafficking from the TGN to the basolateral cell surface in polarized
MDCK cells. We find that the AP-1 adaptor complex associates with the
wild-type pIgR cytoplasmic tail in vivo and that the
interactions are diminished when Ser-726 is mutated to Ala. The
interactions with AP-1 seem to commence in the TGN, persist, and/or are
even enhanced in post-TGN compartments. Interestingly, the TGN to
basolateral delivery of pIgR-Ser-726 to Ala mutant appears to be
significantly slower than that of the wild-type receptor. In addition,
unlike the wild-type receptor, the basolateral pathway of this pIgR
mutant is completely insensitive to the action of BFA. Our results
indicate that AP-1 interactions play a regulatory role in prompting
efficient basolateral transport of the pIgR from the TGN, and they also
suggest the existence of multiple mechanisms that direct membrane
proteins from the TGN to the basolateral surface in MDCK cells.
Characterization of MDCK Cells Stably Expressing the
pIgR--
MDCK cells expressing the wild-type or mutant receptors were
maintained for up to 10 passages in minimal Eagle's medium (MEM, Biological Industries Co, Beit Haemek, Israel) supplemented with 5%
(v/v) fetal bovine serum (Biological Industries Co, Beit Haemek, Israel), 100 units/ml penicillin, and 0.1 mg/ml streptomycin in 5%
CO2, 95% air. In our experiments we used three MDCK cell
lines that stably express the wild-type pIgR ("pIgR-WT"). One
previously described cell line generated by cell transfection with the
retroviral pWE vector has been used (26). Two additional cell lines
were generated by transfecting MDCK cells with the wild-type pIgR
cDNA subcloned into the BglII sites of
cytomegalovirus-based pCB6 or pCB7 vectors, as described previously
(19, 46). In some cases, MDCK cells expressing pIgR mutants at levels
comparable to those of the wild-type receptor were generated by
transfecting the cells with cDNA encoding mutant receptors
subcloned into the pCB6 vector. Polarized MDCK cells expressing
receptors were isolated and characterized as described (19, 20). Two
clones expressing the "pIgR R654stop" mutant, where all but two
residues of the cytoplasmic tail have been deleted (47), have been
isolated and their exocytic transport characteristics were analyzed and
found to be identical to those described for the earlier characterized
expressors (47). The pIgR- Fixation and Fluorescent Labeling of Cells--
MDCK cells
expressing the wild-type receptor were grown on Transwell filters
(Costar, 0.4 µm) for 3-4 days. Cells on filters were fixed for 10 min in methanol at Confocal Microscopy and Co-localization--
A Bio-Rad MRC-1024
confocal scanhead coupled to a Zeiss Axiovert 135M inverted microscope
was used to acquire images of the stained cells, with a 63× oil
objective (numerical aperture 1.4). Excitation light was provided by a
100-milliwatt air-cooled argon ion laser run in the multi-line mode.
The excitation wavelength was 514 nm and was selected with a suitable
interference filter. The relative excitation power level was set to
10% with a neutral density filter. The images presented in this paper
were obtained by accumulating (summing) three scans. In order to detect
three dyes (FITC, TRITC, and Texas Red) simultaneously, the three
detection channels were configured as follows.
The fluorescence emission was first split between two channels by a
dichroic mirror (555DRLP, 50% point at 550 nm). The low wavelength
side of the dichroic was followed by an HQ535/20 (535 nm ± 10 nm)
band pass filter. The iris aperture was 2.5-3.0 mm. This channel
detected FITC. However, when the TRITC emission was much stronger than
the FITC emission, TRITC emission from the tight junctions was also
detected in this channel. In post-processing, the output of the TRITC
channel was subtracted from the FITC channel to eliminate the
contribution of TRITC to this channel. This was particularly effective
because the TRITC only stained tight junctions and was therefore well
localized and distinct from the FITC emission.
The long wavelength side of the first dichroic mirror was split by a
second dichroic mirror (605DRHP, 50% point at 605 nm). The short
wavelength side of this dichroic mirror was followed by an HQ570/30
(570 nm ± 15 nm) band pass filter. The iris aperture was 0.7-1.7
mm. This channel detected TRITC, as well as FITC. When necessary, the
FITC signal as detected in the short wavelength channel described above
was subtracted from the output of the TRITC channel. This subtraction
was done using the mixer controls on the Bio-Rad MRC-1024. In some
cases the TRITC emission was much stronger than the FITC emission, and
this subtraction was not required.
The long wavelength side of the second dichroic mirror was followed by
an HQ655/90 (655 nm ± 45 nm) band pass filter. The iris aperture
was 3.5 mm, and this channel was optimized for Texas Red. All filters
were from Chroma Technology Corp. (Brattleboro, VT). Since this channel
also detected TRITC and some FITC, worst case cross-talk from each of
these sources was estimated and then subtracted from this channel, as follows.
Areas on the images where FITC was dominant were used in order to
establish an upper limit to any such bleed-through. Similarly, the
tight junctions were used to determine bleed-through of TRITC to the
other two channels. The assumption that such areas are due only
to one fluorophore, and that appearance of a signal at that point in
the other channels is caused entirely by bleed-through, sets an upper
limit on the bleed-through. Once a bleed-through factor
Co-localization of the FITC and Texas Red is visualized using the
following processing steps. First, a 3 × 3 median filter was
applied to remove point noise. Then, cross-talk was eliminated as
described above. Finally, the image contrast was stretched. The
contrast-enhanced images were then merged into a single true color
image (green for FITC, red for Texas Red, blue for TRITC). Areas of
overlap of Texas Red and FITC appear yellow. There is no attempt to
quantitate the relative strengths of the FITC and Texas Red emission.
Immunoisolation of MDCK Membranes Expressing the pIgR--
A
post-nuclear supernatant (PNS) fraction was prepared from pIgR
expressing MDCK cells as follows: confluent MDCK culture grown on a
150-mm culture dish was washed twice with ice-cold PBS and once with
homogenization buffer (HB, 250 mM sucrose, 3 mM
imidazole, pH 7.4). Cells were then scraped from the dish with a rubber
policeman, and a mixture of protease inhibitors (25 µg/ml pepstatin,
50 µg/ml chymostatin, 25 µg/ml leupeptin, 50 µg/ml antipain, 2.5 µg/ml aprotinin) was added. Cells were homogenized through a 21-gauge
needle connected to a 1-ml syringe. A PNS fraction was obtained after
centrifugation at 600 × g for 15 min (4 °C). Streptavidin-coated magnetic beads (30 µl, DynaBeads M-280, Dynal, Oslo, Norway) were reacted first with biotinylated rabbit anti-mouse IgG (3 µg, 1 h, 4 °C; Sigma), washed to remove unbound
antibodies, and then incubated (16 h, 4 °C) with 3 µg of protein
G-Sepharose-purified monoclonal antibody SC166 directed against the
cytoplasmic tail of the pIgR (49). Immunobeads were extensively washed
of unbound antibodies and then incubated with PNS for 16 h at
4 °C. Immunobeads were then washed three times with cold HB buffer,
and bound (immunoisolated) membranes were extracted with 1% Triton
X-100 in HB buffer (10 min at 22 °C). One volume of 2× concentrated
SDS sample buffer (1× sample buffer: 15 mM Tris-HCl, pH
6.8, 25 mM EDTA, pH 7.0, 0.25% urea mixed with bromphenol
blue, 6% v/v glycerol, 65 mM dithiothreitol) was added,
and the SDS/Triton mixture was subjected to SDS-PAGE analysis on 10%
acrylamide gels, using the Hoefer Mighty Small II gel system (Hoefer
Scientific Instruments, San Francisco). After SDS-PAGE, proteins were
electrophoretically transferred onto a nitrocellulose membrane for
2 h at 90 mA per single gel. Membranes were then incubated in
blocking buffer (PBS containing 0.5% Tween, 10% glycerol, 0.025% w/v
BSA, 0.01% w/v dry milk, 40 mM glucose, 0.01% sodium
azide) for 1 h at room temperature and subsequently incubated for
16 h at 4 °C with sheep anti-pIgR serum diluted 1:1000 in PBS
supplemented with 0.05% Tween and 5% milk. After washings the
membranes were incubated for 1 h in PBS containing 0.05% Tween
and 1% milk and appropriate horseradish peroxidase-labeled secondary
antibodies (Jackson ImmunoResearch). Horseradish peroxidase-labeled
secondary antibodies were detected using the SuperSignal®
chemiluminescent reagent (Pierce), according to the manufacturer's protocol. The signal on blots was then quenched by incubating the
nitrocellulose sheets in PBS containing 0.1% sodium azide for 30 min,
and the same nitrocellulose sheet was re-probed with 100/3 mouse
antibodies (Sigma) followed by horseradish peroxidase-labeled anti-mouse antibodies (Jackson ImmunoResearch) for the detection of
Detection of pIgR·AP-1 Complexes by Chemical
Cross-linking--
MDCK cells expressing the wild-type pIgR were
cultured for 3-5 days on a 100-mm Transwell porous filter and rinsed
twice with ice-cold TGH, and the basolateral surface of the cells was
selectively permeabilized by placing the filter onto 1 ml of TGH buffer
(50 mM Hepes-NaOH, pH 7.4, 1 mM EGTA-NaOH, 10 mM MgCl2, 150 mM NaCl, 1 mM sodium orthovanadate, 10% v/v glycerol) containing 50 µg/ml digitonin (Sigma) for 20 min on ice, as described (50). Cells were washed with ice-cold TGH, and the permeabilized surface was exposed to 2 mM DTSSP
(3,3'-Dithiobis[sulfosuccinimidyl-propionate, from Pierce), diluted
from a 100 mM stock in N,N-dimethylformamide into Hepes buffer (25 mM Hepes, pH 7.4, 1 mM
MgCl2, 0.25 M sucrose, 1 mM sodium
orthovanadate). Following incubation for 120 min at 4 °C with the
cross-linker, the activity of the cross-linker was quenched by
incubation with 150 mM glycine, pH 7.0, in Hepes buffer for
20 min at 22 °C. The buffer was aspirated leaving the cells on
filter as dry as possible, and cells were then scraped off the filter
using a rubber policeman. Detached cells were resuspended into 1 ml of
2.5% Triton dilution buffer (TDB: 100 mM triethanolamine chloride, pH 8.6, 100 mM NaCl, 5 mM EDTA,
0.025% w/v NaN3) containing 2.5% w/v Triton X-100,
phenylmethylsulfonyl fluoride and a mixture of protease inhibitors,
homogenized through a 1-ml pipette tip, and cleared with CL-2B (50%
slurry, Pharmacia, Uppsala, Sweden) as described (19). The pIgR was
immunoprecipitated, and co-immunoprecipitated adaptor subunits were
analyzed by SDS-PAGE and immunoblotting. The 100/3 monoclonal antibody
was used for detecting Co-immunoprecipitation Detected by Electrophoresis and Western
Blotting--
The co-immunoprecipitation protocol is principally
adapted from Sorkin and Carpenter (52), with the modifications applied by Fire et al. (53), who co-immunoprecipitated the AP-2
adaptors with the epidermal growth factor receptor, or influenza HA,
respectively. All the following steps were performed at 4 °C.
Confluent MDCK cell culture grown on a 100-mm Petri dish was split 1:10
onto three 150-mm dishes, 3-4 days prior to the experiment. Cells in each dish were rinsed twice with ice-cold PBS and once with TGH buffer.
The buffer was then aspirated, leaving the cell monolayer as dry as
possible. Cells were scraped into 300 µl of ice-cold TGH containing
freshly diluted Triton X-100 (1% w/v), phenylmethylsulfonyl fluoride
(5 mM), and a mixture of protease inhibitors. Cell lysate from each dish was pooled into a single Eppendorf tube, and the combined lysate was gently homogenized through a 1-ml pipette tip.
Extracts were centrifuged (13,200 rpm, 15 min, 4 °C; IEC Microfuge
RF) to remove nuclei and detergent-insoluble membranes, and the
resultant supernatant was further spun (75,000 rpm, 30 min, 4 °C;
TL-100 Beckman centrifuge) to remove any remaining Triton-insoluble
materials. The protein concentration of the high speed supernatant was
determined to be 7-10 mg/ml, using the bicinchoninic acid (BCA) assay
kit from Pierce. The pIgR was immunoprecipitated from this supernatant
by incubation with sheep anti-rabbit SC covalently coupled to protein
A-Sepharose for 3 h at 4 °C (19, 54). Immunocomplexes were
washed of unbound proteins with cold TGH/Triton X-100, and pelleted
beads were boiled for 5 min in 2× sample buffer. About one-tenth of
the sample buffer volume was excluded for pIgR analysis and the
remainder for the detection of Co-immunoprecipitation of 35S-Labeled pIgR with
Co-immunoprecipitation of 32P-Labeled pIgR with
Determination of the Rate of Traffic of Newly Synthesized pIgR to
the Cell Surface by Protease-based Assay for Cell-surface
Delivery--
A protease-based delivery assay was used to determine
quantitatively the fraction of newly synthesized pIgR targeted from the
Golgi to either the apical or basolateral surface of MDCK cells. The
methodology has been described in detail, and the data obtained by this
assay were consistent with results achieved by the independent
cell-surface biotinylation-based delivery assay (19, 20, 42). In some
experiments cells were treated with 10 µg/ml BFA (EpiCenter, stored
as a 10 mg/ml stock solution in Me2SO at The AP-1 Adaptors Interact with the Wild-type pIgR--
We employed
cross-linking to examine the interactions between pIgR and AP-1
adaptors in filter-grown cells. The basolateral plasma membrane of
pIgR-expressing MDCK cells was permeabilized with digitonin, and the
permeabilized surface was then incubated with the hydrophilic
cross-linker DTSSP as described under "Experimental Procedures."
Cells were solubilized, and pIgR was immunoprecipitated. Immunoprecipitates were analyzed on Western blots for the presence of
To confirm that the putative interaction between AP-1 and pIgR
demonstrated by cross-linking is a reflection of in vivo
interactions, we next developed a co-immunoprecipitation
approach to study the interactions between the two molecules. This
methodology has been successfully used by others (52, 53) to
demonstrate the interactions between endocytic membrane proteins and
AP-2 adaptors. MDCK cells expressing the wild-type pIgR were
solubilized in buffer containing Triton X-100, and the pIgR was
immunoprecipitated with an anti-ectodomain antibody (anti-SC).
Immunoprecipitates were analyzed by immunoblotting for
co-immunoprecipitating
We compared the efficiency with which BFA Inhibits AP-1 Association with the pIgR--
To characterize
further the specificity and physiological relevance of pIgR-AP-1
adaptor interactions, we examined whether cell treatment with BFA will
reduce the efficiency of pIgR-
To address this point, cells were pulse-labeled and incubated at
18 °C, or pulse-labeled, incubated at 18 °C and then subsequently chased for 10, 30, or 60 min at 37 °C. After 10 min of chase, metabolically labeled pIgR accumulated in the TGN barely reaches the
basolateral cell surface, yet the temperature shift did not result in
significant changes in the amount of co-precipitated 35S-pIgR compared with pIgR blocked in the TGN (Fig.
6B, upper panel), thus diminishing the possibility that
higher co-immunoprecipitation levels observed for 35S-pIgR
after chase at 37 °C is due to more efficient metabolic labeling of
pIgR at 37 versus 18 °C. Interestingly, however, after incubation at 18 °C and chase for 30 or 60 min at 37 °C,
higher levels of 35S-pIgR appeared to
co-immunoprecipitate with the coat protein (Fig. 6B,
lower panel). Co-immunoprecipitation of the pIgR and
Finally, it is important to note that only a small fraction (<10%) of
metabolically labeled pIgR has been co-immunoprecipitated with
Phosphorylated pIgR Co-immunoprecipitates with
However, the persistence of a fraction of phosphorylated S726-A mutant
pIgR that co-immunoprecipitates with The pIgR S726-A Mutant Is Delivered to the Basolateral Surface with
Slower Kinetics Than the Wild-type pIgR and via a BFA-insensitive
Route--
It has been previously demonstrated that mutation of
Ser-726 to Ala does not impair basolateral targeting of the pIgR (43). Since the same mutation seems to impair pIgR interactions with
The data presented in Fig. 9B also indicate that apical
delivery of pIgR- Evidence from several laboratories now strongly argues that both
AP-2 and AP-1 clathrin adaptors recognize tyrosine-based signals
conforming to the YXXØ type cytoplasmic signals (where X is any amino acid and Ø is an amino acid with bulky
hydrophobic group), which mediate endocytosis from the plasma membrane
and may also confer intracellular sorting in the TGN (30). Intact AP-1
and AP-2 adaptors have been shown to interact in vitro with an artificially created Tyr-based basolateral sorting signal that also
functions in endocytosis (39). Nonetheless, the functional role of
these interactions in polarized sorting remains speculative. Other
sorting signals, such as the acidic cluster comprising the CKII site
(ESEER) juxtaposed to a di-leucine motif in the CD-MPR cytoplasmic tail
is also essential for high affinity binding of AP-1 adaptors in
vitro (45) and therefore probably acts as a dominant determinant
controlling CD-MPR sorting in the TGN. The cytoplasmic tail of the pIgR
appears to possess putative AP-1-binding motifs that resemble the known
Tyr-based motifs and the CKII/di-leucine motifs (Fig. 1). In this study
we investigated the interactions between We have used four different methodologies (co-localization,
co-immunoisolation, cross-linking, and co-immunoprecipitation) to
address the possibility that the pIgR interacts with the AP-1 adaptor
complex in MDCK cells. Our data suggest that the entire AP-1 adaptor
complex interacts directly, or indirectly (namely via other proteins),
with the cytoplasmic tail of the pIgR. These interactions are inhibited
by BFA, suggesting that pIgR- Immunofluorescent analysis of pIgR and With respect to the regulation of the putative interaction of pIgR with
AP-1 adaptors, we currently do not know whether Ser-726 phosphorylation
occurs in the TGN or in post-TGN compartments, but our data clearly
suggest that Ser-726, and perhaps its phosphorylated form, is an
important element contributing to the establishment of pIgR- Interactions between the wild-type cytoplasmic domain and the µ1
chain could not be resolved at the resolution provided by the yeast
two-hybrid assay.3 Thus, a
distinct subunit of the AP-1 complex may interact with the cytoplasmic
tail. Although µ2 and µ1 chains recognize Tyr-based signals (60)
and some leucine-based signals (61, 62), the adaptor subunit that
interacts with Ser-based determinants has not yet been identified.
Recent studies have even suggested the N-terminal trunk domain of the
BFA inhibits the binding of the coat protein and basolateral delivery
of the pIgR (41, 42). BFA treatment does not cause receptor
mistargeting to the apical surface, nor does the Ser-726 to Ala
mutation, which also inhibits receptor association with the coat
protein. Thus, basolateral sorting of the pIgR is probably determined
by other cytosolic factors that may interact with the 17-residue
basolateral sorting signal. Consistent with this are recent studies by
Distel et al. (65), demonstrating that deletion of the
AP-1-binding site in a chimeric protein made of the luminal domain of
the influenza virus hemagglutinin and the cytoplasmic tail of CD-MPR
does not affect its polarized sorting to the basolateral surface in
MDCK cells. The basolateral sorting machinery may decode the
basolateral sorting signal and incorporate the pIgR into the basolateral pathway irrespective of the interactions of pIgR with AP-1.
Another possibility is that basolateral sorters interact with the
basolateral sorting signal of pIgR in post-TGN compartments, for
example after possible targeting of exocytic receptors from the TGN to
endosomes (see below). Consistent with this hypothesis is the
characterization of the mammalian Sec6/Sec8 homologues involved in the
regulation of basolateral targeting of membrane proteins in MDCK cells
(66).
The analogy to the MPR raises the hypothesis that the putative CKII/PKA
phosphorylation site and the juxtaposed di-leucine motif mediates pIgR
targeting from the TGN to endosomes in the course of exocytosis. As
indicated before, classical plasma membrane proteins have been
suggested to pass through endosomes en route to the cell
surface in non-polarized cells (22, 23), but the involvement of AP-1
binding or cytoplasmic motifs in this process has not been
investigated. When Ser-726 is inactivated by mutation to Ala,
interactions with the AP-1 are compromised, and receptor exocytosis is
probably mediated by alternative mechanisms that do not involve AP-1
and targeting to endosomes. In this context it is worth pointing out
that exposure of cells to high BFA concentrations did not result in
complete inhibition of pIgR delivery to the basolateral cell surface
(42). This result suggests that the pIgR utilizes parallel biosynthetic
routes to reach the basolateral cell surface; one route depends on AP-1
binding and is BFA-sensitive (and possibly involves passage through
endosomes), and the other route is The physiological significance of multiple redundant pathways is not
clear, but it may provide means for regulation of protein expression by
a given organelle. The existence of mechanistically distinct
alternative pathways may also serve as a salvage mechanism to maintain
membrane trafficking to the cell surface in cases where one of the
pathways is paralyzed, e.g. as a result of cell exposure to
toxins. It is worth noting that two distinct transport pathways for
soluble and membrane-bound proteins from the Golgi to basolateral
plasma membrane of liver cells have been reported by Boll et
al. (67), although the coat proteins regulating these pathways
have not been identified. Another interesting case of two parallel
pathways has been described for the sorting of hydrolases to the
vacuole in yeast; one of these pathways is regulated by the recently
discovered adaptor-like AP-3 coat protein (68). Multiple redundant
pathways for protein delivery thus appear to exist in higher as well as
in lower eukaryotes.
Finally, our results provide significant insight into physiological
mechanisms regulating Golgi-to-basolateral surface transport, as they
suggest that interaction of membranes and cargo with AP-1 adaptors
might be a target for cellular signals regulating constitutive vesicle
formation and membrane traffic in polarized cells. Signal transduction
processes are suggested to be involved in controlling clathrin adaptor
and non-clathrin coat recruitment on membranes (69-72). For example,
phospholipase D, a phospholipid-hydrolyzing enzyme whose activation has
been implicated in signal transduction pathways, cell growth, and
membrane trafficking (73), stimulates the release of nascent secretory
vesicle budding from the TGN (70). ARF1 has been shown to stimulate
endogenous phospholipase D activity, a process that correlated with
enhancement of vesicle budding. A possible model to explain these data
suggests that hydrolysis of phosphatidylcholine in the TGN mediated by
ARF-activated phospholipase D results in the production of high local
concentration of phosphatidic acid; this process could alter local
composition and physical properties of the lipid bilayer in a manner
that facilitates the recruitment of AP-1 adaptors or other coats,
resulting in the budding of nascent secretory vesicles from the TGN
(71). Superimposed upon this regulatory mechanism, modulation of
phosphorylation levels of Ser-726 may determine the extent of pIgR
sequestration into coated areas and consequently determine the kinetics
of its appearance on the basolateral surface. This process can
indirectly affect its subsequent transcytosis. Further characterization
of the regulation of the trafficking of the pIgR in other cell types, including those found in secretory organs, may provide additional insight into the physiological role(s) of Ser-726 in pIgR trafficking.
-adaptin
subunit of the adaptor complex and the polymeric immunoglobulin
receptor partially co-localize in polarized and semi-polarized cells.
-Adaptin is co-immunoisolated with membranes expressing the
wild-type receptor. The entire AP-1 adaptor complex could be chemically
cross-linked to the receptor in filter-grown cells.
-Adaptin could
be co-immunoprecipitated with the wild-type receptor, with reduced
efficiency with receptor mutant whose basolateral sorting motif has
been deleted, and not with receptor lacking its cytoplasmic tail.
Co-immunoprecipitation of
-adaptin was inhibited by brefeldin A. Mutation of cytoplasmic serine 726 inhibited receptor interactions with
AP-1 but did not abrogate the fidelity of its basolateral targeting
from the trans-Golgi network. However, the kinetics of
receptor delivery to the basolateral cell surface were slowed by the
mutation. Although surface delivery of the wild-type receptor was
inhibited by brefeldin A, the delivery of the mutant receptor was
insensitive to the drug. Our results are consistent with a working
model in which phosphorylated cytoplasmic serine modulates the
recruitment of the polymeric immunoglobulin receptor into
AP-1/clathrin-coated areas in the trans-Golgi network. This
process may regulate the efficiency of receptor targeting from the
trans-Golgi network.
INTRODUCTION
Top
Abstract
Introduction
References
-turn secondary structure, which
might constitute a general feature of endocytotic and other sorting
signals (7). Residues involved in basolateral sorting of the pIgR from
the TGN also control polarized sorting in endosomes (20), suggesting
that common mechanisms regulate polarized trafficking in the TGN and in
endosomes. The pIgR thus appears to contain multiple cytoplasmic signals that mediate distinct intracellular transport events; these
signals are probably decoded by a cytoplasmic sorting machinery located
at specific compartments through which the pIgR passes en
route to a target organelle.
and
1 for AP-1 or
and
for AP-2), a medium-sized subunit
(µ1 or µ2), and a small subunit (
1 and
2). Although in a few
cases the interactions between AP-binding and membrane proteins have
been resolved (for a recent review see Ref. 30), the mechanistic
relations between coat binding and membrane protein targeting has not
been fully elucidated. One common feature is that many of the coat
proteins involved in protein sorting are sensitive to the action of the
fungal metabolite brefeldin A (BFA). This drug is thought to act, in
part, by blocking the binding to Golgi membranes of at least four coat
proteins as follows: coatomer (e.g. COP-I) involved in
ER-Golgi transport and in endocytosis (8, 29, 31); the TGN
clathrin-coated vesicle-associated protein AP-1 adaptor complex (32)
involved in sorting of the mannose 6-phosphate receptor (MPR) to
endosomes; p200, a protein of 200,000 daltons that is now known to be
type II myosin (33, 34); and the recently discovered AP-3 adaptor protein (35, 36) whose association with clathrin coats in the Golgi is
controversial (36, 37). Both, AP-1 and COP-I bind to target membranes
in association with a small GTPase ARF1 (for ADP-ribosylation factor 1)
(38). GDP-GTP exchange on ARF occurs concomitant with binding;
inhibition of exchange by BFA blocks the assembly of these coat
proteins. Do coat proteins participate in polarized membrane
trafficking? Recent in vitro binding studies revealed that
AP-1 adaptors interact with tyrosine-based basolateral sorting motif
and with a di-leucine signal artificially introduced into the influenza
hemagglutinin's (HA) C-terminal domain (39), but the function of these
interactions in basolateral trafficking of the HA mutant is unknown.
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Fig. 1.
Schematic representation of putative
-adaptin interaction sites in the cytoplasmic tail of pIgR, pIgR
mutants (A), and pIgR expression levels
(B). A, amino acids are indicated by
single letter code. The 17 amino acids proximal to the
transmembrane domain (TM) shown to contain an autonomous and
dominant basolateral sorting motif (27) are indicated with an
underline. The two main phosphorylation sites of the
cytoplasmic tail are contributed by Ser-664 and Ser-726 (marked with an
asterisk); these residues have been shown to play a role in
constitutive transcytosis and endocytosis, respectively (26, 43).
Ser-726 resides in a putative CKII/PKA phosphorylation site, located
upstream to a double leucine-based motif (underlined).
Typical examples of CKII and PKA phosphorylation motifs are
SXXE and KRXS, where X denotes any
amino acid, and are indicated in parentheses. In the pIgR
mutant, pIgR-
655-668, an internal deletion (
) has been
constructed from Arg-655 to Tyr-668, leaving the C-terminal 89 amino
acids intact and fused to Ala-654 (27). A stop codon has been inserted
instead of arginine 655 to produce a pIgR mutant lacking all but two
residues of the cytoplasmic tail (pIgR R654stop (47)). In the
pIgR-S726-A mutant, serine at position 726 has been substituted with an
alanine (43). The Ser-726 to Ala mutation has been shown to inhibit
pIgR endocytosis, without affecting its basolateral delivery from the
TGN (43). B, pIgR expression level was determined by
SDS-PAGE and immunoblotting analyses of equal protein amounts derived
from cell lysates as indicated under "Experimental Procedures." The
expression levels of pIgR mutants were compared with those of the
pIgR-WT. Cell clones expressing comparable or somewhat higher
expression levels than the wild-type receptor were chosen for analysis
of interactions with
-adaptin.
EXPERIMENTAL PROCEDURES
655-668 mutant whose 15 of the 17 residues comprising the basolateral sorting signal have been deleted
in-frame is delivered from the TGN directly to the apical surface (27).
Newly isolated pIgR-
655-668 expressing MDCK clones revealed
identical exocytic transport properties to the originally described
clone (27). The previously described MDCK clones expressing pIgR S726A,
whose Ser at position 726 in the cytoplasmic tail was mutated to Ala, have been used. This mutation does not abrogate pIgR sorting from the
TGN to the basolateral surface, but it inhibits its endocytosis from
that surface (43). Expression level was determined by SDS-PAGE analysis
of equal protein amounts derived from SDS-cell lysates followed by
immunoblotting and probing with polyclonal sheep anti-SC antibodies and
appropriate horseradish peroxidase-labeled secondary antibodies.
Protein bands were detected using the SuperSignal® chemiluminescent
reagent (Pierce), according to the manufacturer's protocol.
Autoradiograms were scanned at 300 dpi resolution using the HP ScanJet
IIcx scanner, and band intensity was quantified by the NIH image 1.61 software. Mounting of figures was performed using the Adobe
PhotoshopTM 3.05 (Adobe Systems, Inc., Mountain View, CA)
and Aldus Freehand 5.02, Macromedia Inc. Autoradiograms showing the
expression of selected pIgR mutants relative to the wild-type pIgR are
depicted in Fig. 1B.
20 °C, blocked, immunostained for pIgR and
-adaptin, mounted, and stored as described previously (24). The
primary sheep anti-rabbit SC antibody was purified on protein-A
Sepharose and used at 20 µg/ml concentration to label the pIgR. The
100/3 (Sigma Immunochemicals, Rehovot, Israel) antibody was used at
1:200 dilution to label the
-adaptin. The R40.76 anti-ZO-1 rat
monoclonal antibody (48) was used at 1:250 dilution to label the tight
junction-associated protein ZO-1. In some experiments, cells were
treated with 10 µg/ml BFA for 15 min at 37 °C prior to fixation.
Anti-sheep secondary antibodies conjugated to either fluorescein
isothiocyanate (FITC), anti-rat-conjugated tetramethylrhodamine isothiocyanate (TRITC), or anti-mouse coupled to Texas Red were obtained from Jackson ImmunoResearch and used at recommended dilutions as described (24). It should be noted that according to the manufacturer's comments, secondary antibodies were tested for minimal
cross-reactivity for IgG and serum proteins of other species. In
several experiments, the co-localization between
-adaptin and
internalized IgA was examined. In these experiments IgA was internalized into apical recycling endosome either from the basolateral or the apical plasma membrane as described previously (24). Cells on
filters were then fixed and stained with anti-human IgA antibodies
(Sigma), and appropriate secondary antibodies were conjugated to FITC.
-Adaptins were stained with 100/3 monoclonal antibodies and
anti-mouse coupled to Texas Red.
was
determined between two channels, the bleed-through was eliminated by
applying Equation 1.
where CA is the channel to be corrected and
CB is the channel from which the bleed-through
occurs. Note that the areas used for correction were obtained in the
linear portion of the PMT response (gray level under 200).
(Eq. 1)
-adaptin. Autoradiograms were scanned and band intensity was quantified by NIH image.
-adaptins; monospecific rabbit antibodies
directed against
'-adaptin,
-1, or µ-1 subunits kindly provided
by Prof. Margaret S. Robinson (Cambridge, UK) were used as documented
(51). Blots were incubated with horseradish peroxidase-labeled
secondary antibodies, and protein visualization was obtained by the
SuperSignal® chemiluminescent reagent.
-adaptins. Samples were analyzed by
SDS-PAGE and Western blotting, and signals contributed by the pIgR and
-adaptins were quantified as above.
-Adaptin--
MDCK cells expressing the wild-type pIgR were
cultured for 3 days on 24 mm Transwell filters prior to the experiment.
To co-immunoprecipitate pIgR located in the ER, cells were starved for
10 min at 37 °C in Dulbecco's modified Eagle's medium lacking
L-Cys and L-Met (Sigma) but supplemented with
Hanks' balanced salts and 20 mM Hepes, pH 7.4, 5%
dialyzed fetal bovine serum, and pulse-labeled for 8 min with 4.5 mCi/ml [35S]Met-Cys (Pro-Mix, Amersham Corp.,
Buckinghamshire, UK). To co-immunoprecipitate pIgR accumulated in the
TGN (i.e. after TGN block), cells after the pulse were
incubated for 120 min at 18 °C with MEM/BSA (MEM containing Hanks'
balanced salt solution (Sigma), 20 mM Hepes, pH 7.4, 0.6%
BSA) present on the apical and basolateral chambers of the Transwell;
after the 18 °C chase the pIgR becomes insensitive to digestion by
endoglycosidase H (41). To co-immunoprecipitate pIgR chased into
post-TGN compartments, cells subjected to TGN block incubation were
further incubated in warm (37 °C) MEM/BSA for 10, 30, or 60 min.
Cells were then lysed in ice-cold TDB containing 1.25% Triton X-100,
protease inhibitors, and 1 mM sodium orthovanadate, pre-cleared once with Sepharose CL-2B (19) (Pharmacia, Uppsala, Sweden), and
-adaptin was immunoprecipitated at 4 °C for 16 h with the 100/3 antibodies bound to protein A-Sepharose. Beads were
washed three times with "mixed micelle" buffer (MMB: 20 mM triethanolamine Cl, pH 8.6, 150 mM NaCl, 5 mM EDTA, pH 8.0, 8% w/v sucrose, 0.1% NaN3,
1% w/v Triton X-100, 0.2% SDS), once with "Final Wash" buffer
(MMB lacking Triton X-100 and SDS), and bead pellet was boiled in 80 µl of 1% SDS. 35S-pIgR was re-immunoprecipitated from
the released proteins with anti-pIgR antibodies coupled to protein
A-Sepharose. The total amount of radiolabeled pIgR was determined by
pIgR immunoprecipitation from cells that have been metabolically
labeled under identical conditions. Samples were analyzed on 10% SDS
gels; dried gels were exposed to Fuji Imaging Plates, and pIgR
radioactivity levels were determined by the FUJIX BAS 1000 imaging
system. Gels onto which co-immunoprecipitated pIgR was analyzed were
exposed for 3-days, whereas gels with total pIgR were exposed for
15 h.
-Adaptin--
Cells were grown on 35-mm dishes and labeled with 0.5 mCi/ml [32P]orthophosphate for 3 h. Cells were lysed
in cold TDB containing 1.25% Triton X-100, a mixture of protease
inhibitors, and 1 mM vanadate. Lysates were precleared with
Sepharose CL-2B and incubated with anti-
-adaptin 100/3 antibodies
and protein G-Sepharose for 16 h at 4 °C with continuous
end-to-end rotation. Immunoprecipitates were washed three times with
ice-cold MMB buffer and one time with "Final Wash" buffer.
Immunoprecipitates were treated with 80 µl of 1% SDS, boiled for 5 min, and diluted with 1 ml of TDB containing 2.5% Triton X-100. The
pIgR was re-immunoprecipitated using sheep anti-rabbit SC coupled to
protein G-Sepharose. Samples were analyzed on 10% gels, and dried gels
were autoradiographed using Kodak Bio-Max x-ray film for 72 h with
intensifying screen (for 32P-pIgR
re-immunoreprecipitates), or for 2.5 h (for total
32P-pIgR immunoprecipitation). The total amount of
radiolabeled pIgR and the amount of radiolabeled pIgR that has been
co-immunoprecipitated with
-adaptins was determined by autoradiogram
scanning and imaging with the NIH Image program.
20 °C), as
described (42).
RESULTS
-Adaptin Partially Co-localizes with the pIgR in Polarized and
Semi-polarized MDCK Cells--
To provide qualitative analysis of the
interactions between pIgR and
-adaptin, we analyzed the
co-localization between these molecules in MDCK cells grown on filter
supports (i.e. in polarized cells, Fig.
2A) or in cells grown on
coverslips (i.e. in semi-polarized cells, Fig.
2B) using confocal immunofluorescence microscopy. Clear and
dense overlapping fluorescent signals (yellow in the merged
panel) contributed by the pIgR (green) and
-adaptin
(red) are seen primarily in the center of the apical region
of many cells (an example is given in Fig. 2A). In addition
to the trans-Golgi network,
-adaptin has been recently
reported to be localized to basolateral endosomal structures (50);
thus, the co-localization of
-adaptin and pIgR could represent
co-localization of the two proteins in endosomal membranes. However,
the
-adaptin-positive structures observed here are apical, and, in
double-labeling experiments, dIgA internalized into apical endosomes
from either the apical or basolateral membrane does not significantly
co-localize with
-adaptin.2 Thus, we
conclude that the majority of co-localization of
-adaptin and pIgR
is associated with the trans-Golgi network. In addition, in
immunostained MDCK cells grown on coverslips, significant
co-localization between
-adaptin and pIgR could be observed in
perinuclear structures, a distribution characteristic of the
trans-Golgi network in semi-polarized cells (32).
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Fig. 2.
Confocal immunofluorescence microscopy
analysis of co-localization of -adaptin with transfected pIgR in
filter-grown (A) or coverslip-grown (B) MDCK
cells. MDCK cells expressing the wild-type pIgR were cultured for
4 days on 12.5-mm diameter filter supports or for 3 days on a
coverslip. Cells were triple-labeled for pIgR (purified sheep
anti-rabbit pIgR followed by FITC-conjugated anti-sheep immunoglobulin
G),
-adaptin (100/3 monoclonal antibody followed by Texas
Red-conjugated anti-mouse antibodies), and for the tight-junction ZO-1
protein (R40.76 anti-ZO-1 rat monoclonal antibodies followed by
TRITC-conjugated anti-rat antibodies) (blue). In the overlay
images obtained for pIgR (green) and
-adaptin
(red), yellow indicates areas of co-localization.
Arrows point to structures where pIgR and
-adaptins
co-localize.
-Adaptin Subunit of the AP-1 Complex Is Co-immunoisolated
with Membranes Expressing the pIgR--
We have used MDCK monolayers
that express the wild-type pIgR and various pIgR mutants to identify
the association between the pIgR and
-adaptin by immunoisolation of
membranes from MDCK cells. A PNS fraction was prepared from MDCK cells
expressing the wild-type pIgR, and the crude membrane preparation was
incubated with purified antibodies directed against the cytoplasmic
tail of the pIgR (49) coupled to magnetic beads. Immunoisolated
membranes were solubilized and analyzed for the presence of pIgR and
-adaptin by immunoblotting. Data presented in Fig.
3A demonstrate that
-adaptin is co-immunoisolated with membranes bearing the wild-type pIgR. The antibodies specifically immunoisolate membranes expressing an
intact cytoplasmic tail since only residual amounts of pIgR are
detected upon incubation of immunobeads with membranes containing a
pIgR mutant which lacks all but two cytoplasmic amino acid residues (pIgR-R654stop, Fig. 3B, upper panel).
-Adaptin did not
bind to immunobeads exposed to MDCK cells that do not express the pIgR (Fig. 3A), further suggesting that the coat protein is
specifically co-isolated with pIgR-expressing membranes. To evaluate
the relative extent of
-adaptin association with membranes
expressing pIgR mutants, the
-adaptin/pIgR ratio was determined in
the immunoisolated membranes, and the value obtained was normalized to
that determined for the wild-type receptor (Fig. 3B, lower
panel). The results reveal a significant reduction in
-adaptin/pIgR ratio in membranes isolated from MDCK cells expressing
pIgR-
655-668, or pIgR-S726-A. Note that although the expression
levels of pIgR mutants were similar to those of the wild-type receptor
(Fig. 1B), membranes expressing pIgR mutants were more
efficiently immunoisolated since compared with the expression of the
wild-type pIgR, higher pIgR levels are associated with them (compare
band intensity of pIgR-
655-668 or pIgR-S726-A to band intensity of
pIgR-WT in Fig. 3B, upper panel). The apparently inefficient
isolation of membranes expressing the wild-type receptor might be
attributed to inaccessibility of the antibody to the cytoplasmic tail
by associated cytosolic proteins, e.g. by
-adaptin, which
may be more efficiently recruited to the cytoplasmic tail of the
wild-type pIgR but not to its mutants, making these membranes less
accessible to the anti-tail antibodies. These results suggest that
-adaptin associates more extensively with membranes containing the
wild-type pIgR than those with the indicated pIgR mutants. Our next aim
was to characterize the interaction between
-adaptin and the
pIgR.
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Fig. 3.
Immunoisolation of pIgR-containing
membranes. A PNS fraction was prepared from the
following: MDCK cells that do not express the pIgR; MDCK cells that
express the wild-type receptor (A); or from MDCK cells that
express the pIgR mutants pIgR- 655-668, pIgR-S726-A, or
pIgR-R654stop (B). Purified anti-tail monospecific
antibodies were reacted with streptavidin-coupled magnetic beads
pre-coated with biotinylated rabbit anti-mouse IgG. Immunobeads were
exposed to equal protein amounts of PNS in the cold and washed to
remove unbound membranes, and bound (immunoisolated) membranes were
solubilized in Triton X-100-containing buffer, mixed with SDS sample
buffer, and subjected to SDS-PAGE followed by immunoblotting with
anti-pIgR or anti-
-adaptin antibodies. Signals were visualized by
enhanced chemiluminescence. For quantitative determination of band
intensity, exposures were always in the linear range, and the mean band
density was quantified using the NIH Image program. Intensity levels
contributed by
-adaptins were divided by those contributed by the
pIgR, and the ratio determined for each pIgR mutant was calibrated to
the ratio measured for the wild-type receptor (B, lower
panel). Two different MDCK clones expressing each pIgR construct
were analyzed in three separate experiments. Results are mean ± S.E.
-adaptin,
1-adaptin,
1, and µ1 subunits of the AP-1 adaptor complex. The results presented in Fig. 4
demonstrate that all four subunits of the AP-1 complex co-precipitate
with the pIgR in the cross-linked cells but not in cells that were not
exposed to the cross-linker. Neither pIgR nor adaptor chains were
co-precipitated with plain protein A. These results hence suggest that
the pIgR is complexed with the entire AP-1 adaptor in polarized MDCK
cells.
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Fig. 4.
Cross-linking of AP-1 adaptors to the
pIgR. The basolateral surface of pIgR-expressing MDCK cells was
selectively permeabilized with digitonin and subsequently incubated
with the hydrophilic cross-linker DTSSP (+ cross-linking).
In one experiment cells were not treated with the cross-linker
( cross-linking). After quenching with glycine, cells were
solubilized in buffer containing Triton X-100, and the pIgR was
immunoprecipitated from the lysate using sheep anti-pIgR antibodies. In
a control experiment, cells exposed to the cross-linker were
subsequently incubated with plain protein A-Sepharose (Protein
A-Seph). Immunoprecipitates were separated by SDS-PAGE,
immobilized on a nitrocellulose membrane, and probed with sheep
anti-pIgR antibodies, mouse monoclonal anti-
-adaptin 100/3
antibodies, and rabbit monospecific antibodies directed against
peptides derived from the
1,
1, and µ1 subunits of the AP-1
complex.
-adaptin. Negligible levels of pIgR and
-adaptin were associated with plain protein A-Sepharose beads or
with protein A-Sepharose coupled to irrelevant sheep IgG (Fig. 5A, upper panel).
In contrast, only when the pIgR was immunoprecipitated by anti-SC antibodies were significant amounts of
-adaptin
co-immunoprecipitated (Fig. 5A, upper panel). The
amount of co-immunoprecipitated
-adaptin correlated with the amount
of pIgR that had been immunoprecipitated from cell lines expressing
different levels of pIgR (not shown), further suggesting that AP-1
interacts with the wild-type pIgR in MDCK cells.
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Fig. 5.
-Adaptin co-immunoprecipitates
(Co-IPs) with the pIgR in a BFA-sensitive fashion.
A, MDCK cells expressing the wild-type pIgR were solubilized
in the cold with buffer containing Triton X-100. Triton-insoluble
materials were removed by high speed centrifugation. Solubilized
proteins were subjected to immunoprecipitation (IP) with
anti-pIgR antibodies. Negative controls include lysates exposed to
protein A-Sepharose (Protein A Seph) or to protein
A-Sepharose coupled to non-relevant sheep IgG. Immunocomplexes were
analyzed by SDS-PAGE and immunoblotting for the presence of pIgR
(Anti-pIgR) and
-adaptins (Anti-
-adaptin)
(upper panel). Note that immunoprecipitated pIgR often
appears as two major bands, one at 110 kDa represents the intact
receptor and the other at 85 kDa represents cell-associated SC. In the
lower panel, the relative efficiencies of
co-immunoprecipitation of
-adaptin with pIgR in the absence
(Co-IP) and presence of chemical cross-linker
(Cross-linking) is also shown. B, MDCK cells
expressing the wild-type pIgR were either treated (+) or not treated
(
) with 10 µg/ml BFA for 1 h at 37 °C. Cells were lysed in
ice-cold Triton X-100 containing buffer, and subjected to pIgR
immunoprecipitation. Immunoprecipitates and 5-µl sample from each
lysate were subjected to SDS-PAGE and Western blotting, which were
subsequently probed with anti-pIgR (Anti-pIgR, upper
panel) or anti-
-adaptin antibodies
(Anti-
-adaptin, lower panel). Approximately
equal amounts of pIgR were immunoprecipitated by protein A-Sepharose
coupled to sheep anti-pIgR IgG, as judged by quantifying pIgR band
intensity (pIgR level, arbitrary units (a.u.),
upper panel). A reduction of approximately 50% in the
amount of co-immunoprecipitated
-adaptin was observed in BFA-treated
cells relative to untreated cells, after normalizing the amount of
co-precipitated adaptins to the level of precipitated pIgR (lower
panel). This result is the mean of three experiments.
Representative results are shown.
-adaptin was
co-immunoprecipitated with the pIgR after cross-linking
versus after co-immunoprecipitation. The cross-linker
covalently links the AP-1 adaptor with the pIgR, thus higher efficiency
of
-adaptin co-precipitation is expected to be observed in the
cross-linking experiment. Indeed, data presented in Fig. 5A
(lower panel) are consistent with this expectation. The
amount of co-immunoprecipitated
-adaptin normalized to the level of
precipitated pIgR in a typical cross-linking experiment was about 20 times higher than the ratio determined for a co-immunoprecipitation experiment. This result suggests that co-immunoprecipitation
experiments are limited in their ability to provide a quantitative
estimation on pIgR fraction that interacts with
-adaptin in the
cell. Flexible and weak interactions probably mediate pIgR association
with proteins involved in its trafficking, and the majority of these
interactions are likely to be disrupted upon cell solubilization under
conditions of co-immunoprecipitation. Even the cross-linking
approach cannot provide such information as it is impossible to
determine the absolute efficiency at which AP-1 complexes are
cross-linked to the receptor. Nonetheless, since co-immunoprecipitation
is relatively easy to perform, and as it enables the detection of
specific and physiological interactions, further analysis of
-adaptin-pIgR interactions was performed using this approach.
-adaptin co-immunoprecipitation. Cells
expressing the wild-type pIgR were either not treated (
BFA) or
treated with BFA (+BFA) and were subsequently subjected to pIgR
immunoprecipitation. Approximately equal amounts of pIgR have been
immunoprecipitated from each preparation (as determined by quantitative
immunoblotting; see Fig. 5B, upper panel). The amount of
co-immunoprecipitated
-adaptin was quantified as well and normalized
to the amount of precipitated pIgR (Fig. 5B, lower panel).
Compared with untreated cells, a reduction of approximately 50% in
co-immunoprecipitated
-adaptin was observed in BFA-treated cells
(Fig. 5B, lower panel). BFA treatment has no effect on total
-adaptin levels in the cells, indicating that the reduction in
co-immunoprecipitated coat protein is not due to fluctuations in
expression level of endogenous
-adaptin. The BFA-resistant
interactions could be explained by previous data suggesting that even
after long periods of exposure to BFA, a small amount of
-adaptin
remains concentrated in the perinuclear region of MDCK cells, possibly
representing
-adaptin that did not dissociate from the Golgi
apparatus (32).2 Nevertheless, the reduction in
-adaptin
co-immunoprecipitation by BFA argues that BFA-sensitive interactions
between pIgR and
-adaptin are detected by this approach, reflecting
specific and physiologically relevant interactions between these
molecules in the cell.
-Adaptin Interactions Are Initiated in the TGN and Persist
in Post-TGN Compartments--
We combined pulse-chase with
co-immunoprecipitation to characterize the intracellular sites at which
pIgR-
-adaptin interactions are initiated. Filter-grown MDCK cells
expressing the wild-type receptor were metabolically pulse-labeled for
8 min at 37 °C with [35S]Cys-Met. Under these labeling
conditions, the cohort of metabolically labeled pIgR is located in the
ER, as judged by the sensitivity of the pulse-labeled receptor form to
digestion with endoglycosidase H (41). Upon a subsequent chase at
18 °C, pulse-labeled receptors accumulate in the TGN and become
resistant to digestion by endoglycosidase H (41). To chase
metabolically labeled receptor into post-TGN compartments, cells were
pulse-labeled and chased for 30 min at 37 °C or cells were
pulse-labeled, chased at 18 °C, and subsequently chased for 10, 30, or 60 min at 37 °C. Cells expressing 35S-pIgR in the ER,
in the TGN, or in post-TGN compartments were solubilized, and
-adaptin was immunoprecipitated. Immunoprecipitates were analyzed
for the presence of co-precipitated 35S-pIgR. Fig.
6A shows that pulse-labeled
receptors are not co-immunoprecipitated with
-adaptin, confirming
that newly synthesized receptors in the ER are not associated with AP-1
adaptors. In contrast, pIgR accumulated in the TGN appears to
co-immunoprecipitate with AP-1 adaptors. Similarly,
35S-pIgR chased into post-TGN compartments
co-immunoprecipitates with
-adaptin. These results suggest that the
coat protein interacts with the pIgR after its delivery to
the TGN and that these interactions persist after pIgR exit from the
TGN and/or are either re-initiated in or persist in post-TGN
compartments. After 30 and 60 min chase, the majority (75%) of the
35S-pIgR cohort reaches the basolateral surface (19, 20,
27),2 and a significant fraction of the surface-arriving
molecules is likely internalized into endosomal and transcytotic
elements. Note that the signal contributed by 35S-pIgR
co-immunoprecipitated following 30 min chase at 37 °C is significantly greater than the signal contributed by
35S-pIgR after "TGN block" (Fig. 6A),
suggesting that pIgR-
-adaptin interactions could be augmented when
35S-pIgR is present in post-TGN compartments. An
alternative explanation is that incubation at 37 °C increases the
affinity of pIgR interactions with the adaptor complex or merely
enhances the efficiency of pIgR labeling with the radioactive amino
acids.
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Fig. 6.
35S-pIgR is co-immunoprecipitated
with -adaptins after its accumulation in the TGN. A,
filter-grown MDCK cells expressing the wild-type pIgR were either
pulse-labeled for 8 min at 37 °C with [35S]cysteine
and methionine alone, pulse-labeled and subsequently chased for 120 min
at 18 °C (TGN Block), or pulsed and chased for 30 min at
37 °C. Cells on filters were lysed in cold buffer containing Triton
X-100, and
-adaptin was immunoprecipitated using the 100/3
monoclonal antibodies coupled to protein A-Sepharose.
Immunoprecipitated complexes were released from the beads by boiling
the beads in SDS, and the pIgR was re-immunoprecipitated from the
released protein mixture by anti-pIgR antibodies coupled to protein
A-Sepharose. In the control experiment, lysates prepared from cells
that have been pulse-labeled and chased for 30 min at 37 °C were
incubated with irrelevant mouse IgG2b that does not precipitate
-adaptin and protein A-Sepharose. Immunoprecipitates were analyzed
by SDS-PAGE, and autoradiography of co-immunoprecipitated pIgR is
shown. B, PIgR-expressing MDCK cells were pulse-labeled and
subsequently incubated under conditions that block the pIgR in the TGN.
Cells were either not chased (0 min) or chased for 10, 30, or 60 min at
37 °C. Cells were then subjected to immunoprecipitation of
-adaptin and analyzed for co-immunoprecipitated
35S-labeled pIgR as above. Total amounts of radiolabeled
pIgR after each chase period were immunoprecipitated from parallel
samples and analyzed by SDS-PAGE, and autoradiography is presented. The
amount of 35S-pIgR co-immunoprecipitated after 10 min chase
at 37 °C is identical to 35S-pIgR co-precipitated after
TGN block (upper panel). In contrast, compared with the
amount of 35S-pIgR co-precipitated with
-adaptin after
TGN block, the level of co-precipitated 35S-pIgR is about
3.5-fold greater after TGN block and chase for 30 or 60 min at 37 °C
(lower panel). Note that the levels of total
35S-pIgR immunoprecipitation after 30- and 60-min chase
periods is reduced compared with the amount of 35S-pIgR
immunoprecipitated after TGN block. This reduction is attributed
to 35S-pIgR transcytosis and arrival at the apical surface
where it is cleaved to soluble SC.
-adaptin seems to be specific since metabolically labeled receptors were not immunoprecipitated if protein A-Sepharose coupled to irrelevant IgG2b (the 100/3 monoclonal antibody is of mouse IgG2b isotype) were used (see Control in Fig. 6A).
Since after 30 or 60 min chase at 37 °C, the majority of labeled
receptors likely reached the basolateral surface and
endocytic/post-endocytic/compartments, these data indicate that
pIgR-AP-1 interactions are enhanced in these compartments.
-adaptin. We believe that this figure does not necessarily indicate
that only a small fraction of pIgR interacts with AP-1 adaptors.
Rather, inefficient co-immunoprecipitation may reflect a limitation of
this experimental protocol in detecting the real fraction of pIgR
molecules that interacts with
-adaptin, as previously discussed.
-Adaptin Interacts with Reduced Efficiency with Apically
Targeted pIgR and with pIgR Mutated in Ser-726--
We employed
co-immunoprecipitation to examine directly the level of interactions
between pIgR-
655-668 (Fig.
7A) or pIgR-S726-A (Fig.
7B) and
-adaptin. The amount of co-precipitated adaptins was normalized to the levels of immunoprecipitated pIgR, and the relative association of
-adaptin with pIgR mutants was
quantitatively determined with respect to the wild-type receptor (Fig.
7C). To confirm the specificity of
-adaptin
co-immunoprecipitation with pIgR mutants, co-immunoprecipitation from
MDCK cells that do not express the pIgR (MDCK), or from
cells that express the wild-type pIgR (pIgR-WT), or from cells
expressing the pIgR-R654stop mutant was conducted in parallel as
negative and positive controls.
-Adaptin could not be efficiently
co-immunoprecipitated with pIgR-R654stop or with pIgR-S726-A mutants.
The amount of
-adaptin present in cell lysates was equal, indicating
that the reduction in co-immunoprecipitated coat protein is not due to
fluctuations in endogenous expression levels of
-adaptin in the
different clones. This latter result, which is in agreement with the
co-immunoisolation data shown above (Fig. 3), argues that apically
targeted pIgR-R654stop or basolaterally targeted receptors with
inactivated Ser-726 largely avoid interactions with AP-1 adaptors.
Yet, pIgR-
655-668, which is also apically targeted, seems to maintain higher levels of interaction
with
-adaptin than the other mutant pIgRs. This result supports the contention that Ser-726 is involved in AP-1 association since pIgR-
655-668 contains an intact Ser-726 motif that is potentially free to interact with AP-1 complexes. Previous in vitro
binding experiments indeed demonstrated that unlike the wild-type
influenza HA (an apical protein in MDCK cells), which does not
efficiently interact with AP-1 adaptors, a mutant HA bearing C-terminal
di-leucine sequence is capable of interacting with purified AP-1
adaptors (39). Hence, apically targeted pIgR-
655-668 may still
maintain significant degree of interactions with the coat protein,
which suggests that AP-1 adaptors may still be involved in certain
steps of apical targeting of the pIgR mutant.
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Fig. 7.
-Adaptin interacts with the cytoplasmic
tail of the pIgR, possibly through the Ser-726 motif.
A, the pIgR was immunoprecipitated from the following: MDCK
cells; from MDCK cells that express the wild-type receptor; from cells
that express the pIgR-
655-668 mutant; or from cells that express
the pIgR-R654stop mutant. B, the pIgR was immunoprecipitated
from cells that express the wild-type pIgR or cells that express the
pIgR-S726-A mutant. In all cases comparable amounts of the pIgR were
immunoprecipitated under conditions described in Fig. 4 and under
"Experimental Procedures." Immunoprecipitated pIgR and
co-immunoprecipitated
-adaptin were visualized by Western blotting
followed by enhanced chemiluminescence, and representative exposures
are presented. The experiment was performed at least six times on three
different MDCK clones expressing the wild-type pIgR and on two
different cell lines expressing each of the indicated pIgR mutants.
Band intensity of
-adaptin was normalized to the intensity of
precipitated pIgR. C, the
-adaptin/pIgR ratio obtained
for each mutant receptor is calibrated to that of the wild-type
receptor. Data are presented as mean ± S.E.
-Adaptin--
If
phosphorylation of Ser-726 is important for binding to AP-1 adaptors,
one would expect that phosphorylated receptors would associate with the
coat protein. To address this point, cells were labeled with
[32P]orthophosphate to steady-state levels and lysed in
Triton-containing buffer, and AP-1 adaptors were
immunoprecipitated via
-adaptin. The amount of co-immunoprecipitated
32P-labeled pIgR was assessed by SDS-PAGE and
autoradiography quantified by densitometric analysis after normalizing
the amount of co-immunoprecipitated 32P-pIgR to the total
amount of 32P-pIgR immunoprecipitated from the cell
lysates. 32P-pIgR co-immunoprecipitated with
-adaptin
(Fig. 8, upper panel). Less
than 5% of total labeled pIgR co-precipitated with
-adaptin. Beads
coupled to irrelevant mouse IgG2b did not immunoprecipitate
-adaptin
nor co-precipitate the pIgR (not shown). BFA treatment significantly
reduced the level of co-precipitated 32P-pIgR (Fig. 8).
32P-pIgR-
-adaptin interactions with the coat protein
also seem to be inhibited by mutational inactivation of Ser-726 as a
relatively smaller fraction of 32P-labeled pIgR-S726-A
co-immunoprecipitated with
-adaptin (Fig. 8). Together, the results
indicate that
-adaptin interact with phosphorylated wild-type pIgR
in a BFA-sensitive manner and that Ser-726 is involved in these
interactions.
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Fig. 8.
-Adaptin interacts with
32P-pIgR. MDCK cells expressing the wild-type pIgR or
pIgR-S726-A were labeled with [32P]orthophosphate; equal
amounts of
-adaptin were immunoprecipitated, and pIgR was
re-precipitated from the immunocomplexes as described in Fig. 5 and
under "Experimental Procedures." Total amount of
32P-pIgR was estimated by immunoprecipitating the entire
32P-pIgR population from the lysates. In one experiment,
cells that express the wild-type receptor were treated with 10 µg/ml
BFA for 1 h at 37 °C prior to immunoprecipitation.
Immunoprecipitates were analyzed by SDS-PAGE, and representative
autoradiograms are presented in the upper panel. Note that
band intensity of co-precipitated pIgR-WT appears to be greater
than the corresponding band of total 32P-pIgR in cell
lysate. This apparent discrepancy is due to much shorter autoradiogram
exposure times for total radiolabeled pIgR as indicated under
"Experimental Procedures." Band intensity was quantitatively
determined in five independent experiments. The signal of
co-immunoprecipitated 32P-pIgR was normalized to the signal
contributed by total 32P-pIgR, and the ratio obtained for
each experiment was calibrated with respect to pIgR-WT (lower
panel). Results are mean ± S.E.
-adaptin suggests that whereas
Ser-726 is apparently critical for the association of AP-1 adaptors
with the pIgR, it may not be the sole factor in this interaction.
Since Ser-664, the residue that regulates the constitutive transcytosis
of the pIgR (26), is likely to be the phosphorylated residue in the
S726-A mutant pIgR, these data raise the possibility that
phosphorylated pIgR in the constitutive transcytotic pathway may also
interact with AP-1 adaptors.
-adaptin, we conclude that the interactions are not involved in
basolateral targeting of the molecule. We have reasoned, however, that
interactions with
-adaptin may play a regulatory role in exocytosis
of pIgR from the TGN. Thus, the pIgR-S726-A might display basolateral
delivery kinetics that differ from those exhibited by wild-type pIgR.
The results in Fig. 9, A and
B, indeed reveal that pIgR-S726A is directly delivered to
the basolateral surface with slower kinetics than the wild-type
receptor. In addition, unlike the case for the wild-type
pIgR, whose biosynthetic pathway is inhibited by BFA, basolateral
delivery of newly synthesized pIgR-S726-A is not inhibited by the drug.
These results further emphasize the importance of Ser-726 in mediating
the interactions with AP-1 adaptors and that interactions with AP-1
adaptors could elicit a BFA-sensitive exocytic step that concentrates
pIgR molecules into AP-1/clathrin-coated areas of the TGN. This process
may result in efficient delivery of pIgR molecules from the TGN to the
basolateral surface.
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Fig. 9.
Kinetics and BFA sensitivity of delivery of
wild-type pIgR, pIgR-S726-A, pIgR- 655-668, and pIgR-R654stop from
the TGN to the basolateral and apical cell surface. A,
delivery of nascent receptors to the respective cell surface was
measured over 15-, 30-, and 60-min chase periods at 37 °C using the
protease-based delivery assay, as described under "Experimental
Procedures" and elsewhere (19, 20). B, targeting of
receptors to the apical and basolateral surfaces was measured after 60 min chase at 37 °C in the absence (
) or presence (+) of BFA. In
BFA-treated cells, BFA (10 µg/ml) was included in the media bathing
both the apical and basolateral surfaces of the cells throughout the
starvation, pulse, and chase steps (42). Results are mean ± S.E.
of six independent experiments performed on two different clones
expressing each pIgR construct.
655-668 and pIgR-R654stop is significantly
inhibited by BFA. These results are consistent with previous findings
that, whereas BFA may not inhibit exocytosis of some
basolaterally targeted membrane proteins, in all cases reported so far,
BFA inhibited exocytosis of apical membrane proteins. In
addition, this result is intriguing as it suggests that,
particularly in the case of pIgR-R654stop, interactions
between pIgR cytoplasmic tail and BFA-sensitive coat proteins are not
required for mediating apical targeting. Apical transport of
transmembrane proteins occurs either by proteinaceous determinants (15)
or is mediated by widely distributed carbohydrate
determinants (13, 14, 55, 56). It is hence possible that
BFA-sensitive coat proteins are general regulators of trafficking of
apical membrane proteins. An alternative scenario is that unlike
basolateral membrane proteins, transmembrane proteins destined to the
apical surface all pass through a common compartment whose sorting
activity is impaired by BFA.
DISCUSSION
-adaptin and the pIgR, and
we asked whether a cytoplasmic Ser-726, residing in a putative CKII/PKA
phosphorylation site upstream to a di-leucine motif, is important for
these interactions. Ser-726 is required for rapid internalization of
the pIgR from the basolateral plasma membrane of MDCK cells but not for
basolateral targeting from the TGN (43). We investigated whether
Ser-726 plays a role in the mechanism of pIgR exocytosis
from the TGN to the basolateral cell surface by virtue of its
interactions with the AP-1 clathrin adaptor.
-adaptin interactions might be
regulated by the small GTPase ARF1 (57). Hence, like in the case of
MPRs bound to lysosomal enzymes, specific determinants in the
cytoplasmic tail, perhaps more particularly a CKII phosphorylation
site, bind AP-1 adaptors, a process that leads to pIgR sorting in the
TGN into clathrin-coated vesicles.
-adaptin suggests that at
steady-state the pIgR partially co-localized with
-adaptin, possibly
on the surface of the trans-Golgi network. Our
co-immunoprecipitation data further suggest that the pIgR interacts
with
-adaptin and that these interactions are initiated in the Golgi
since metabolically labeled pIgR could be co-immunoprecipitated with
-adaptin only after pIgR accumulation in the TGN. Interestingly,
however, the co-immunoprecipitation experiments also suggest that
-adaptin may interact with pIgR located in post-TGN compartments,
possibly in basolateral endosomes or transcytotic compartments. This
interpretation is consistent with previous studies showing the
association of
-adaptin with endosomes in non-polarized cells (58)
and with basolateral endosomes containing internalized transferrin or
dIgA in polarized cells (50).
-adaptin
interactions, in addition to regulating the interaction of pIgR with
endocytotic machinery (38). Phosphorylation of Ser-726 may therefore
serve as a bifunctional biological switch that prompts the interactions
with either AP-1 adaptors in the TGN or AP-2 adaptors at the
basolateral membrane. Alternatively, the detection of
pIgR-AP-1 interactions in the TGN presented here may be a consequence
of "cross-talk" with a degenerate sorting signal for AP-2 adaptors
regulated by Ser-726 or vice versa. Even if phosphorylated Ser-726 is
not directly involved in the binding of adaptors, phosphorylation may
cause conformational changes in the cytoplasmic tail which expose other
motifs, such as the downstream di-leucine motif, for interactions with
the coat protein, as was recently proposed for the CD3
chain of the
T cell receptor (59).
-subunit of membrane bound AP-1 adaptors to mediate the interactions
with the bovine papillomavirus E6 protein (63), indicating that adaptin
subunits other than medium chains may mediate interactions with signal
motifs. Of course, an alternative scenario is that other proteins may
contribute to the interactions between AP-1 and the cytoplasmic domain
of pIgR, such as the recently cloned PACS family of TGN-associated coat
proteins involved in the trafficking of furin and MPRs (64).
-adaptin independent and
BFA-insensitive (and possibly bypasses the endosomal system).
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ACKNOWLEDGEMENTS |
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We thank Prof. Gerard Apodaca (University of Pittsburgh) and Dr. Vanda Reich (Hebrew University) for critical reading of the manuscript and M. S. Robinson for providing the antibodies directed against each of the AP-1 adapter subunits.
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FOOTNOTES |
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* This work was supported by grants from the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities-The Dorot Science foundation, a grant from the Ministry of Science (to B. A.), and the American Association of the Colleges and Pharmacy, the Zumberge Research and Innovation Fund, and National Institutes of Health Grant DK RO1-51588 (to C. T. O).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Fax: 972-2-5617918; E-mail: aroeti{at}cc.huji.ac.il.
The abbreviations used are: pIgR, polymeric immunoglobulin receptor; BFA, brefeldin A; TGN, trans-Golgi network; MDCK, Madin-Darby canine kidney; ER, endoplasmic reticulum; SC, secretory component; CD, cation-dependent; MPR, mannose 6-phosphate receptor; ARF, ADP-ribosylation factor; PAGE, polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; PNS, post-nuclear supernatant; PBS, phosphate-buffered saline; MEM, minimal Eagle's medium; HA, hemagglutinin; BSA, bovine serum albumin; dIgA, dimeric IgA; PKA, protein kinase A; DTSSP, 3,3'-dithiobis[sulfosuccinimidyl propionate.
2 E. Orzech and B. Aroeti, unpublished observations.
3 D. Stephens, G. Banting, and B. Aroeti, unpublished data.
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REFERENCES |
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