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(Received for publication, November 28, 1994; and in revised form, August 1, 1995) From the
Hepatocytes must transport newly synthesized apical membrane
proteins from the basolateral to the apical plasma membrane. Our
earlier morphological study showed that the apical proteins share a
late (subapical) part of the transcytotic pathway with the well
characterized polymeric immunoglobulin A receptor (Barr, V. A., and
Hubbard, A. L.(1993) Gastroenterology 105, 554-571).
[Abstract]
Starting with crude microsomes from the livers of
[
The plasma membrane (PM) ( Fortunately, the transcytosis of one PM protein has been studied
extensively in hepatocytes(14, 15) ; this protein is
the polymeric IgA receptor (pIgA-R), which transports IgA from blood to
bile. Fig. 1shows a current view of the intracellular itinerary
of newly synthesized pIgA-R. Mature pIgA-R is delivered to the
basolateral PM from the Golgi. Both free and ligand-bound receptor are
internalized in clathrin-coated
vesicles(16, 17, 18) . Then ligand and
receptor are taken to early endosomes; in uptake experiments ligand
appears there very quickly(16, 19, 20) .
Later the ligand (presumably still traveling with the receptor) can be
visualized in a subapical tubulovesicular
compartment(16, 17, 21, 22) .
Following a brief delay, pIgA is released into bile, complexed to the
ectodomain of the pIgA-R (now called secretory component). Because
pIgA-R is cleaved at the apical PM, most of the receptor in the cell is
less than 2 h old(23, 24) . pIgA-R can be seen in both
early endosomes and in the subapical compartment by
immunofluorescence(25, 26, 27) , showing that
at steady state there are significant amounts of receptor in both
compartments, but not at the apical PM(17, 23) . Work
in MDCK cells transfected with pIgA-R cDNA has identified multiple
signals on the 103-amino acid cytoplasmic tail of pIgA-R that guide the
receptor along its complicated journey (27, 28, 29) .
Figure 1:
The life cycle of pIgA-R in
hepatocytes. After synthesis in the ER and processing in the Golgi,
mature pIgA-R is transported from the trans-Golgi network to the
basolateral PM. Receptors are internalized via clathrin-coated vesicles
and delivered to early basolateral endosomes. Ligand (pIgA presumably
bound to receptor) is next found in a subapical tubulovesicular
compartment. Because this compartment is not continuous with
basolateral endosomes, transport is probably mediated by vesicular
transcytotic carriers. After delivery to the apical PM, pIgA-R is
cleaved quickly and the ectodomain bound to pIgA is released into bile.
However, there is a delay between the arrival of pIgA in the subapical
compartment and release into bile, indicating that there may also be a
vesicle mediated delivery step between the subapical compartment and
the apical PM. These putative vesicular transport steps are indicated
by question marks.
In contrast to the pIgA-R,
many apical PM proteins either have very short cytoplasmic tails with
no obvious sorting signals (e.g. dipeptidyl peptidase IV (DPP
IV) and aminopeptidase N; (30, 31, 32, 33, 34, 35) )
or have no cytoplasmic sequences whatsoever (e.g. 5`
nucleotidase, a glycophosphatidylinositol (GPI)-anchored
protein)(36, 37) . Yet all of these proteins are
transcytosed in hepatocytes(6, 7) . Do they travel
with pIgA-R? In an earlier study, we found that bile duct ligation
(BDL) slowed the transport of vesicles to the apical PM, and led to
accumulation of newly synthesized pIgA-R and apical PM proteins in a
common subapical tubulovesicular compartment(1) . This result
supports the idea that apical PM proteins share at least some of the
pIgA-R pathway. Our goal in this study was to determine if newly
synthesized pIgA-R and apical PM proteins are found in the same
transcytotic vesicles under normal conditions(38) . In this
study, we have immunoadsorbed vesicles from rat liver with a monoclonal
antibody that recognizes the tail of pIgA-R. Advantages to the use of
pIgA-R as ``bait'' are its abundance in hepatocytes and the
availability of well characterized anti-tail
antibodies(24, 39) . One disadvantage is that the
pIgA-R has a very short half-life (t
The protein synthesis inhibitor, cycloheximide (CHX) was given as an
intraperitoneal injection of 1.0 mg/100 g of body weight (from 3 mg/ml
solution in 10% EtOH-saline). Rats were given 20 mCi of
Tran
Protein concentrations were determined with the BCA
assay (Pierce).
Immunoadsorptions were performed at 4 °C as shown in Fig. 2. For most experiments, 5.6 mg of microsomes or 1.4 mg of
CVF diluted into 14 mls of 0.25 M sucrose, 150 mM NaCl, PBS, pH 7.4, 1% bovine serum albumin (Suc/PBS/BSA), were
immunoadsorbed in 15-ml conical tubes. Generally, immunoadsorptions
from microsomes used 4 times more protein than immunoadsorptions from
CVF. Variations are noted in the figure legends. The fractions were
first precleared by addition of fixed Staphylococcus aureus (from ATCC)(43) , that had been washed three times in
Suc/PBS/BSA (700 µl of 22.5% SA/14 ml of microsomes or CVF). This
mixture was incubated on a platform shaker at 200 oscillations/min for
1.25 h. The samples were spun at 3000
Figure 2:
The protocol for immunoadsorption of
vesicles. Vesicular fractions were sequentially incubated once with
fixed S. aureus, twice with anti ASGP-R antibodies and fixed S. aureus, and then with SC166 or GAM immunoadsorbent. After
each incubation, the samples were centrifuged and the pellets were
washed as described under ``Materials and Methods.'' The
preclear contains vesicles that adhere to S. aureus nonspecifically, A1 and A2 contain vesicles with ASGP-R, and SC166
contains any vesicles with pIgA-R but without ASGP-R. GAM serves as a
control for vesicles that adhere to the linker antibody or Sepharose
beads.
The washed pellet from each sample: the preclear, A1, A2, SC166
immunoadsorbent, and GAM immunoadsorbent, was divided into portions
while resuspended in the last rinse. One portion was then analyzed for
DPP IV content, while another portion of the same sample was analyzed
for pIgA-R. After centrifugation, the final pellets were resuspended in
1 ml of 20 mM octyl-
Figure 3:
Characterization of samples immunoadsorbed
from microsomes. A, immunoblot of pIgA-R in the immunoadsorbed
samples. 400 µg of microsomes were used in the immunoisolation
protocol as described under ``Materials and Methods'' and in Fig. 2, except that gel samples were made directly from unwashed
pellets at each step. 50% of the preclear and A1 and 25% of the unbound
sedimentable material are shown on this immunoblot. B,
recovery of pIgA-R in the various samples. Bands from immunoblots like
the one shown in Fig. 2A were quantified by video
densitometry. This graph shows the average of three experiments. 25% of
the pIgA-R from microsomes was in the preclear, 36% was in the A1
sample, 12% in the SC166 sample, but only 2% was in the GAM sample. 6%
of the pIgA-R remained unbound; most of this was the immature ER form.
We could account for 80-100% of the starting pIgA-R in unwashed
samples; however, washing the vesicles after each immunoadsorption
resulted in the loss of as much as 50% of the pIgA-R, indicating the
adsorbed vesicles probably break and lose membranes during the washes. C,
We also determined the distributions of
DPP IV and Tf-R throughout the immunoadsorption. The SC166 sample
contained
Figure 4:
Electron micrographs of the immunoadsorbed
samples. Immunoadsorbed samples were fixed and processed for EM without
washing. A, small vesicles (60-150 nm in diameter) were
bound to the SC166 immunoadsorbent. The inset shows a random
selection of bound vesicles. Usually only single vesicles were bound to
the beads, although occasionally aggregates were present. B, a
few vesicles were seen on the GAM immunoadsorbent. Bar = 0.5 µm.
Figure 5:
Immunoadsorption of DPP IV (A and B) and pIgA-R (C and D) from liver
microsomes of animals given Tran
There was a surprisingly large
amount of both As a positive control, we also
performed immunoadsorptions using microsomes obtained from the livers
of BDL animals, since increased amounts of both DPP IV and pIgA-R are
found in the subapical compartment after BDL(1) . More
Figure 6:
Vesicles containing sialyl transferase do
not bind to SC166 immunoadsorbent after in vivo CHX treatment.
Animals were treated with CHX for 95 min before the preparation of
microsomes. 400 µg of these microsomes were used in the
immunoadsorption protocol, gel samples were made without
immunoprecipitation, and the resulting blots were probed with
polyclonal anti-sialyl transferase antibodies. A, ST was
present in immunoadsorbed SC166 samples from control microsomes,
indicating that Golgi-derived vesicles were present. B, ST was
substantially depleted from SC166 samples immunoadsorbed from
microsomes of CHX-treated animals. The amount of ST in microsomes was
slightly increased by CHX treatment. (40 ± 9% of the homogenate
ST was in control microsomes; 60 ± 20% of homogenate ST was in
microsomes from CHX-treated animals.)
When we examined the distribution of DPP IV immunoadsorbed from
CHX-treated microsomes (Fig. 7), we found that the amount of
mature
Figure 7:
The immunoadsorption of DPP IV and HA321
from liver microsomes of CHX-treated animals given
Tran
We reasoned that a bona
fide transcytotic compartment should not contain newly synthesized
basolateral PM proteins, while earlier compartments (including the
Golgi complex) might contain newly synthesized proteins en route to
both membrane domains. So we looked for a newly synthesized basolateral
PM protein (HA321) in the samples immunoadsorbed from CHX-treated
microsomes (Fig. 7). We did not
use CHX routinely because it altered the subcellular distribution of
pIgA-R and changed the morphology of many hepatocytes (data not shown).
Figure 8:
Quantification of DPP IV in samples
immunoadsorbed from microsomes. Bands from fluorograms and immunoblots
like those shown in Fig. 5and Fig. 7were quantified by
video densitometry (data from BDL animals are also shown). The darkness
of each band was compared to a standard curve of homogenate to obtain
the percentage of the starting homogenate found in the immunoadsorbed
samples.
Microsomes contained about half the
Fig. 9shows the
distribution of DPP IV and pIgA-R immunoadsorbed from CVF. The
immunoadsorbed A1+A2 samples were similar to those adsorbed from
microsomes. However, we found very little newly synthesized DPP IV in
the SC166 sample.
Figure 9:
Immunoadsorption of DPP IV and pIgA-R from
liver CVF of animals given Tran
We also performed immunoadsorptions using CVF from
BDL rats. There was no increase in the amount of newly synthesized DPP
IV immunoadsorbed by SC166, showing that the subapical compartment was
not in this fraction. In addition, the specific activity of the DPP IV
was low in all of the samples that were immunoadsorbed from CVF. (The
quantification of DPP IV in the SC166 samples from CVF is given in Fig. 10.) We also examined immunoadsorptions from animals
labeled for 90 min, to look for transcytotic vesicles that might have
already passed through a compartment found in the 110-min CVF. However,
the results matched those of the 110-min labeling experiments (data not
shown).
Figure 10:
Quantification of DPP IV in SC166 samples
immunoadsorbed from CVF. Bands from fluorograms and immunoblots like
those shown in Fig. 9were quantified by video densitometry
(data from BDL animals are also shown) and analyzed as described in Fig. 8. Each bar shows data from a different animal. Very little
newly synthesized DPP IV was found in the SC166 samples; only 0.1% of
the homogenate DPP IV. Furthermore the specific activity of this DPP IV
was low (1.4 times homogenate). Thus, we did not find newly synthesized
DPP IV being transported with pIgA-R in vesicles found in the
CVF.
The SC166 samples from CVF consistently contained 1-2%
of both the
Figure 11:
Analysis of the transcytosis of DPP IV
using a Stella model. A, a schematic drawing of the model. The boxes represent intracellular compartments, and the arrows represent the movement of DPP IV from one compartment to the next.
The cloud at the beginning signifies the input of newly
synthesized protein, while the cloud at the end signifies
degradation. The ER conveyor is a mathematical construct that retains
newly synthesized DPP IV for a set period (4 min) before allowing
movement to the Golgi. All other transport steps are modeled as first
order processes. There is only one long lived transcytotic compartment. B, comparison of the model to the in vivo kinetics of
DPP IV biosynthesis. These graphs show data from earlier studies on the
maturation and movement of newly synthesized DPP IV from the ER to the
apical PM in comparison to the kinetics calculated from the model. The left graph shows movement from the ER through the Golgi, while
the right side shows movement through the basolateral PM to
the apical PM. The model matches the kinetics of DPP IV biosynthesis
fairly well, although the model brings DPP IV to the apical membrane a
little quickly. The model also predicts a smaller amount of newly
synthesized DPP IV at the basolateral membrane at 70 min. However, the
data show only that the peak of basolateral DPP IV occurs around 70
min; we obtained the peak amount by subtracting the amounts in the ER
and Golgi at this time from 100%. If a substantial amount of newly
synthesized DPP IV is basolateral endosomes, as is indicated by the
immunoadsorption data, then the amount at the basolateral PM decreases.
Adding basolateral endosomes to the kinetic model also delays the
arrival of newly synthesized DPP IV at the apical PM, so the revised
model fits the data better. C, characteristics of DPP IV at
110 min after synthesis. The model predicts that 6% of the newly
synthesized DPP IV should be in the subapical compartment at this time.
We found about 3% of the
We determined the steady state
distribution of DPP IV and the distribution of a pulse 110 min after
synthesis in a model that included the known biosynthetic compartments
and only one transcytotic compartment between the basolateral and
apical PM (Fig. 11A). This initial choice was based on
our identification of the SC166-immunoadsorbed vesicles as the only
obvious compartment that was involved in the transcytosis of newly
synthesized DPP IV. The movement of DPP IV through the biosynthetic
pathway predicted by this model is slightly different from that found
in the experimental studies (6, 7) (Fig. 11B). In particular, DPP
IV arrives at the apical PM more quickly than was found experimentally,
and the peak at the basolateral PM is smaller than the data indicates,
although it occurs at the correct time. It is important to remember
that the experimental data are from in vivo studies that
involved single point determinations from a series of animals; this
means that the exact distribution of The amount of newly synthesized DPP IV in the
transcytotic compartment and the specific activity calculated from the
model are shown in Fig. 11C. The model predicts that
5.6% of the DPP IV made in a 15-min pulse should be in this compartment
110 min after synthesis. We found an average of 3% of the labeled DPP
IV from homogenate in the SC166 sample from microsomes. Since we may
have lost bound membrane during the washes, the amount we found is in
good agreement with the model. Although we did not find much There are several reasons to think that the transcytotic
compartment is the subapical compartment described in the transcytosis
of pIgA-R. First, the SC166 samples contain a significant amount of the
homogenate pIgA-R, both immunoreactive and
The A1 and A2 samples contain
markers of several compartments, including Golgi (about 5% of the
homogenate ST), basolateral PM (1-2% of the homogenate HA321), ( If the model is adjusted to
show transit of newly synthesized DPP IV through basolateral endosomes,
newly synthesized DPP IV leaves the basolateral PM earlier and arrives
at the apical PM later. Thus, the revised model fits the experimental
kinetic data better. It also predicts the movement of pIgA-R from the
Golgi to the apical PM fairly accurately (data not shown).
Figure 12:
Transcytosis of DPP IV and pIgA-R.
Compartments containing DPP IV are shown in black, those with
pIgA-R are shown in white, and compartments where the two
proteins are together are shown in gray. Since DPP IV lacks a
signal for inclusion in clathrin-coated vesicles, it is probably
internalized from the basolateral surface by vesicles that do not
contain pIgA-R. DPP IV is then transported to early endosomes, which do
contain pIgA-R, and both proteins are transcytosed across the cell to
the subapical compartment. Finally, they are delivered to the apical
PM; we do not know if the same delivery vesicles take both proteins to
the apical PM.
Our data suggest that a single
transcytotic pathway could carry all proteins from basolateral
endosomes to the apical PM in hepatocytes. Different proteins may be
internalized by different mechanisms at the basolateral PM:
clathrin-mediated uptake for pIgA-R and
ASGP-R(16, 17, 66, 67) ,
non-clathrin-mediated uptake for DPP IV(68, 69) ; and
perhaps caveolae-mediated uptake for GPI-anchored proteins like 5`
nucleotidase (70, 71) . However, they all arrive at
early endosomes(72) ; presumably they can then be sorted into
transcytotic vesicles. A comparison of our kinetic models for
transcytosis of
Volume 270,
Number 46,
Issue of November 17, 1995 pp. 27834-27844
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
S]methionine-labeled rats, we sequentially
immunoadsorbed first vesicles containing the endocytic
asialoglycoprotein receptor and then (from the depleted supernatant)
vesicles containing the polymeric IgA receptor. Biochemical
characterization indicated that early basolateral and late endosomes
were present in the first population but not in the second. Neither
Golgi-, apical plasma membrane (PM)-, nor basolateral PM-derived
vesicles were significant contaminants of either population. Both
vesicle populations contained S-labeled receptor andS-labeled-dipeptidyl peptidase IV. Importantly, the
elevated relative specific activity of the dipeptidyl peptidase (% of S-labeled/% immunoblotted) in the second population
indicated that these vesicles must transport newly synthesized
dipeptidyl peptidase IV. A distinct kind of vesicle was immunoadsorbed
from a ``carrier-vesicle fraction''; surprisingly, these
vesicles contained little S-receptor and virtually no
dipeptidyl peptidase IV. These results, together with previous kinetic
data from in vivo experiments, are consistent with a
computer-generated model predicting that newly synthesized dipeptidyl
peptidase IV is delivered to basolateral endosomes, which also contain
newly synthesized polymeric immunoglobulin A receptor. The two proteins
are then transcytosed together to the subapical region.
)of polarized epithelial
cells is separated into distinct domains that have different functions
and compositions(2, 3) . Newly synthesized integral PM
proteins must be delivered to the correct domain to maintain such
functional polarity. The route used to deliver newly synthesized apical
PM proteins is particularly interesting because it varies in different
epithelia(3, 4, 5) . In hepatocytes, all
newly synthesized PM proteins studied so far are transported from the
Golgi first to the basolateral PM domain. Apical PM proteins must then
be internalized, sorted, and transcytosed to the apical
domain(6, 7, 8) . Caco-2 cells use this
indirect route for a few proteins(9, 10) , while
Madin-Darby canine kidney (MDCK) cells deliver their PM proteins
directly from the Golgi to the correct domain(11) . However,
even MDCK cells can transcytose apical PM proteins if they are
missorted to the basolateral surface (12, 13) . We
want to define this transcytotic pathway; because hepatocytes rely so
heavily on transcytosis, we have focused on this cell type.
= 2
h), so it is present in all of the biosynthetic compartments leading to
its ultimate destination, the bile(24) . Consequently,
immunoadsorptions using anti-pIgA-R antibodies will bind membranes
derived from several different compartments. Pulse labeling protocols
help alleviate this problem by allowing us to follow a cohort of newly
synthesized molecules. Even so, as time passes the cohort becomes
unsynchronized, so that pulse labeled pIgA-R is found in multiple
intracellular compartments(24) , making it difficult to
determine the origin of the membranes being immunoadsorbed from a
homogenate. In this study, we describe a protocol that differentiates
transcytotic vesicles from basolateral endosomes and Golgi-derived
vesicles, two other compartments that contain pIgA-R. Using this
approach, we isolated transcytotic vesicles that contain S-labeled DPP IV and S-labeled pIgA-R. We
also found S-labeled DPP IV in vesicles that contain the
asialoglycoprotein receptor (ASGP-R), a marker of basolateral
endosomes. Based on these results, we believe that newly synthesized
DPP IV is delivered to basolateral endosomes, which also contain newly
synthesized pIgA-R; the two proteins are then transcytosed together.
Preliminary results have been presented elsewhere (40) .
Chemicals
All chemicals were purchased from
Sigma and were of reagent grade unless otherwise noted.
TranS-label and ultrapure sucrose were obtained from ICN
Radiochemicals (Irvine, CA). Trasylol was from FBA Pharmaceuticals
(West Haven, CT). Octyl-
-D-glucopyranoside was acquired
from Boehringer Mannheim.Animals
Young adult male Sprague-Dawley rats
(200-250 g; CD strain; Charles River Breeding Laboratories,
Wilmington, MA) were housed with free access to laboratory chow and
water. All animals were fasted during the dark cycle before sacrifice.
Bile duct ligation was performed as described previously(1) . S-label by saphenous vein injection. No chase was
used. After various times, the animals were sacrificed by decapitation
and the livers were excised and perfused with ice-cold 0.9% saline in
preparation for subcellular fractionation. 
I-Asialoorosomucoid (ASOR) was prepared by
neuraminidase treatment of orosomucoid, iodinated using chloramine T as
described (41, 42) and was administered by saphenous
vein injection. The animals were sacrificed 2 min later, and the livers
were excised and perfused(43) . Approximately 5 min elapsed
between the ASOR injection and the homogenization of the livers.Preparation of Fractions
Livers were homogenized
in 4.3 volumes of 0.25 M sucrose, 3 mM imidazole, pH
7.4 (0.25 M Suc/Im) containing protease inhibitors (100
units/ml Trasylol, 1 mM phenylmethylsulfonyl fluoride, and 5
µg/ml each of antipain, leupeptin, and benzamidine) by 7 strokes in
a Potter homogenizer at 4200 rpm. Microsomal fractions and carrier
vesicle fractions (CVF) were prepared as described by Sztul et al.(38) with the following modifications. The filtered
homogenate was centrifuged at 3500 
g for 10 min (60Ti
rotor, Beckman L7-55) and the supernatant was centrifuged at 180,000
g for 60 min (60Ti rotor, Beckman L7-55). The
resulting pellet was resuspended in 20 ml of 0.25 M Suc/Im for
use in immunoadsorptions and/or analysis. To make CVF, the microsomal
pellet was resuspended in 14 ml of 1.22 M Suc/Im and 7 ml was
successively overlaid with 8.5 ml of 1.15 M Suc/Im, 0.86 M Suc/Im, and 0.25 M Suc/Im. These gradients were
centrifuged at 82,500 g for 3 h (SW28 rotor, Beckman
L7-55), and the 1.15 M Suc/Im fraction below the white band of
protein at the 1.15 M Suc/Im/0.86 M Suc/Im interface
was collected.Preparation of Immunoadsorbents
Immunoadsorbents
were prepared by a modification of the method given by Sztul et
al.(38) . 1 ml of 50% Protein A-Sepharose bead slurry was
incubated with 2 mg/ml goat anti-mouse IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA) overnight at 4 °C and was then washed
twice with 137 mM NaCl, 8.5 mM
Na
HPO
, 2.6 mM KCl, 1.5 mM
KHPO (PBS). This will be called GAM
immunoadsorbent. To produce SC166 immunoadsorbent, 1 ml of GAM
immunoadsorbent was incubated with 4 mg of SC166 IgG overnight (39) and then washed again in PBS. Both immunoadsorbents were
cross-linked with dimethylpimelimidate as described (44) and
stored as 50% slurries in PBS/NaN
.Immunoadsorption of
After an overnight fast, rats were given 20 mCi
of TranS-Labeled
FractionsS-label and sacrificed at 25 or 110 min. Then
either microsomes or CVF were prepared. Similar fractions were obtained
from rats whose bile ducts had been ligated 24 h before injecting S-label; these rats were sacrificed at 110 min. To prevent
Golgi-derived vesicles from binding to the SC166 immunoadsorbent, two
rats were given 1.0 mg of CHX/100 g of body weight 15 min after
injection of radioactivity and fractions were prepared 95 min later.
The combined labeling and CHX chase time was 110 min. g for 15 min
(3750 rpm, Beckman GS-6R centrifuge, GH-3.8 rotor). The pellet was
rinsed three times by resuspension-centrifugation, first in 7 ml of
Suc/PBS/BSA, then in 7 ml of Suc/PBS (twice). All the rinses were
discarded. The final pellet was designated ``preclear.'' The
precleared supernatant (from the initial centrifugation) was
transferred to a new tube and incubated as described above, but with
affinity-purified anti ASGP-R antibody (84 µg/14 ml). Fixed S.
aureus was added, and the incubation was continued for a second
hour. The sample was centrifuged and rinsed as above. This washed S. aureus pellet was designated the ``first ASGP-R
immunoadsorption'' (A1). The A1 supernatant was transferred to a
fresh tube containing affinity-purified anti ASGP-R antibody, and the
process was repeated. The resulting S. aureus pellet was
designated the ``second ASGP-R immunoadsorption'' (A2). The
A2 supernatant was split into two 7-ml aliquots, and 490 µl of
either SC166 or GAM immunoadsorbent (50% slurry) was added to an
aliquot. The mixtures were incubated overnight (12-16 h) with end
over end rotation, centrifuged at 300 g for 3 min and
were rinsed as described above. The SC166 and GAM samples were
centrifuged at the lowest possible g force to avoid
nonspecific pelleting of membranes. The S. aureus immunoadsorbents required stronger centrifugation because of the
difference in size. (S. aureus has an average diameter of 1
µm, while Sepharose beads range from 45 to 160 µm in diameter.)
-D-glucopyranoside, 0.5%
(w/v) Triton X-100, 300 mM NaCl, 25 mM
NaPO, pH 7.4, and 0.02% NaN (solubilization
buffer) plus protease inhibitors. After incubating on ice for 30 min,
the solubilized material was saved for immunoprecipitation or analysis.
Because the solubilization buffer did not release the pIgA-R attached
to SC166 immunoadsorbent, the immunoadsorbent beads were added to the
immunoprecipitation mixture to insure recovery of all the pIgA-R.Immunoprecipitations
Immunoprecipitations were
performed as described previously(7, 45) , except for
pIgA-R. All steps were performed at 4 °C. The solubilized samples
were added to 70 µl of anti-DPP IV IgG-Sepharose or anti-HA321
IgG-Sepharose and incubated overnight. pIgA-R was immunoprecipitated by
incubating the solubilized sample and the SC166 or GAM immunoadsorbent
with 10 µl of anti-secretory component polyclonal rabbit serum for
4 h, followed by incubating 6-15 h with 70 µl of Protein
A-Sepharose beads. At the same time pIgA-R, DPP IV, and HA321 were
immunoprecipitated from various amounts of homogenate (2-900
µg), microsomes (60-240 µg), or CVF (25-240
µg). Finally all the immunoprecipitated samples were washed and
made into reduced gel samples(7, 45) , except for
HA321, which was not reduced. To quantify the amount of S-labeled protein, 80% of each sample was separated on a
7.5% SDS-PAGE gel, the gel was treated with 2,5-diphenyloxazole dried,
and exposed to x-ray film for 5-6 weeks. The remaining 20% of the
immunoadsorbed SC166 and GAM samples were brought to 100 µl in gel
buffer and then run on another 7.5% gel. Smaller fractions of the
remaining preclear, A1, and A2 samples were brought to 100 µl in
gel buffer and applied to gels. After electrophoresis, polypeptides
were transferred to nitrocellulose by standard methods(46) ,
incubated with antibodies and the resulting bands visualized using the
ECL detection system (Amersham Corp.). Band intensities were quantified
on a Microcomputer Imaging Device (MCID) (Imaging Research Inc., St.
Catharines, Ontario, Canada). The relative amount of protein in a
fraction (% of homogenate) was determined by comparing the intensity of
the band of interest to a standard curve of homogenate consisting of 3
or 4 points.Electron Microscopy
Immunoadsorbed samples and
membrane fractions were fixed for 1 h in 1.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) at 4 °C. SC166 and GAM
samples were then embedded in agarose(47) , while the other
samples were pelleted by centrifugation at 10,000 g for 10 min (SW 50.1 rotor, Beckman L7-55). The samples were
postfixed in OsO and uranyl acetate, dehydrated, and
embedded in epon according to standard
procedures(43, 48) . Membrane contrast was enhanced by
staining with lead citrate for 5 min(49) .Enzyme Assays
-Glucuronidase activity was
assayed by monitoring the release of free phenolphthalein from
phenolphthalein mono--glucuronic acid, using a diagnostic kit
(Sigma). Cathepsin D activity was determined from the release of
trichloroacetic acid-soluble material from hemoglobin at pH 3,
following the quality control test procedure from Sigma.Computer Modeling
Computer models were constructed
with the Stella II program (High Performance Systems, Inc. Hanover,
NH). Models consisted of reservoir ``stocks'' connected by
first order ``flows'' that transferred material from one
stock to the next. The ER compartment also contained a conveyor
function to model a required residence time. A pulse of protein
synthesis was modeled as a graphical time input. This input was 0 at 0
min, rose linearly to 1 at 3 min, remained at 1 until 10 min, and then
decreased linearly to 0 at 15 min. Data from previous studies (6, 7) were used to determine the kinetic parameters
for movement through the biosynthetic pathway. We used the numbers of
molecules of DPP IV determined in a previous study to calculate the
number of molecules in each compartment. This study showed that there
are 5 10
molecules of DPP IV/hepatocyte and that
1.2 10 molecules would be synthesized in a 15-min
pulse(24) .
Characterization of the Immunoadsorbed
Samples
We used in vivo metabolic labeling combined
with vesicle immunoadsorption to test the hypothesis that apical PM
proteins are transcytosed with pIgA-R. Our starting material, the
microsomal fraction, contained Golgi-derived vesicles (indicated by the
presence of sialyl transferase (ST)), basolateral endosomes (indicated
by the presence of ASGP-R, transferrin receptor (Tf-R), and I-ASOR internalized for 5 min) and transcytotic vesicles
(indicated by the presence of I-pIgA internalized for 60
min). Because pIgA-R, our target for immunoadsorption, is most likely
present in all of these compartments(50, 51) , we
designed a protocol that specifically isolated transcytotic vesicles (Fig. 2). The preclear step removed vesicles that stuck to S. aureus nonspecifically. The next two immunoadsorption steps
removed membranes that contained ASGP-R. ASGP-R is abundant in early
endosomes, but it is also at the basolateral PM (52) and in
late endosomes(41) . Thus, vesicles derived from these
compartments would be found in the A1 and A2 samples. Any remaining
vesicles that contained pIgA-R but lacked ASGP-R would then be bound by
the SC166 immunoadsorbent. The GAM immunoadsorbent served as a control
for nonspecific binding. Comparison of the amounts of a particular
protein in each of the immunoadsorbed samples gave us a quantitative
picture of its distribution in the biosynthetic pathway. S. aureus was used for the first immunoadsorptions because we knew from
previous work that we could remove all the endosomes in this manner.
However, we did not use S. aureus for selecting pIgA-R
vesicles, because it shows higher nonspecific binding than does
Sepharose.Recovery of pIgA-R
Fig. 3A shows
an immunoblot of the pIgA-R that was found in samples which were
analyzed after the initial sedimentation (i.e. no washing).
pIgA-R was present in each immunoadsorbed sample, but the amount varied
dramatically (Fig. 3B). The preclear and A1 samples
contained >65% of the pIgA-R. Of the total microsomal pIgA-R, the
SC166 sample consistently contained >10%, which was 6-fold more than
in the GAM control. 
7% of the pIgA-R remained unbound. Overall, we
could account for 80-100% of the pIgA-R through the four steps of
the immunoadsorption protocol. However, only 30% of the microsomal
pIgA-R was recovered in comparable samples that had been washed prior
to analysis (A1 + A2 + SC166 23%; unbound 7%).
The difference between washed and unwashed samples was predominantly in
the A1 and A2 samples, suggesting to us that immunoadsorbed vesicles
may have broken during the S. aureus rinsing procedure, with
consequent pinching off and loss of unattached membrane. We used washed
samples in most experiments despite this loss, because we wanted to
avoid contamination by nonspecific sticking of membranes, particularly
contamination with PM.
I-ASOR was removed by immunoadsorption with S. aureus and anti ASGP-R antibodies. Animals were given I-ASOR for 2 min before sacrifice; the labeled ASOR
should be in early endosomes at this time. Microsomes were prepared and
used in the immunoisolation protocol described in A. The graph
shows the distribution of the sedimentable ASOR in unwashed
immunoadsorbed samples (100% = the sedimentable I-ASOR found in microsomes, 90% of the total ASOR).
Essentially no ASOR is left after A2, showing that early endosomes have
been efficiently removed. The pattern seen here, preclear < A1 
A2 binding, is characteristic of the binding of membranes containing
ASGP-R. As seen in B, 12% of the microsomal pIgA-R was found
in immunoadsorbed SC166 samples, demonstrating the presence of vesicles
containing pIgA-R but not ASGP-R.
The Material Immunoadsorbed by SC166 Is Not from
Endosomes
To be sure that early endosomes were substantially
depleted from the SC166 sample, we examined microsomes from animals
that had been given I-ASOR 5 min before sacrifice. At
this time, 94% of the labeled ASOR had been taken up by the liver (data
not shown). According to previous work, the ligand is still bound to
ASGP-R and is found in early endosomes(43, 53) . 77%
of the I-ASOR present in homogenate was found in the
starting microsomes, and of this, 90% was sedimentable (100,000 g for 1 h) (data not shown). When we used these microsomes in
our immunoadsorption protocol, more than 98% of the sedimentable ASOR
was in unwashed preclear, A1, and A2 samples (Fig. 3C),
confirming our earlier studies(43) . Essentially no ASOR was
found in the immunoadsorbed SC166 sample (i.e. 0.7% of the
ASOR was in each sample (SC166, GAM, and unbound)). ASGP-R appeared to
be substantially depleted from the unbound sedimentable material, but
we could not quantify the amount in our immunoadsorbed samples, because
IgG heavy chains (40-50 kDa) migrated at the same molecular
weight range as the receptor and interfered with the immunoblot
analysis (data not shown).0.1% of the homogenate DPP IV, which was 2.5 times
more DPP IV than was found in the GAM sample (range 2-3.3-fold).
In contrast, only 0.03% of the homogenate Tf-R was found in either the
SC166 or GAM sample. We found that Tf-R was predominantly in the A1
sample (data not shown), suggesting that this recycling receptor was
present in the vesicles that contained the ASGP-R. Neither cathepsin D
nor -glucuronidase activity, markers of late endosomes and
lysosomes(41) , was present in the immunoadsorbed SC166 sample.
However, both activities were found in the immunoadsorbed A1 and A2
samples. These results indicate that the SC166 samples did not include
either early or late endosomes, in contrast to the A1 and A2 samples,
which contained both.Electron Microscopy of the Immunoadsorbed
Samples
Examination of the immunoadsorbed samples by electron
microscopy revealed the presence of small vesicles bound to SC166 (Fig. 4A); very few vesicles were seen in the GAM
control (Fig. 4B). The SC166 vesicles ranged from 60 to
150 nm in diameter and were smaller and more uniform than the tubules
and vesicles present in the A1 and A2 samples (43; data not shown).
These samples also contained a few vesicles with attached ribosomes.
However, both the endosomal (A1 and A2) and transcytotic (SC166)
vesicles were minor components of total microsomes, because there was
no obvious difference between the starting and unbound material (data
not shown).
Newly Synthesized DPP IV Is in Vesicles That Contain
Newly Synthesized pIgA-R
Next, we analyzed the distributions of
DPP IV and pIgA-R in samples immunoadsorbed from liver microsomes of
rats labeled with TranS-label for 110 min. The choice of
labeling time was based on results from our earlier metabolic studies,
which indicated that most of the newly synthesized apical PM proteins
should have moved beyond the basolateral PM and would be en route to
the apical surface at this time(6, 7) . The
difficulties in following a ``pulse''-labeled cohort in
vivo have been discussed(7, 24) . The results
presented in Fig. 5(A and C) show that newly
synthesized pIgA-R and DPP IV were both present in vesicles bound by
the SC166 immunoadsorbent. (Only the upper band, which represents
mature DPP IV, was quantified.) The dramatic difference in the signals
between the SC166 and GAM samples shows that pIgA-R containing vesicles
specifically adhered to the SC166 immunoadsorbent and that these
vesicles contained S-labeled DPP IV. Although the presence
of immature proteins indicated that some ER- derived membranes were
also bound to the SC166 immunoadsorbent (ER does contain pIgA-R), we
analyzed only the mature DPP IV, which we know is derived from post-ER
compartments(7) . Furthermore, the electron micrographs
indicate that most of the immunoadsorbed vesicles were free of bound
ribosomes and, conversely, most of the vesicles with attached ribosomes
remained in the unbound material.
S-label for 110 min in
vivo. After 110 min of labeling, microsomes were prepared and 2600
µg were used in the immunoisolation protocol as described under
``Materials and Methods'' and in Fig. 2. The vesicles
bound to the immunoadsorbents were solubilized, divided into portions,
either DPP IV or pIgA-R was immunoprecipitated and gel samples were
made from the immunoprecipitates. The preclear, A1, and A2 samples were
run on 1 gel, while the SC166 and GAM samples were run on another. Each
gel contained a standard curve of immunoprecipitated homogenate and 2
dilutions of microsomes. A, fluorogram showing S-labeled DPP IV. Various amounts of each immunoadsorbed
sample (indicated by Ad) were loaded to obtain bands of
similar densities; the caption shows how much sample was loaded on the
gel (equivalent µg of starting microsomes used in the protocol).
Lanes showing DPP IV directly immunoprecipitated from microsomes are
indicated by Ad:None. Newly synthesized DPP IV is seen in all
samples; much less is found in the GAM and A2 samples. The lower band
is immature DPP IV, which is still present at this time. B,
immunoblot showing the total amount of DPP IV in the various samples.
There was a surprisingly large amount of total DPP IV found in the A1
sample. The substantial decrease in A2 suggests that this DPP IV was in
vesicles that also contain ASGP-R. C, fluorogram showing S-labeled pIgA-R. The immunoadsorbed A1 and SC166 samples
contained the most newly synthesized pIgA-R, although a small amount
was present in the A2 and GAM samples. D, immunoblot showing
the distribution of pIgA-R in the various immunoadsorbed samples. A
substantial amount of pIgA-R is found in the SC166 sample, while very
little is seen on GAM immunoadsorbent. Presumably the pIgA-R found in
the A1 and A2 samples is in early basolateral endosomes, a compartment
known to be involved in the transcytosis of
pIgA-R.
S- and total DPP IV in the A1 sample.
Furthermore, much more DPP IV and pIgA-R were found in it than in the
A2 sample, which was suggestive of specific binding of both proteins in
the A1 immunoadsorption step.S-labeled DPP IV was in the SC166 samples from ligated
animals ()(data not shown), which is consistent with the
idea that we immunoadsorbed vesicles from the subapical compartment.
The increase was modest, as we expected from our previous
immunoelectron microscopic quantification(1) , and was within
the variation in our measurements.The Enrichment in Specific Activity of DPP IV in the
SC166 Sample Is Not Due to Golgi Contamination
Analysis of the
SC166 sample showed that ST was present, indicating that Golgi-derived
vesicles containing pIgA-R were being immunoadsorbed (Fig. 6).
Furthermore, in studies of animals labeled for 25 min, when most of the
newly synthesized DPP IV should still be in the Golgi
complex(7) , S-labeled DPP IV was easily detected
in the SC166 sample (data not shown). Because some DPP IV remains in
the Golgi complex even at 110 min after labeling, we were concerned
that the newly synthesized DPP IV detected in the immunoadsorbed SC166
samples was in Golgi-derived vesicles, not in vesicles from
compartments involved in transport between the basolateral and apical
PM. Therefore, after a 15-min pulse, we treated rats with CHX for the
next 95 min. This reduced the Golgi contamination in two ways. First,
the amount of pIgA-R left in the Golgi under these conditions should
have been very small(24) , which would decrease Golgi
contamination of the SC166 immunoadsorbent. Second, there should have
been less reincorporation of the S label, which would
reduce the amount of labeled DPP IV in the Golgi complex. Fig. 6shows that the amount of ST found in the SC166 sample was
reduced to background levels by CHX treatment, indicating that
Golgi-derived vesicles were not bound to the SC166 immunoadsorbent.
S-labeled DPP IV in the SC166 sample was not
changed by CHX treatment. Thus the newly synthesized DPP IV in the
SC166 samples was not from Golgi vesicles.
S-label for 110 min in vivo. Microsomes from
CHX-treated animals were used in the immunoadsorption protocol and
analyzed like those in Fig. 5. A, fluorogram showing S-labeled DPP IV. After CHX treatment, the
immunoadsorption of Golgi-derived vesicles was greatly reduced, but the
amount of labeled DPP IV in the immunoadsorbed SC166 sample did not
decrease. The distribution of S-labeled DPP IV in the
immunoadsorbed samples was not affected by CHX treatment. B,
immunoblot showing the distribution of DPP IV. The distribution of
total DPP IV in the immunoadsorption was unchanged by CHX treatment.
Because the amount of labeled DPP IV in vesicles bound to SC166 was not
decreased by CHX treatment, the newly synthesized DPP IV in this sample
was not from Golgi-derived vesicles. C, fluorogram showing
newly synthesized HA321. All of these samples were run on a single gel.
Newly synthesized HA321 was found in the A1 sample, not in the SC166
sample. D, immunoblot showing the distribution of HA321, these
samples were also run on a single gel. There was no S-labeled and little immunoreactive HA321 in the SC166
sample (in fact, an examination of the blot shows mainly background
staining). This suggests that the vesicles containing DPP IV and pIgA-R
that bind to SC166 immunoadsorbent arise from a compartment involved in
transcytosis of apical proteins, after separation from basolateral
proteins.
S-Labeled HA321 was never
detected in the SC166 sample. (A small amount of HA321 (0.05% of
homogenate) was detected in the immunoblot shown in Fig. 7, but
in another sample there was no immunoreactive protein.)The SC166 Samples Are Enriched in Newly Synthesized DPP
IV
We quantified the amounts of S-labeled and
immunoreactive DPP IV in the samples from the sequential
immunoadsorptions (Fig. 8). Since we would predict that
transcytotic vesicles would contain predominantly newly synthesized
molecules, the ratio of newly synthesized to total protein should be
higher in these vesicles than in homogenate. This ratio will be higher
for a protein with a long half-life and low synthetic rate, like DPP
IV; most of the DPP IV protein is at the apical PM and very few newly
synthesized molecules are in transit to that surface(24) . In
contrast, older pIgA-R is removed once it reaches the apical
PM(22, 54, 55) . Thus, most of the pIgA-R
within the hepatocyte is newly synthesized; this means that the
specific activity of pIgA-R within transport vesicles is never much
greater than the specific activity of homogenate. The ratio (S-labeled protein, % of homogenate/immunoblotted protein,
% of homogenate), which we called the specific activity, indicates the
relative enrichment of newly synthesized molecules in a sample in
comparison to homogenate.
S values were calculated from fluorograms, and
total amounts were calculated from immunoblots. The specific activity
was then calculated by taking the ratio of S-labeled
protein to total protein for each sample. Each bar shows data from a
different animal. A, DPP IV in the starting microsomes. About
40% of the S-labeled DPP IV from homogenate was in the
microsomal fraction; slightly less was found in microsomes from
CHX-treated animals. The specific activity of the DPP IV in microsomes
was about the same as homogenate (1.2 times homogenate overall), so the
microsomal fraction was not enriched in newly synthesized DPP IV. B, DPP IV in the A1 and A2 samples. Membranes that contained
ASGP-R also contained about 10% of the newly synthesized DPP IV
(A1+A2, 80% contributed by A1); however, the specific activity of
these samples was low, about 1.8 homogenate. C, DPP IV
in the SC166 samples. 3% of the newly synthesized DPP IV from
homogenate was found in the immunoadsorbed SC166 samples. But the
specific activity of this DPP IV was 25 times that of homogenate (note
the change of scale on the right y axis), indicating these
samples do contain a compartment(s) transporting newly synthesized DPP
IV. Moreover, neither the amount nor the specific activity was
decreased by CHX treatment, indicating that this DPP IV was in a
post-Golgi compartment.
S-labeled DPP IV from homogenate, but showed little
increase in specific activity (Fig. 8A). Although the
early endosomal samples (A1+A2) contained 10% of S-labeled DPP IV, the specific activity did not increase (Fig. 8B). In contrast, the SC166 samples had less S-labeled DPP IV (3%), but this DPP IV had a specific
activity 25 times higher than homogenate (Fig. 8C, note
the change of scale on the specific activity axis). Such a high
specific activity indicated that we had immunoadsorbed vesicles
involved in the transport of newly synthesized DPP IV 110 min after
synthesis. It is formally possible that these vesicles contained older
unlabeled pIgA-R, while other vesicles in the SC166 samples contained
the newly synthesized pIgA-R. However, the short half-life of pIgA-R in
hepatocytes predicts that no membranes would contain primarily
unlabeled pIgA-R.The Vesicles Containing Newly Synthesized DPP IV Are Not
Present in a ``Carrier'' Vesicle Fraction
We also
examined immunoadsorptions from the CVF, because previous studies
indicated that it was enriched in transcytotic
vesicles(38, 56) . By immunoblot analysis, CVF also
contains early endosomes, Golgi derived vesicles and a small amount of
PM (data not shown). Approximately 50% of the pIgA-R from the CVF was
found in A1+A2 samples and another 20% bound to SC166
immunoadsorbent, accounting for the majority of the pIgA-R in the
starting fraction (data not shown).
S-label for 110 min in
vivo. After 110 min of labeling, a CVF was prepared, used in the
immunoisolation protocol and analyzed as described under
``Materials and Methods'' and Fig. 5. A,
fluorogram showing S-labeled DPP IV. These samples were
all run on a single gel. Newly synthesized DPP IV was found in the
immunoadsorbed A1 sample, but very little was in the immunoadsorbed
SC166 sample. B, immunoblot showing the distribution of DPP
IV. These samples were also run on a single gel. The A1 sample
contained a significant amount of immunoreactive DPP IV. C,
fluorogram showing S-labeled pIgA-R. There were vesicles
containing labeled pIgA-R bound to the SC166 immunoadsorbent. D, immunoblot showing the distribution of pIgA-R. A
substantial amount of pIgA-R from CVF, both S-labeled and
immunoreactive, was found in the immunoadsorbed A1 and SC166 samples.
There was very little newly synthesized DPP IV in the immunoadsorbed
SC166 sample.
S-labeled and total pIgA-R. Moreover, there
was less S-labeled pIgA-R in immunoadsorptions of CVF from
rats labeled for 25 min than in similar immunoadsorptions from rats
labeled for 90 or 110 min. Finally, SC166 samples from CVF did not
contain HA321 (data not shown). These characteristics indicated that
the SC166 samples from CVF contained vesicles involved in the transport
of pIgA-R after it had passed through the basolateral PM; however,
these vesicles lacked newly synthesized DPP IV.The Characteristics of the Immunoadsorbed Samples Depend
on the Fractions Used, Not on the Treatment of the Animal
The
amounts of pIgA-R and DPP IV found in SC166 samples immunoadsorbed from
the same fraction appears to be similar in all our treatment groups.
Because the variation between animals given different treatments was as
great as the variation between groups, we combined the data from all
the groups. In immunoadsorptions of microsomes from control, BDL-, or
CHX-treated animals labeled for 110 min, 3% of the S-labeled DPP IV was found in the SC166 samples with a
specific activity of 25-times homogenate (Table 1) (average of
data from 6 animals). These vesicles also contained an average of 13%
of the S-labeled pIgA-R. In contrast, in immunoadsorptions
of CVF from control or BDL animals labeled for 110 or 90 min, only
0.07% of the S-labeled DPP IV was found in the SC166
samples with a specific activity of 1.4-times homogenate (Table 2) (average of data from 5 animals). These vesicles
contained 2% of the S-labeled pIgA-R, much less than was
found in the samples from microsomes.
Three Kinds of Vesicles Transport pIgA-R from
Basolateral to Apical PM in Hepatocytes; Only Two of These Carry DPP
IV
We were able to immunoadsorb three biochemically distinct
vesicles that all appeared to be involved in the transport of newly
synthesized pIgA-R between the basolateral and apical PM. First, we
found vesicles that contained ASGP-R, a large amount of newly
synthesized pIgA-R (18% of homogenate), and a substantial amount of
newly synthesized DPP IV (10% of homogenate). Next we found vesicles
that lacked ASGP-R but contained a substantial amount of newly
synthesized pIgA-R (13% of homogenate) and some newly synthesized DPP
IV (3% of homogenate). Finally, there were vesicles in the CVF that
contained a small amount of newly synthesized pIgA-R (2% of
homogenate), but lacked both ASGP-R and DPP IV. Only the middle group,
the vesicles with pIgA-R and DPP IV, contained DPP IV with a high
specific activity, a feature we expected in vesicles transporting newly
synthesized apical PM proteins. What subcellular compartments involved
in the transcytosis of pIgA-R give rise to these three kinds of
vesicles?A Computer Model of the Transport of Newly Synthesized
DPP IV
We used the Stella modeling program to explore the
movement of DPP IV through the biosynthetic pathway (Fig. 11).
The model is based on simple assumptions. We assumed that exit from a
compartment is random and proportional to the amount in the
compartment, so we have used first order rate constants to model
movement between compartments. However, synthesis in the ER required a
discrete residence time, because most proteins cannot leave the ER
until they have attained the proper
conformation(57, 58) . The kinetics of the maturation
and movement of DPP IV determined in earlier in vivo studies
from this laboratory were used to define the rate constants used in the
model(6, 7) .
S-labeled DPP IV in the
immunoadsorbed SC166 samples, so the model indicates that this small
amount is not unreasonable. The model predicts that DPP IV in all the
biosynthetic compartments should have a high specific activity and that
in the transcytotic compartment the specific activity should be even
higher than the 25-fold increase we found in the SC166 samples.
However, even a very small amount of apical PM in our immunoadsorbed
samples would decrease the specific activity dramatically because of
the large amount of unlabeled DPP IV at the apical
surface.
S-labeled DPP IV is
not well defined experimentally. In addition, the amounts in the
various compartments are often estimates based on the recoveries in the
fractions. Given the uncertainty in the experimental data, the model
does a good job of predicting the rate of DPP IV maturation and arrival
at the PM.S-labeled DPP IV, it is not an unreasonably small amount.
The specific activity in the transcytotic compartment in the model is
100 times that of homogenate, while the specific activity in the SC166
samples was 25 times homogenate, indicating that the experimental
sample has more unlabeled DPP IV than would be predicted, based on our
simple model. However, if we had immunoadsorbed even a small amount of
apical PM (as little as 0.05%), it would lower the experimental
specific activity dramatically. The generation (during homogenization)
of a few vesicles derived from the apical PM with the tail of pIgA-R in
the correct orientation would result in the immunoadsorption of some
apical PM.S-labeled. The
two compartments that contain observable pIgA-R in hepatocytes are
basolateral endosomes and the subapical
compartment(23, 24, 25) . Since the SC166
samples lack basolateral endosomal markers, the most likely source is
the subapical compartment. The modest increase in newly synthesized DPP
IV immunoadsorbed from BDL animals is consistent with this
identification, as the two molecules are together in the subapical
compartment after BDL(1) .DPP IV Also Passes through Basolateral
Endosomes
The characteristics of the DPP IV found in the
immunoadsorbed A1 and A2 samples (10% of the S-labeled DPP
IV and 5% of the total DPP IV, with a relative specific activity of 2
times homogenate) cannot be fit into our current kinetic model. The
model predicts that the relative specific activity of all the
biosynthetic compartments, including the ER, should be about 30 times
that of homogenate. Moreover, putting a significant amount of the total
DPP IV (more than 2%) into any biosynthetic compartment at steady state
prevents the pulse of newly synthesized DPP IV from reaching the apical
PM for 6 h. Since we know that ASGP-R is present in many intracellular
compartments, it is likely that the A1 and A2 samples contain a mixture
of vesicles and that the DPP IV in these samples is present in two
kinds of vesicles, some that contain mainly labeled DPP IV and some
that contain older, unlabeled DPP IV.)early endosomes (80% of the sedimentable ASOR in
microsomes), and late endosomes (2.5% of cathepsin D, 1.5% of
-glucuronidase). Only early endosomes are present in sufficient
quantity in the A1+A2 samples to account for 10% of the newly
synthesized DPP IV. We speculate that the unlabeled DPP IV in
A1+A2 is from late endosomes. Internalized material from the
basolateral and apical surfaces meet at the level of late endosomes in
MDCK cells(59) , so this compartment could contain proteins
from both PM domains in hepatocytes. Thus, the unlabeled DPP IV found
in A1 and A2 could be older DPP IV internalized from the apical PM and
on its way to degradation(23) .There Is a Transport Step Where pIgA-R and DPP IV Are
Separate
In this study we also found vesicles that contained
labeled pIgA-R, little labeled DPP IV, and yet had many characteristics
of vesicles involved in the late transport of pIgA-R. This is
consistent with earlier studies showing that vesicles immunoadsorbed
from CVF were enriched in mature forms of pIgA-R, contained dimeric IgA
and therefore were transcytotic vesicles(38) . Antibodies
generated against components of samples immunoadsorbed from CVF
recognize a 108-kDa protein that has been implicated in vesicle fusion
throughout the biosynthetic
pathway(38, 56, 60) ; thus, these vesicles
could be carriers of newly synthesized pIgA-R. However, the low
recovery of labeled pIgA-R from a similar starting fraction (CVF) in
our study (2% of homogenate), together with our failure to find labeled
DPP IV in immunoadsorptions of CVF from BDL animals, suggests that
these vesicles are not part of the subapical compartment. They could be
vesicles involved in internalization of pIgA-R from the basolateral PM,
in delivery of pIgA-R to the apical PM from the subapical compartment,
or in retrieval of pIgA-R from the apical PM. It is possible that
pIgA-R and DPP IV are internalized from the basolateral PM separately,
because DPP IV lacks the cytoplasmic sequences associated with rapid
uptake into clathrin-coated
vesicles(61, 62, 63) . At present, we favor
the notion that SC166 samples from CVF contain pIgA-R internalized from
the basolateral PM in vesicles without sufficient ASGP-R to be bound in
the A1 or A2 steps.DPP IV and pIgA-R Share the Same Transcytotic
Pathway
Our analysis of the immunoadsorbed samples, combined
with kinetic modeling, suggests that DPP IV uses the same transcytotic
pathway that carries pIgA-R (Fig. 12). DPP IV is internalized
from the basolateral PM and delivered to early endosomes where it is
sorted into transcytotic carriers. Then it arrives at the subapical
compartment on the way to the apical PM. After several days at the
apical PM, DPP IV is retrieved and sent to late endosomes where a
significant amount accumulates before being degraded. We believe these
are all ``permanent'' compartments; the nature of the
transport intermediates is still unknown. While it seems likely that
all transport intermediates are vesicles, this has not been proved.
Mostov (64) has suggested that in MDCK cells the connection
between basolateral and apical endosomes is tubular, based on the
presence of basolateral receptors in apical endosomes. However, other
researchers have reported contradictory findings(65) , and we
have no evidence of basolateral PM proteins in the subapical
compartment of hepatocytes.
I-pIgA ligand and S-DPP IV
indicates that the transcytosis of both proteins proceeds with the same
kinetics. This suggests that DPP IV is both internalized and sorted
into the transcytotic pathway efficiently. In contrast, 5` nucleotidase
is transcytosed very slowly, as if internalization or sorting is
difficult for GPI-anchored proteins. Thus, while the same transcytotic
pathway may be used by all proteins, the interactions with the
machinery may be quite different. Finally, if DPP IV is internalized
separately from pIgA-R, there should be another class of endocytic
vesicles that lack both pIgA-R and ASGP-R. Clearly, more study is
needed to determine the exact pathway used by the multiple types of
apical membrane proteins going from the basolateral to the apical PM.
))S-labeled DPP
IV from homogenate was in BDL samples, 1.3% in control samples. Within
paired sets of animals sacrificed at the same time, samples from the
BDL animal always had more S-labeled DPP IV than the
control sample (1.5% BDL versus 1.0% control in set 1 and 7.8%
BDL versus 1.6% control in set 2).)
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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 D. Cohen, E. Rodriguez-Boulan, and A. Musch Par-1 promotes a hepatic mode of apical protein trafficking in MDCK cells PNAS, September 21, 2004; 101(38): 13792 - 13797. [Abstract] [Full Text] [PDF]
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 P. L. TUMA and A. L. HUBBARD Transcytosis: Crossing Cellular Barriers Physiol Rev, July 1, 2003; 83(3): 871 - 932. [Abstract] [Full Text] [PDF]
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 L. K. Nyasae, A. L. Hubbard, and P. L. Tuma Transcytotic Efflux from Early Endosomes Is Dependent on Cholesterol and Glycosphingolipids in Polarized Hepatic Cells Mol. Biol. Cell, July 1, 2003; 14(7): 2689 - 2705. [Abstract] [Full Text] [PDF]
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 M. Cuchacovich, H. Gatica, P. Vial, J. Yovanovich, S. V. Pizzo, and M. Gonzalez-Gronow Streptokinase Promotes Development of Dipeptidyl Peptidase IV (CD26) Autoantibodies after Fibrinolytic Therapy in Myocardial Infarction Patients Clin. Vaccine Immunol., November 1, 2002; 9(6): 1253 - 1259. [Abstract] [Full Text] [PDF]
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C. J. Soroka, M. K. Pate, and J. L. Boyer Canalicular Export Pumps Traffic with Polymeric Immunoglobulin A Receptor on the Same Microtubule-associated Vesicle in Rat Liver J. Biol. Chem., September 10, 1999; 274(37): 26416 - 26424. [Abstract] [Full Text] [PDF]
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M. Verges, R. J. Havel, and K. E. Mostov A tubular endosomal fraction from rat liver: Biochemical evidence of receptor sorting by default PNAS, August 31, 1999; 96(18): 10146 - 10151. [Abstract] [Full Text] [PDF]
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 P. L. Tuma, C. M. Finnegan, J.-H. Yi, and A. L. Hubbard Evidence for Apical Endocytosis in Polarized Hepatic Cells: Phosphoinositide 3-Kinase Inhibitors Lead to the Lysosomal Accumulation of Resident Apical Plasma Membrane Proteins J. Cell Biol., May 31, 1999; 145(5): 1089 - 1102. [Abstract] [Full Text] [PDF]
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S. C.D. van IJzendoorn and D. Hoekstra (Glyco)sphingolipids Are Sorted in Sub-Apical Compartments in HepG2 Cells: A Role for Non-Golgi-Related Intracellular Sites in the Polarized Distribution of (Glyco)sphingolipids J. Cell Biol., August 10, 1998; 142(3): 683 - 696. [Abstract] [Full Text] [PDF]
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G. Ihrke, G. V. Martin, M. R. Shanks, M. Schrader, T. A. Schroer, and A. L. Hubbard Apical Plasma Membrane Proteins and Endolyn-78 Travel through a Subapical Compartment in Polarized WIF-B Hepatocytes J. Cell Biol., April 6, 1998; 141(1): 115 - 133. [Abstract] [Full Text] [PDF]
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M. Jin, L. Saucan, M. G. Farquhar, and G. E. Palade Rab1a and Multiple Other Rab Proteins Are Associated with the Transcytotic Pathway in Rat Liver J. Biol. Chem., November 22, 1996; 271(47): 30105 - 30113. [Abstract] [Full Text] [PDF]
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