(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
[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 and
S-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.
The plasma membrane (PM) ()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.
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 = 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) .
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
TranS-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.
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 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.)
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--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.
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, 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.
We also determined the distributions of
DPP IV and Tf-R throughout the immunoadsorption. The SC166 sample
contained 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.
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 TranS-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.
There was a surprisingly large
amount of both 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.
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 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.
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 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.
Figure 7:
The immunoadsorption of DPP IV and HA321
from liver microsomes of CHX-treated animals given
TranS-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.
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). 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.)
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. 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.
Microsomes contained about half the 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.
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 TranS-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.
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 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.
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 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.
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 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.
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 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.
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 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) .
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), ()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) .
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 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.