Localization of Metallocarboxypeptidase D in AtT-20 Cells
POTENTIAL ROLE IN PROHORMONE PROCESSING*
Oleg
Varlamov,
Francis J.
Eng,
Elena G.
Novikova, and
Lloyd D.
Fricker
From the Department of Molecular Pharmacology, Albert Einstein
College of Medicine, Bronx, New York 10461
 |
ABSTRACT |
Carboxypeptidase D (CPD) is a recently discovered
metallocarboxypeptidase that is predominantly located in the
trans-Golgi network (TGN), and also cycles between the cell
surface and the TGN. In the present study, the intracellular
distribution of CPD was examined in AtT-20 cells, a mouse anterior
pituitary-derived corticotroph. CPD-containing compartments were
isolated using antibodies to the CPD cytosolic tail. The immunopurified
vesicles contained TGN proteins (TGN38, furin, syntaxin 6) but not
lysosomal or plasma membrane proteins. The CPD-containing vesicles also contained neuropeptide-processing enzymes and adrenocorticotropic hormone, a product of proopiomelanocortin proteolysis. Electron microscopic analysis revealed that CPD is present within the TGN and
immature secretory granules but is virtually absent from mature granules, suggesting that CPD is actively removed from the regulated pathway during the process of granule maturation. A second major finding of the present study is that a soluble truncated form of CPD is
secreted mainly via the constitutive pathway in AtT-20 cells,
indicating that the lumenal domain does not contain signals for the
sorting of CPD to mature secretory granules. Taken together, these data
are consistent with the proposal that CPD participates in the
processing of proteins within the TGN and immature secretory vesicles.
 |
INTRODUCTION |
Most peptide hormones and neurotransmitters are produced from
larger precursors by limited proteolysis. Initially, the prohormone precursors are processed at multiple basic amino acid cleavage sites by
a family of endoproteases collectively known as prohormone convertases
(PC)1 (1, 2). The subsequent
processing step is mediated by carboxypeptidases, which remove the
basic amino acids from the C terminus of the peptide to generate either
the bioactive product, or a precursor for the formation of the
C-terminal amide group (3, 4).
Carboxypeptidase E (CPE, also known as carboxypeptidase H and
enkephalin convertase) is the major carboxypeptidase involved with the
processing of many peptides (3, 4). Within several tissues, CPE has
been localized to the peptide-containing secretory granules (5-7). The
involvement of CPE in peptide processing is evident from the finding
that fat/fat mice have a reduced ability to convert
proinsulin into insulin (8). A missense mutation in the CPE gene is the
molecular basis for the fat mutation (8). Although a
full-length CPE protein is translated, the protein is inactive due to a
point mutation in the coding region of the gene (8, 9). Despite the
absence of functional CPE, the mature forms of numerous neuropeptides
are detectable in brain and other tissues of fat/fat mice
(8, 10-13). The presence of correctly processed peptides indicates
that an additional enzyme is able to compensate for the deficiency of
CPE.
Recently, a second carboxypeptidase designated metallocarboxypeptidase
D (CPD) has been identified in the secretory pathway of bovine
pituitary glands (14). This protein was independently discovered as
gp180, a duck protein that binds duck hepatitis B virus particles (15).
In duck and rat, CPD is present in many tissues (15-19) suggesting a
broad function. CPD cDNA has been cloned and sequenced from human,
rat, duck, Drosophila, and Aplysia (16, 18,
20-22). In general, the protein is highly conserved among species, and
shows similar enzymatic properties (14, 17, 22, 23). Human, rat, and
duck CPD cDNAs encode proteins that contain three
carboxypeptidase-like domains, followed by a transmembrane domain and a
58-residue cytosolic tail. The cytosolic tail contains several
consensus routing motifs found in other integral membrane peptide
processing enzymes such as furin and peptidylglycine-
-amidating monooxygenase (PAM) (24).
CPD is predominantly located in the TGN, and also cycles between the
cell surface and the TGN (25). The co-localization of CPD and the TGN
endoprotease furin (25) supports the hypothesis that these two enzymes
may function in the same pathway. We were previously unable to detect
CPD in mature secretory granules by light microscopy. As mature
secretory vesicles contain many of the peptide processing enzymes
(prohormone convertases 1 and 2, CPE, PAM), the lack of detectable CPD
in this compartment raised doubts as to whether CPD can participate in
peptide processing. Although the processing of some neuropeptides
begins in the TGN, immature vesicles appear to be the most productive
proteolytic compartments. For example, the processing of proinsulin
begins in immature secretory granules in
-cells (26). Also, the
generation of ACTH and
-endorphin from the larger precursor
proopiomelanocortin (POMC) in AtT-20 cells is predominantly a post-TGN
event (27, 28), although POMC processing may be initiated in the TGN
(29-31).
Soluble forms of CPD have been identified in various bovine and rat
tissues (17). The intracellular distribution of the soluble CPD was not
previously examined. Interestingly, both the soluble and
membrane-associated forms of PAM are packaged into mature secretory
granules in AtT-20 cells (32), indicating that the lumenal domain of
PAM contains sorting information. In the present study, we further
characterized the intracellular distribution of CPD using
immunoisolation and electron microscopic approaches. We have also
examined the intracellular distribution of a soluble truncated form of
CPD. Although neither the full-length or the truncated form of CPD are
efficiently routed to mature secretory vesicles, both forms are present
in immature secretory vesicles. These findings strongly support the
proposal that CPD functions in the processing of prohormones and other
proteins that transit either the regulated or constitutive secretory pathways.
 |
MATERIALS AND METHODS |
Generation of Constructs and Expression of Proteins in AtT-20
Cells--
gp180 and gp170 constructs previously described for
baculovirus expression (23) were subcloned into the pcDNA3
expression vector (Invitrogen) and transfected into AtT-20 cells using
the standard calcium phosphate procedure (33). Stable cell lines were
selected using 0.6 mg/ml Geneticin (G418). Cells expressing both
constructs were identified by Western blot analysis. For each of these
constructs, several positive clones were selected and analyzed as
described below; the data shown are representative of two or three
separate clones.
Antibodies--
The antiserum to duck CPD recognizes both the
soluble and membrane-associated forms of the protein (i.e.
gp170 and gp180) but does not cross-react with mouse CPD (24). The
antiserum to the C-terminal cytosolic tail of CPD (17) recognizes both rat and duck proteins. The antiserum raised against a C-terminal peptide of bovine CPE recognizes the mouse protein (9). Antisera to
ACTH and
-endorphin that recognize both precursor and processed peptide (31) were a gift of Dr. Richard Mains (Johns Hopkins University, Baltimore, MD). The antiserum to TGN38 was a gift of Dr.
Sharon Milgram (University of North Carolina, Chapel Hill, NC), the
antiserum to the furin C-terminal tail was a gift of Dr. Ruth Angeletti
(Albert Einstein College of Medicine, New York, NY), the monoclonal
antibody against syntaxin 6 was a gift of Jason Bock and Dr. Richard
Scheller (Stanford University, Stanford, CA), the antiserum to PC1 was
a gift of Dr. Iris Lindberg (Louisiana State University, New Orleans,
LA), the antiserum to cathepsin B was a gift of Dr. Regina Kuliawat
(Albert Einstein College of Medicine, Bronx, NY), the monoclonal
antibody to SNAP-25 was a gift of Dr. Peter Davies (Albert Einstein
College of Medicine, New York, NY), and the antiserum to calnexin was a
gift of Dr. Ari Helenius (Yale University, New Haven, CT).
For immunoisolation, polyclonal antibodies against the C-terminal
cytosolic tail of CPD were subjected to affinity purification using
glutathione S-transferase-CPD cytosolic tail peptide coupled to Sepharose 4B by the standard cyanogen bromide method (Sigma). Affinity-purified antibodies were concentrated to 1 mg/ml in PBS, and
used for the immunoisolation procedure as described below.
Immunoaffinity Isolation of CPD/gp180-containing
Vesicles--
Typically, six confluent 10-cm plates of AtT-20 cells
expressing gp180 were used for the immunoisolation. Cells were washed three times with cold PBS, scraped from the plates, and gently pelleted
at 700 × g. The cell pellet was resuspended in 4 ml of cold 0.25 M sucrose, 10 mM HEPES, pH 7.4, containing 1 mM EDTA, 3 mM EGTA, and a mixture
of protease inhibitors (1 µM aprotonin, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 15 µM pepstatin A, 22 µM leupeptin, 14 µM E-64, and 40 µM bestatin). The suspension was passed five times
through a 22-gauge needle, and then homogenized in a tight-fitting
Dounce homogenizer (25 strokes). The resulting homogenate was
centrifuged for 7 min at 1000 × g twice, and the supernatant was layered onto 4 ml of 1.6 M sucrose and
centrifuged in a SW41 rotor (Sorvall) for 1 h at 200,000 × g. Typically, 1 ml of a microsomal fraction was collected
from the interface of the 0.25 and 1.6 M sucrose, and then
subjected to immunoisolation. The affinity resin for the
immunoisolation was prepared by incubation of 100 µl of Pansorbin
(Calbiochem) with 100 µg of affinity-purified antibodies to the CPD
C-terminal cytosolic tail. An aliquot (300 µl) of the microsomal
fraction was incubated for 3 h at 4 °C with gentle agitation
with 100 µl of the affinity resin in a final volume of 500 µl
containing 140 mM NaCl and the protease inhibitors described above. The Pansorbin resin was recovered by low speed centrifugation and was washed three or four times with the same buffer,
and then vesicle-associated proteins were subjected to a detergent
elution using 1% Triton X-100. IgG-associated gp180 was eluted from
the beads by boiling in 1% SDS gel loading buffer for 5 min. Aliquots
of each fraction were subjected to Western blot analysis using antisera
to various secretory pathway proteins at 1:1000 dilution.
Labeling of AtT-20 Cells with [35S]Met--
AtT-20
cells expressing gp170 were labeled with [35S]Met (100 µCi/ml) for 15 min, washed twice with PBS, and then incubated in
Dulbecco's modified Eagle's medium for different periods of time.
Media were removed, cells were washed with PBS, and then frozen in 10 mM NaAc, pH 5.5, with 1 mM phenylmethylsulfonyl
fluoride. The cells and the media were then subjected to
immunoprecipitation using the antiserum to either duck CPD or CPE as
described (30). To test whether CPD-containing compartments contain the
neuropeptide precursor POMC and its proteolytic fragments, AtT-20 cells
expressing gp180 were either continuously incubated with
[35S]Met (100 µCi/ml) for 8 h, or pulsed for only
20 min and then chased for 0 or 90 min in the presence of unlabeled
Met. Following the labeling, the cells were washed three times with
cold PBS and then subjected to the immunoisolation procedure described above. The resulting fractions were subjected to immunoprecipitation using antisera to either ACTH or
-endorphin, and analyzed on 10%
polyacrylamide Tricine gels.
Regulated Secretion--
To examine whether gp170 is secreted
via the regulated pathway, the cells expressing gp170 were grown on
35-mm cell culture dishes to 90% confluence. The cells were washed
twice with PBS, then treated with the secretagogue 5 mM
8-Br-cAMP or control media for 30 min and the secreted proteins
analyzed by Western blotting. To examine the regulated secretion of
[35S]Met-labeled proteins, cells were labeled for 15 min
with [35S]Met, chased for 2 h, washed three times
with PBS, and then incubated for 30 min in either control media or with
5 mM 8-Br-cAMP. The media were subjected to
immunoprecipitation using the antiserum to either CPE or duck CPD.
Immunofluorescence Analysis--
Transfected AtT-20 cells were
cultured on growth-supporting glass coverslips (Fisher Scientific).
Cells were washed with PBS, fixed in 4% paraformaldehyde for 15 min,
and then permeabilized for 15 min in 0.1% Triton X-100 in PBS. After
1 h of blocking in 3% bovine serum albumin, the cells were
immunostained for 1 h with the primary antisera (1:1000 dilution).
Cells were washed with PBS containing 0.2% Tween 20 and then incubated
with fluorescein-labeled anti-mouse or rhodamine-labeled anti-rabbit
secondary antibody (Vector Laboratories Inc., 1:200 dilution) for
1 h, followed by extensive PBS washing. Immunofluorescence
staining was examined using a Bio-Rad confocal microscope.
Electron Microscopy Analysis--
Stably transfected AtT-20
cells expressing either gp180 or gp170 were fixed for 30 min at room
temperature in 4% formaldehyde, 0.2% glutaraldehyde in PBS, then
dehydrated, embedded in LR-White resin according to standard procedure,
sectioned, and mounted on either gold or nickel grids. Each grid was
incubated for 30 min in blocking solution containing 10% goat serum,
2.5% bovine serum albumin, 0.1% Tween 20 in PBS at pH 8.2, and then
incubated for 1 h in the same solution containing the antiserum to
either duck CPD or CPE (final dilution 1:200). Cells were extensively washed in 0.1% Tween 20 in PBS, and incubated for 2 h with 10-nm colloidal gold (BioCell) coupled to anti-rabbit antibodies in blocking
solution (dilution 1:10). After extensive washes, sections were stained
for 2 min with uranyl acetate in 30% ethanol and examined with a Jeol
100CX electron microscope. For conventional electron microscopy, the
cells were fixed in 2% glutaraldehyde, then postfixed in 1%
OsO4 and embedded in LR-White resin.
To quantitate the intracellular distribution of gp180, gp170, and CPE,
electron micrographs that were immunogold-labeled with antibodies to
either duck CPD or CPE were scanned in random order. In total, about
1000 gold particles/grid were analyzed, and the percentage of total
label that was found in specific compartments was determined. The main
morphological criterion to distinguish different subclasses of the
secretory granules was the size of the vesicles and the size of the
dense cores (34), which were measured from negatives taken at
magnification ×54,000. An additional morphological criteria was the
presence of the lighter core in immature granules surrounded by a broad
electron-lucid peripheral zone (this zone was much broader in granules
at the low state of condensation). Light aggregates of condensing
proteins were observed in the dilated cisterns of the TGN, consistent
with previous studies that formation of the dense core aggregates
begins at the level of the TGN in AtT-20 cells (35). These
electron-dense granules at the low state of condensation, which may
represent cross-sections of the TGN or early immature vesicles, had an
average diameter of 280 nm and dense cores that were too variable to
accurately determine an average. Electron-dense granules at an
intermediate state of condensation had an ovoid or spherical shape, and
were in the process of detaching or already detached from the TGN. The
average diameters of these granules were 170 nm, and their dense cores
were 120 nm. These vesicles closely resemble immature secretory
granules previously described in AtT-20 cells (35). Another type of
vesicle was found in close proximity to the plasma membrane; these
vesicles had a regular spherical shape and highly condensed dense
cores, which are characteristic features of mature secretory granules
in endocrine cells (35). The average diameters of mature secretory
granules were typically 150 nm and their dense cores were 110 nm, which
is consistent with previous studies on AtT-20 cells (28, 36, 37).
 |
RESULTS |
We have previously demonstrated that endogenous CPD is
predominately localized to the perinuclear region in AtT-20 cells (25). To biochemically determine the proteins that co-localize with CPD, we
used an immunoisolation technique. For these studies, microsomes from
cells stably transfected with full-length duck CPD (gp180) were
isolated at the interface between a 1.6 M and a 0.25 M sucrose layer (Fig.
1A, Ms). Typically,
about 30% of the total gp180 in the cell homogenate was recovered in
the microsomal fraction (Fig. 1B). Similarly, 20-30% of
the calnexin (an ER membrane protein), SNAP-25 (a plasma membrane
protein), and syntaxin-6 (a TGN protein) were also recovered in this
microsomal fraction (Fig. 1B). None of these proteins were
detected in the 0.25 M or the 1.6 M sucrose
layers (Fig. 1B), indicating no enrichment or loss of these
organelles during the preparation. For immunoisolation, Pansorbin was
precoated with the affinity-purified antibodies to the cytosolic tail
of CPD, incubated with the microsomal fraction from AtT-20 cells, and
then both bound and non-bound materials were analyzed by Western blot
using antibodies to various secretory pathway markers (Fig.
1C, right panels). In control
experiments, when rabbit IgG was used instead of antibodies to the CPD
tail, most of the gp180 immunoreactivity and secretory markers were recovered in non-bound fractions (Fig. 1C, left
panels), indicating that nonspecific binding of microsomes
to the affinity carrier is very low. When using antibodies to the CPD
tail, most of the gp180 (typically >95%) is recovered in the bound
fraction (Fig. 1C, top). Furin, TGN38, and
syntaxin 6, which have been previously localized to the TGN and
immature secretory granules (38-40), are exclusively associated with
gp180-containing vesicles (Fig. 1C, right
panels). In contrast to the TGN proteins, CPE and PC1 which were previously found within mature secretory granules (5, 41, 42),
demonstrate only a partial co-localization with the gp180 compartment
(Fig. 1C, right panel). Typically,
20-30% of CPE, and 40-50% of PC1 were recovered in non-bound
fractions. To further validate the specificity of our immunoisolation
technique, we probed bound and non-bound fractions with antibodies to
cathepsin B, calnexin, and SNAP-25 (Fig. 1C). Cathepsin B
and SNAP-25 are exclusively recovered in the non-bound fraction (Fig.
1C), indicating that lysosomes and plasma membrane are not
present in the immunoisolates. Approximately 30% of the calnexin is
associated with the CPD-containing compartment (Fig. 1C),
consistent with the expectation that the ER contains newly synthesized
CPD. The finding that the plasma membrane and ER membrane proteins are
not efficiently isolated in the CPD-containing organelles further
suggests that co-sedimentation of the TGN proteins and gp180 is not due
to nonspecific binding of membrane proteins to the affinity resin.
Identical immunoisolation procedure using wild type AtT-20 cells
demonstrated a similar distribution of the various secretory pathway
markers (data not shown), indicating that this pattern of distribution
is not due to overexpression of gp180 in AtT-20 cells.

View larger version (55K):
[in this window]
[in a new window]
|
Fig. 1.
Immunoisolation of the gp180-containing
compartment. A, AtT-20 cells were homogenized
(H) and then subjected to centrifugation at 1000 × g, as described under "Materials and Methods." The
supernatant from this (S1) was then layered onto 4 ml of 1.6 M sucrose and centrifuged for 1 h at 200,000 × g. Microsomes were collected from the interface of the two
layers. B, equivalent aliquots of the homogenate, the S1
fraction, the 0.25 M sucrose layer (0.25), the
1.6 M sucrose layer (1.6), and the microsomal
fraction (Ms) were analyzed by Western blotting using
antisera to the indicated proteins. C, microsomes prepared
from AtT-20 cells expressing gp180 were incubated for 3 h at
4 °C with Pansorbin precoated with either affinity-purified
antibodies to the CPD C-terminal cytosolic tail ( CPD) or
rabbit IgG (IgG). The aliquots of bound (B) and
non-bound (NB) fractions were subjected to Western blot
analysis using antisera either to the N-terminal portion of duck CPD or
to different secretory pathway markers. The positions of prestained
protein standards are indicated. Except for the analysis of cathepsin
B, calnexin, and SNAP-25, which was performed twice, the entire
experiment was repeated five times with similar results.
|
|
To test whether the CPD-containing compartments contain the
neuropeptide precursors or their proteolytic fragments, AtT-20 cells
were labeled with [35S]Met for 8 h and then
subjected to the immunoisolation procedure with antibodies to the CPD
cytosolic tail. To reduce the possibility of nonspecific proteolysis, a
mixture of protease inhibitors was included throughout the isolation
procedure. Typically, 30-40% of the total POMC and POMC-derived
material was recovered in the microsomal fraction (data not shown).
Pansorbin-bound and non-bound materials were subjected to
immunoprecipitation using antibodies either against ACTH or
-endorphin. The antisera against ACTH used in the present study
recognizes both POMC and its processed product (31). Typically 70-80%
of the ACTH (both glycosylated and non-glycosylated forms) was
recovered in the bound fraction (Fig.
2A, right
panel). In experiments with control IgG, most of the ACTH is
found in the non-bound fraction (Fig. 2A, left panel), although the 30-kDa POMC shows some nonspecific
binding (Fig. 2A, left panel). When
immunoisolated material was immunoprecipitated with antibodies to
-endorphin, the bulk of the
-endorphin-reactive material was
recovered in the bound fraction (data not shown). As an additional
control for nonspecific proteolysis, we also immunoprecipitated the
immunoreactive ACTH peptides in the homogenate, intermediate fractions
and microsomes, prior to the vesicle immunoisolation. All fractions
showed similar relative amounts of ACTH versus the larger
peptides, indicating that no significant proteolysis of POMC occurred
during the immunoisolation (data not shown).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 2.
Recovery of POMC-derived peptides within the
gp180-containing compartment. A, AtT-20 cells
expressing gp180 were labeled with [35S]Met for 8 h,
and then subjected to the immunoisolation procedure. Pansorbin-bound
(B) and non-bound (NB) materials were subjected
to immunoprecipitation using an antiserum against ACTH.
CPD, Pansorbin precoated with antibodies to the CPD
C-terminal cytosolic tail; IgG, Pansorbin precoated with
rabbit IgG. The positions of POMC and ACTH are indicated. The entire
experiment was repeated four times with similar results. B,
AtT-20 cells expressing gp180 were labeled with [35S]Met
for 20 min and then chased for either 0 or 90 min and subjected to
vesicle immunoisolation with antibodies to the CPD tail. The aliquots
of the cell homogenate (H) and microsome-derived
(Ms) immunoisolated fractions were subjected to
immunoprecipitation using an antiserum to ACTH. The dried gel was
exposed to x-ray film for 2 h (chase 0) or for 20 h (chase 90).
|
|
To examine the effect of shorter labeling times on the pattern of
immunoreactive ACTH peptides, cells were labeled with
[35S]Met for 20 min, chased for 0 or 90 min in unlabeled
Met, and then subjected to vesicle immunoisolation using an identical
procedure. When cells were labeled for only 20 min, without a chase,
only POMC-sized material was detected in both the initial homogenate and the immunoisolated vesicles (Fig. 2B, H and
Ms). This result further suggests that the POMC processing
does not occur during the vesicle immunoisolation procedure. When the
cells were chased for 90 min prior to vesicle immunoisolation and
immunoprecipitation, ACTH-sized peptides were detected (Fig.
2B). The reduction in the amount of [35S]Met
label recovered upon immunoprecipitation after the 90 min chase is due
to both loss of Met upon cleavage of POMC (ACTH contains 1 of the 3 Met
in POMC) and secretion of POMC peptides from AtT-20 cells.
Approximately 40% of the ACTH immunoreactivity was recovered in the
non-bound fraction after the 90-min chase (Fig. 2B),
suggesting that this pool has left the CPD-containing compartment.
Taken together, these results demonstrate that a substantial portion of
POMC processing by endopeptidases is completed in CPD-containing compartments.
To further investigate the intracellular distribution of CPD, we
performed double-labeling of AtT-20 cells with various antisera (Fig.
3). Co-staining of cells with antibodies
to syntaxin 6 and TGN38 demonstrates complete co-localization of these
proteins (Fig. 3, bottom) indicating that syntaxin 6 is
localized to the TGN in AtT-20 cells, as previously found in other cell
lines (40). The majority of the ACTH-reactive material is co-localized
with syntaxin 6 in the perinuclear region (Fig. 3). In addition, the anti-ACTH antiserum also labels large discrete vesicles distributed throughout the cytoplasm and at the processes of the cells (Fig. 3,
arrows), which presumably represent the mature secretory
granules. gp180 is found exclusively in a perinuclear compartment which overlaps with the distribution of syntaxin 6 (Fig. 3, top),
suggesting that the majority of gp180 does not enter the mature
ACTH-containing vesicles.

View larger version (47K):
[in this window]
[in a new window]
|
Fig. 3.
Immunofluorescence analysis of AtT-20
cells. Stably transfected cells expressing either gp170
(second row) or gp180 (all
other rows) were subjected to dual
immunofluorescent staining as described under "Materials and
Methods" using monoclonal antibodies to syntaxin 6 (right
column) or polyclonal antibodies to different proteins
(left column). All images represent composites of
1-µm optical sections through the cell. To obtain images within the
linear range of exposure, the tonal range of each image was adjusted
appropriately. Arrowheads, perinuclear staining;
arrows, punctate vesicles detected with the ACTH antiserum.
Bar, 10 µm.
|
|
Although CPD was originally described as a 180-kDa integral membrane
protein, a small amount of CPD was previously purified from bovine and
rat tissues as a 170-kDa soluble protein that did not react with the
antiserum raised against the C-terminal tail (17). To further explore
the intracellular distribution of the soluble form of CPD, we created a
deletion within the coding region of gp180 that removes the
transmembrane and cytosolic domains of the protein, and stably
expressed the resulting construct (named gp170) in AtT-20 cells. Cells
expressing gp170 were double-labeled with antibodies against duck CPD
and syntaxin 6 and examined by immunofluorescence. A large portion of
gp170 is localized to the perinuclear region and co-stained with
syntaxin 6 antibodies (Fig. 3). In addition, gp170 also shows a diffuse
distribution throughout the cytoplasm (Fig. 3, second
row). The diffuse pattern of gp170 is distinct from the
punctate distribution of ACTH (Fig. 3).
To obtain further structural information about the CPD-containing
compartments, we used immunogold electron microscopy (Fig. 4). A conventional electron micrograph
reveals secretory granules and TGN-derived vacuoles containing
condensing material (Fig. 4A). Cells expressing gp180 were
processed for immunogold electron microscopy and stained with the
antiserum directed against duck CPD (Fig. 4, B-F). The
majority of the CPD-reactive material is concentrated along the
trans side of the Golgi cisternae (Fig. 4, B and
C, filled arrows), and within vesicles
containing electron-dense core material at an early state of
condensation (Fig. 4, B-D, open
arrows). These vesicles may represent cross-sections of the TGN or early immature secretory granules. CPD staining was also detected within vesicles with dense cores at an intermediate stage of
condensation that resemble immature secretory granules (Fig. 4,
C-E, open arrowheads), and even in a
small number of dense core vesicles located in close proximity to the
plasma membrane that resemble mature secretory granules (Fig.
4F, filled arrowheads). The small
number of gold particles detected associated with dense core vesicles
is likely to reflect the real presence of CPD in these compartments
rather than background labeling, as the gold particles were often
present in clusters of 3 or more. Additionally, virtually no labeling
was detected in mitochondria or nucleus, which are not expected to
contain CPD. In control experiments in which the primary antiserum was
omitted, only background staining was observed in the TGN and vesicles
(data not shown). Quantitation of approximately 1000 gold particles
demonstrates that 84% of gp180 is located in the TGN, 10% is found
within moderately-condensed granules, 4% is located within granules at
an intermediate state of condensation, and only 2-3% is recovered
within mature granules (Table I). Taken
together, the electron microscopy, immunocytochemical, and
immunoisolation results further support the idea that gp180 predominately functions at the level of the TGN, and to a lesser extent
in the immature secretory vesicles.

View larger version (189K):
[in this window]
[in a new window]
|
Fig. 4.
Electron microscopy of AtT-20 cells.
Stably transfected cells expressing either gp180 (A-F) or
gp170 (G-J) were processed for the electron microscopy as
described under "Materials and Methods," and then stained with
either antibodies to duck CPD (B-I) or CPE (J).
A, cells subjected to the conventional electron microscopy.
B-J, immunogold analysis. The criteria for the following
assignments are described under "Materials and Methods":
filled arrows, TGN; open
arrows, TGN dilations containing protein aggregates;
open arrowheads, immature secretory granules;
filled arrowheads, mature granules;
crossed arrows, small vesicles containing gp170.
The entire experiment was repeated using two different clones.
Bar, 200 nm.
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Quantitation of immunogold labeling: intracellular distribution of
carboxypeptidases in AtT-20 cells (percentage of total)
EDGLow, electron-dense granules at the low state of
condensation (these "granules" may represent cross-sections of the
TGN or early immature granules). The dense cores were too variable to
determine the average diameter.
EDGInt, electron-dense granules at an intermediate state of
condensation (these granules presumably represent immature vesicles).
|
|
Immunogold electron microscopic analysis was also performed on the
AtT-20 cell line expressing gp170 (Fig. 4, G-I). In
addition to the TGN localization, gp170 is also found within secretory vesicles containing electron-dense material (Fig. 4I,
open arrowheads, and Table I) that are
morphologically similar to the gp180-containing vesicles. As with
gp180, immunoreactive gp170 was not generally detected in mature
granules (Fig. 4H, Table I). A large fraction of gp170 was
also found in small clusters (40-50 nm in diameter) distributed
throughout the cytoplasm (Fig. 4G, crossed
arrows). These structures may represent small constitutive
vesicles that have been previously described (43, 44), or
cross-sections of the ER. In contrast to the pattern of CPD
distribution, the majority of the immunoreactive CPE was localized in
the dense core granules that are morphologically similar to immature
and mature secretory vesicles (Fig. 4J, Table I).
To further explore the routing pathway of gp170, we determined the
secretion kinetics of the transfected gp170 and the endogenous CPE in
AtT-20 cells. Cells were labeled for 15 min with
[35S]Met, chased for different periods of time, and then
both cells and media were subjected to immunoprecipitation using
antibodies to either duck CPD or mammalian CPE. The secretion rate of
gp170 is fairly rapid; after a 2-h chase approximately 90% of the
labeled gp170 is present in the media (Fig.
5, A and B). In
contrast, a large portion of CPE (50%) is retained within the cells
after 2 h of the chase period, and the CPE secretion approaches a
plateau (Fig. 5, A and B). These results are
consistent with electron microscopy data, demonstrating that the bulk
of gp170 does not usually enter dense core secretory granules, whereas
CPE is targeted to this compartment (Fig. 4).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 5.
Pulse/chase analysis of AtT-20 cells.
A, AtT-20 cells expressing gp170 were labeled with
[35S]Met for 15 min, and then incubated with unlabeled
Met for the indicated time (hours). The cells and the media were
subjected to immunoprecipitation using the antiserum to either duck CPD
or CPE. B, quantitation of the secretion of gp170 and CPE
from AtT-20 cells.
|
|
To further characterize the gp170 pathway, we tested whether the
secretion of gp170 could be induced by secretagogues. Two different
approaches were used to study the secretion of gp170. First, cells were
incubated for 30 min in the presence or absence of Br-cAMP and then
media were analyzed by Western blot (Fig. 6, left). Although the
secretion of CPE was stimulated 2.5-3-fold by the secretagogue
treatment, the secretion of gp170 was not significantly affected by the
secretagogue treatment (Fig. 6, left). Two other
secretagogues (forskolin and a phorbol ester) also did not
significantly stimulate the secretion of gp170 from the AtT-20 cells
(data not shown). These results indicate that the bulk of gp170
undergoes constitutive secretion. As a small amount of radiolabeled
gp170 was detected within the cells after several hours of chase (Fig.
5A), we tested whether this pool of gp170 could undergo
regulated release in response to a secretagogue. Cells were labeled
with [35S]Met for 15 min, chased for 2 h, washed,
and then incubated for an additional 30 min in either control media or
media containing Br-cAMP. Both gp170 and CPE are released to the media
in the regulated fashion (Fig. 6, right). Quantitation of
three independent experiments demonstrated a statistically significant
stimulated release of both gp170 (Fig. 6, right) and CPE
(data not shown) in response to the secretagogue, indicating that a
small fraction of the gp170 undergoes regulated exocytosis. These
findings are consistent with the electron microscopy and
immunocytochemistry data, and taken together suggest that the bulk of
gp170 does not enter mature secretory vesicles in AtT-20 cells.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 6.
Regulated secretion of gp170 and CPE from
AtT-20 cells. Left panels (Western), cells
expressing gp170 were incubated either in control media or media
containing 5 mM Br-cAMP for 30 min and the secreted
proteins analyzed by Western blotting. Right
panels (pulse/chase), AtT-20 cells expressing gp170 were
labeled with [35S]Met for 15 min, chased for 2 h,
washed, and then incubated for an additional 30 min in either control
media or media containing Br-cAMP. Cells and the media were subjected
to immunoprecipitation using the antiserum either to duck CPD or CPE.
Bottom graphs, effect of the secretagogue on
gp170 secretion in Western blot analysis (left) and
pulse/chase analysis (right). Error
bars show standard error of the mean from three independent
experiments. Asterisk (*), statistically different from
control using Student's t test (p < 0.05).
|
|
 |
DISCUSSION |
CPD was previously localized by light microscopy to a perinuclear
compartment that overlaps with the distribution of the TGN endopeptidase furin in AtT-20 cells (25). In the present study, biochemical and electron microscopic analysis supports the localization of CPD to the TGN, and in addition has provided evidence that a
fraction of the CPD is present in immature secretory granules. The
processing of many neuroendocrine peptides begins in the TGN and
continues in the immature secretory granules. The acidification of the
TGN and secretory granules is thought to play an important role in the
activation of the processing enzymes such as prohormone convertase 1 and 2 (28, 45). Importantly, CPD has a broad pH optimum (14, 23) and
could function both in the environment of the TGN and within acidic
secretory granules. In contrast, CPE is active only at acidic pH values
and is essentially inactive at neutral pH (46), consistent
with a role for CPE in the processing of neuroendocrine
peptides that occurs in the late secretory pathway. The
endopeptidases prohormone convertase 1 and 2 have been also localized to mature secretory granules (28, 42), indicating that CPE is likely to be a functional partner for these
endopeptidases whereas CPD is a functional partner for furin.
Co-localization of CPD with the proteolytic fragments of
POMC, together with electron microscopy data, strongly suggest
that CPD enters the regulated secretory pathway in AtT-20 cells.
However, in contrast to CPE, only a small fraction of CPD routes to
mature secretory granules. It has been previously demonstrated that
some TGN membrane proteins including furin and mannose 6-phosphate receptor enter the immature secretory granules of the regulated pathway in neuroendocrine cells (47, 48). As with CPD, both of these
proteins are largely removed from immature granules during the
maturation and are not generally detected in mature secretory granules.
The mannose 6-phosphate receptor is sorted from immature secretory
vesicles to endosomes by an AP-1- and clathrin-dependent process (48). Consistent with this, the furin cytoplasmic tail interacts with AP-1, a component of the TGN clathrin sorting machinery (47). This interaction is dependent on phosphorylation of the furin
cytoplasmic tail by casein kinase II (47); mutation of the casein
kinase II phosphorylation sites results in mistargeting of furin to
mature secretory granules (47). Interestingly, potential casein kinase
II phosphorylation sites are also found within an acidic residue-rich
cluster of the CPD cytosolic tail. Mutation of these sites altered the
trafficking of CPD and resulted in detectable staining of the mutant
protein in the tips of AtT-20 cell processes (24). This suggests that a
similar mechanism of retrieval from immature secretory granules is
involved in CPD trafficking. Our finding that CPD is more abundant
within vesicles with a low degree of condensation than in highly
condensed vesicles (Fig. 4, Table I) supports the idea that CPD is
progressively removed from immature granules in the process of their
maturation. The retrieval of CPD from immature vesicles may be required
for the targeting of CPD to the recycling pathway and its efficient return to the TGN (Fig. 7).

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 7.
Post-TGN routing pathways of CPD in
neuroendocrine cells. The membrane- associated form of CPD
(bullets) is targeted to immature secretory granules
(ISG), containing neuroendocrine peptides (dots).
We propose that CPD is progressively removed from immature granules via
the constitutive-like vesicles (CLV) in the process of
maturation to enter recycling endosomes (RE) and then either
the TGN or the plasma membrane (PM). The soluble form of CPD
(rectangles) may follow a similar immature granule-mediated
sorting pathway, and then is removed from the regulated pathway.
Alternatively, gp170 may directly enter the TGN-derived constitutive
vesicles (CV).
|
|
Another important finding of the present study is that the soluble form
of CPD (gp170) also enters immature secretory vesicles, but is not
abundant in mature vesicles. As a result, the bulk of this protein is
secreted constitutively from AtT-20 cells. A soluble form of CPD has
been previously detected in various tissues including liver, kidney,
and brain (17). It is likely that this form resembles the truncated
gp170 form, as the endogenous soluble form is 170 kDa and does not bind
the antiserum directed against the C-terminal tail of CPD (17). Our
finding that gp170 also enters the regulated secretory pathway supports
the idea that the soluble form of CPD may be also involved in the
processing of neuropeptides in immature vesicles. As the majority of
gp170 is secreted via the constitutive or constitutive-like pathway, it
is also possible that the soluble CPD functions within constitutive vesicles or outside the cell. Our previous finding that gp180/CPD cycles between the plasma membrane and the TGN (24, 25) raises the
possibility that the full-length form of CPD functions within endocytic
compartments. Interestingly, it has been demonstrated that furin
cleaves precursor proteins in both the exocytic and endocytic pathways
(49). Several viral coat proteins including influenza hemagglutinin and
human immunodeficiency virus gp160 are processed by furin when
co-expressed with the endopeptidase (49). Furin-mediated cleavage of
many toxins occurs either at the plasma membrane or in endosomes (49).
As furin cleaves to the C-terminal side of basic amino acids, the
product of furin cleavage will contain C-terminal basic residues. If
these need to be removed for the products to be biologically active, as
is the case for most neuroendocrine peptides, CPD is a likely candidate for this activity based on its tissue distribution, intracellular distribution, and pH optimum. The involvement of CPD in important biological functions is supported by the fact that the mutations in the
Silver gene, the Drosophila homologue of CPD, are
embryonic lethal (21).
The finding that gp170 is primarily secreted in a constitutive manner
from AtT-20 cells also suggests that the lumenal domain of CPD is not
sufficient for the efficient targeting of the protein to mature
secretory granules, although it is sufficient for entry into immature
secretory vesicles. In contrast, soluble forms of PAM are efficiently
packaged into storage granules in AtT-20 cells, indicating that the
lumenal domain of PAM plays a major role in sorting into the regulated
pathway (32). The mechanism of the protein sorting to the regulated
secretory pathway remains unclear, and two alternative models have been
proposed. The active sorting model postulates that regulated proteins
and hormones bind to the sorting receptor in the TGN, and then are
delivered to immature secretory vesicles. Proteins lacking specific
signals for sorting into the regulated pathway follow the TGN-derived
constitutive pathway. In the passive sorting model, protein sorting in
the TGN is not selective and both the constitutive and regulated
proteins enter immature secretory granules. As sorting proceeds,
proteins of the constitutive pathway are selectively removed from
immature granules via constitutive-like vesicles, whereas proteins of
the regulated pathway undergo further condensation and packaging into mature secretory granules. The finding that only a small fraction of
CPD is present in mature granules indicates that CPD may fail to
undergo further condensation during granule biogenesis.
In conclusion, we have previously demonstrated that CPD is
predominantly localized to the TGN, and also cycles between the TGN and
the plasma membrane in AtT-20 cells (25). In the present study, we
demonstrate that CPD enters immature secretory granules containing
neuroendocrine peptides (Fig. 7, ISG). We propose that CPD
is progressively removed from immature granules in the process of their
maturation and enters recycling endosomes (Fig. 7, RE). The
soluble form of CPD (gp170) may follow a similar immature granule-mediated sorting pathway, and then is efficiently removed from
the regulated pathway via constitutive-like vesicles (CLV) (Fig. 7). Alternatively, only a small fraction of gp170 may enter the
regulated pathway, whereas the bulk of it follows a constitutive secretion (Fig. 7, CV).
 |
ACKNOWLEDGEMENTS |
We thank the following people for providing
antisera described in Materials and Methods: Ruth Angeletti, Jason
Bock, Peter Davies, Ari Helenius, Regina Kuliawat, Iris Lindberg,
Richard Mains, Sharon Milgram, and Richard Scheller. We thank Frank
Maceluso and the Analytical Imaging Facility of Albert Einstein College of Medicine for technical assistance. We gratefully acknowledge Sharon
Tooze and Sharon Milgram for helpful suggestions and discussion.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01 DK-51271 and by Research Scientist Development K02 Award DA-00194 (to L. D. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Molecular
Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park
Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-430-8954; E-mail:
fricker{at}aecom.yu.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
PC, prohormone
convertase;
CPE, carboxypeptidase E;
CPD, carboxypeptidase D;
PAM, peptidylglycine-
-amidating monooxygenase;
TGN, trans-Golgi network;
ACTH, adrenocorticotrophic hormone;
POMC, proopiomelanocortin;
PBS, phosphate-buffered saline;
ER, endoplasmic reticulum.
 |
REFERENCES |
-
Steiner, D. F.,
Smeekens, S. P.,
Ohagi, S.,
and Chan, S. J.
(1992)
J. Biol. Chem.
267,
23435-23438[Free Full Text]
-
Seidah, N. G.,
Chretien, M.,
and Day, R.
(1994)
Biochimie
76,
197-209[CrossRef][Medline]
[Order article via Infotrieve]
-
Fricker, L. D.
(1988)
Annu. Rev. Physiol.
50,
309-321[CrossRef][Medline]
[Order article via Infotrieve]
-
Fricker, L. D.
(ed)
(1991)
Peptide Biosynthesis and Processing, pp. 199-230, CRC Press, Boca Raton, FL
-
Fricker, L. D.,
and Snyder, S. H.
(1982)
Proc. Natl. Acad. Sci. U. S. A.
79,
3886-3890[Abstract]
-
Docherty, K.,
and Hutton, J. C.
(1983)
FEBS Lett.
162,
137-141[CrossRef][Medline]
[Order article via Infotrieve]
-
Hook, V. Y. H.,
and Loh, Y. P.
(1984)
Proc. Natl. Acad. Sci. U. S. A.
81,
2776-2780[Abstract]
-
Naggert, J. K.,
Fricker, L. D.,
Varlamov, O.,
Nishina, P. M.,
Rouille, Y.,
Steiner, D. F.,
Carroll, R. J.,
Paigen, B. J.,
and Leiter, E. H.
(1995)
Nat. Genet.
10,
135-142[Medline]
[Order article via Infotrieve]
-
Varlamov, O.,
Leiter, E. H.,
and Fricker, L. D.
(1996)
J. Biol. Chem.
271,
13981-13986[Abstract/Free Full Text]
-
Rovere, C.,
Viale, A.,
Nahon, J.,
and Kitabgi, P.
(1996)
Endocrinology
137,
2954-2958[Abstract]
-
Fricker, L. D.,
Berman, Y. L.,
Leiter, E. H.,
and Devi, L. A.
(1996)
J. Biol. Chem.
271,
30619-30624[Abstract/Free Full Text]
-
Udupi, V.,
Gomez, P.,
Song, L.,
Varlamov, O.,
Reed, J. T.,
Leiter, E. H.,
Fricker, L. D.,
and Greeley, G. H. J.
(1997)
Endocrinology
138,
1959-1963[Abstract/Free Full Text]
-
Cain, B. M.,
Wang, W.,
and Beinfeld, M. C.
(1997)
Endocrinology
138,
4034-4037[Abstract/Free Full Text]
-
Song, L.,
and Fricker, L. D.
(1995)
J. Biol. Chem.
270,
25007-25013[Abstract/Free Full Text]
-
Kuroki, K.,
Cheung, R.,
Marion, P. L.,
and Ganem, D.
(1994)
J. Virol.
68,
2091-2096[Abstract]
-
Kuroki, K.,
Eng, F.,
Ishikawa, T.,
Turck, C.,
Harada, F.,
and Ganem, D.
(1995)
J. Biol. Chem.
270,
15022-15028[Abstract/Free Full Text]
-
Song, L.,
and Fricker, L. D.
(1996)
J. Biol. Chem.
271,
28884-28889[Abstract/Free Full Text]
-
Xin, X.,
Varlamov, O.,
Day, R.,
Dong, W.,
Bridgett, M. M.,
Leiter, E. H.,
and Fricker, L. D.
(1997)
DNA Cell Biol.
16,
897-909[Medline]
[Order article via Infotrieve]
-
Dong, W.,
Fricker, L. D.,
and Day, R.
(1999)
Neuroscience
89,
1301-1317[CrossRef][Medline]
[Order article via Infotrieve]
-
Tan, F.,
Rehli, M.,
Krause, S. W.,
and Skidgel, R. A.
(1997)
Biochem. J.
327,
81-87[Medline]
[Order article via Infotrieve]
-
Settle, S. H. J.,
Green, M. M.,
and Burtis, K. C.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
9470-9474[Abstract]
-
Fan, X.,
Qian, Y.,
Fricker, L. D.,
Akalal, D. B.,
and Nagle, G. T.
(1999)
DNA Cell Biol.
18,
121-132[CrossRef][Medline]
[Order article via Infotrieve]
-
Eng, F. J.,
Novikova, E. G.,
Kuroki, K.,
Ganem, D.,
and Fricker, L. D.
(1998)
J. Biol. Chem.
273,
8382-8388[Abstract/Free Full Text]
-
Eng, F. J.,
Varlamov, O.,
and Fricker, L. D.
(1999)
Mol. Biol. Cell
10,
35-46[Abstract/Free Full Text]
-
Varlamov, O.,
and Fricker, L. D.
(1998)
J. Cell Sci.
111,
877-885[Abstract/Free Full Text]
-
Orci, L.,
Ravazzola, M.,
Storch, M. J.,
Anderson, R. G. W.,
Vassalli, J. D.,
and Perrelet, A.
(1987)
Cell
49,
865-868[Medline]
[Order article via Infotrieve]
-
Fernandez, C. J.,
Haugwitz, M.,
Eaton, B.,
and Moore, H. H.
(1997)
Mol. Biol. Cell
8,
2171-2185[Abstract/Free Full Text]
-
Tanaka, S.,
Yora, T.,
Nakayama, K.,
Inoue, K.,
and Kurosumi, K.
(1997)
J. Histochem. Cytochem.
45,
425-436[Abstract/Free Full Text]
-
Schnabel, E.,
Mains, R. E.,
and Farquhar, M. G.
(1989)
Mol. Endocrinol.
3,
1223-1235[Abstract]
-
Milgram, S. L.,
and Mains, R. E.
(1994)
J. Cell Sci.
107,
737-745[Abstract/Free Full Text]
-
Zhou, A.,
Bloomquist, B. T.,
and Mains, R. E.
(1993)
J. Biol. Chem.
268,
1763-1769[Abstract/Free Full Text]
-
Milgram, S. L.,
Johnson, R. C.,
and Mains, R. E.
(1992)
J. Cell Biol.
117,
717-728[Abstract]
-
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
-
Tooze, S. A.,
Flatmark, T.,
Tooze, J.,
and Huttner, W. B.
(1991)
J. Cell Biol.
115,
1491-1503[Abstract]
-
Tooze, J.,
and Tooze, S. A.
(1986)
J. Cell Biol.
103,
839-850[Abstract]
-
Matsuuchi, L.,
Buckley, K. M.,
Lowe, A. W.,
and Kelly, R. B.
(1988)
J. Cell Biol.
106,
239-251[Abstract]
-
Koedam, J. A.,
Cramer, E. M.,
Briend, E.,
Furie, B.,
Furie, B. C.,
and Wagner, D. D.
(1992)
J. Cell Biol.
116,
617-625[Abstract]
-
Molloy, S. S.,
Thomas, L.,
VanSlyke, J. K.,
Stenberg, P. E.,
and Thomas, G.
(1994)
EMBO J.
13,
18-23[Abstract]
-
Luzio, J. P.,
Brake, B.,
Banting, G.,
Howell, K. E.,
Braghetta, P.,
and Stanley, K. K.
(1990)
Biochem. J.
270,
97-102[Medline]
[Order article via Infotrieve]
-
Bock, J. B.,
Klumperman, J.,
Davanger, S.,
and Scheller, R. H.
(1997)
Mol. Biol. Cell
8,
1261-1271[Abstract]
-
Guest, P. C.,
Ravazzola, M.,
Davidson, H. W.,
Orci, L.,
and Hutton, J. C.
(1991)
Endocrinology
129,
734-740[Abstract]
-
Hornby, P. J.,
Rosenthal, S. D.,
Mathis, J. P.,
Vindrola, O.,
and Lindberg, I.
(1993)
Neuroendocrinology
58,
555-563[Medline]
[Order article via Infotrieve]
-
Hohl, I.,
Robinson, D. G.,
Chrispeels, M. J.,
and Hinz, G.
(1996)
J. Cell Sci.
109,
2539-2550[Abstract/Free Full Text]
-
Thorens, B.,
and Roth, J.
(1996)
J. Cell Sci.
109,
1311-1323[Abstract/Free Full Text]
-
Shennan, K. I. J.,
Taylor, N. A.,
Jermany, J. L.,
Matthews, G.,
and Docherty, K.
(1995)
J. Biol. Chem.
270,
1402-1407[Abstract/Free Full Text]
-
Greene, D.,
Das, B.,
and Fricker, L. D.
(1992)
Biochem. J.
285,
613-618[Medline]
[Order article via Infotrieve]
-
Dittie, A. S.,
Thomas, L.,
Thomas, G.,
and Tooze, S. A.
(1997)
EMBO J.
16,
4859-4870[Abstract/Free Full Text]
-
Klumperman, J.,
Kuliawat, R.,
Griffith, J. M.,
Geuze, H. J.,
and Arvan, P.
(1998)
J. Cell Biol.
141,
359-371[Abstract/Free Full Text]
-
Nakayama, K.
(1997)
Biochem. J.
327,
625-635[Medline]
[Order article via Infotrieve]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.