From the Department of Molecular Pharmacology and the
§ Department of Developmental and Molecular Biology, Albert
Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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Metallocarboxypeptidase D (CPD) is a
membrane-bound trans-Golgi network (TGN) protein. In AtT-20
cells, CPD is initially produced as a 170-kDa endoglycosidase
H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to
180 kDa and is resistant to endoglycosidase H as a result of
carbohydrate maturation. CPD also undergoes an activation step required
for binding to a substrate affinity resin. Blocking the protein exit
from the endoplasmic reticulum inhibits the increase in molecular mass
but not the step required for affinity column binding, suggesting that
enzyme activation precedes carbohydrate maturation and that these
reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which
bud from the TGN of permeabilized AtT-20 and GH3 cells. The
budding efficiency of CPD into vesicles is 2-3-fold lower than that of
endogenous proopiomelanocortin in AtT-20 cells or prolactin in
GH3 cells. In contrast, the packaging of a truncated form
of CPD, which lacks the cytoplasmic tail and transmembrane domain, was
similar to that of proopiomelanocortin. Taken together, the results
support the proposal that CPD functions in the TGN in the processing of
proteins that transit the secretory pathway and that the C-terminal
region plays a major role in TGN retention.
Many bioactive peptides are synthesized from larger proteins by
proteolytic processing that occurs in different organelles. Several
endoproteases, which cleave at basic amino acid containing sites, have
been described: prohormone convertase-1
(PC1)1 (also known as PC3),
PC2, PC4, PACE4, PC5A and -B (also known as PC6A and -B), and PC7 (also
known as PC8 and LPC) (1-6). Following the endoprotease step, a
carboxypeptidase is usually required to remove residual Lys and/or Arg
residues from the C terminus of the peptide (7, 8). In some cases,
further modification results in a C-terminal amide residue; this
reaction is catalyzed by the enzyme peptidylglycine Until recently, carboxypeptidase E (CPE, also known as carboxypeptidase
H, enkephalin convertase, and E.C. 3.4.17.10) was thought to be the
only carboxypeptidase involved in the generation of peptide hormones
and neurotransmitters (7, 8). However, studies on
Cpefat/Cpefat mice have
suggested that another carboxypeptidase contributes to peptide
processing in addition to CPE (10). The
Cpefat/Cpefat mice have a
point mutation in the coding region of the CPE gene, which inactivates
the enzyme resulting in its degradation prior to secretion (10, 11).
These mice have reduced levels of several peptide hormones and
neurotransmitters and elevated levels of proteolytic-processing
intermediates containing C-terminal basic residues (10, 12, 13); this
finding is strong evidence that CPE plays a major role in peptide
processing. However, fully processed peptides are present in moderate
levels in Cpefat/Cpefat
mice, raising the possibility that another carboxypeptidase contributes to peptide processing.
Carboxypeptidase D (CPD) was discovered during a recent search for
enzymes with carboxypeptidase-like enzymatic properties (14). Both CPE
and CPD have substantial activity at pH values in the 5-6 range (14),
which is consistent with the internal pH of the intracellular
compartments where processing is thought to occur (15, 16). However,
the intracellular localization of CPE and CPD differ; CPE is
predominantly in the secretory vesicles of the regulated pathway,
whereas CPD is enriched in the TGN and immature secretory granules (17,
18). The physical properties of the two enzymes are also different. CPE
is a 50-56-kDa protein that is present both as a soluble and
peripheral membrane-associated form, whereas CPD is a type I 180-kDa
transmembrane glycoprotein (14). Recently, rat (19) and human (20) CPD
cDNAs have been sequenced and were found to be homologs of duck
gp180, a protein that binds duck hepatitis B virus particles (21). Duck
gp180 and rat and human CPD contain three repeats of a 50-kDa CPE-like domain, followed by a transmembrane domain and a 58 residue cytosolic tail (19-21). Several other enzymes in the secretory pathway are also
membrane-bound proteins, such as furin, PC5B, and peptidylglycine The purpose of the present study was to examine the biosynthesis and
sorting of endogenous CPD into nascent secretory vesicles in the AtT-20
and GH3 cell lines. These cells express CPD mRNA (19)
and have been extensively used to study the biosynthesis and
intracellular trafficking of peptide hormones and processing enzymes.
The results of the pulse-chase analysis in the present study are
consistent with the previous finding that CPD is present in the TGN and
cycles from the cell surface back to the TGN (17) in AtT-20 cells. In
addition, the finding that newly synthesized CPD is able to enter
nascent secretory vesicles that bud from the TGN, albeit at a reduced
efficiency relative to endogenous prohormones and hormones, is further
evidence that CPD functions in the TGN and immature secretory vesicles
in the processing of peptides and proteins. Our results also
demonstrate that the cytoplasmic tail and/or transmembrane domain of
CPD play an essential role in retaining the enzyme in the TGN.
Pulse-Chase Analysis--
Wild type AtT-20 cells in 60-mm plates
were incubated for 1 h in Dulbecco's modified Eagle's medium
lacking methionine and then radiolabeled (pulsed) with
[35S]Met for 15 min, washed 3 times with media, and
chased for various times at 37 °C. In some experiments, the chase
was performed in media containing brefeldin A (5 µg/ml),
cycloheximide (100 µg/ml), or at 15 or 20 °C. Following the chase,
the media were removed, and the cells were washed once with
phosphate-buffered saline. Cells were then frozen on dry ice after
adding 0.5 ml of 10 mM NaAc buffer at pH 5.5.
For the analysis of the active form of CPD, the cells were thawed and
sonicated, and the buffer was adjusted to 1% Triton X-100 and 1 M NaCl in 50 mM NaAc, pH 5.5. The homogenate
was centrifuged at 30,000 × g for 30 min, and the
supernatant was subjected to purification on a 0.5-ml
p-aminobenzoylarginine-Sepharose 6B affinity column as
described (14, 27). Cell medium was also analyzed on the affinity resin
after first adjusting the pH to 5.5 with 50 mM NaAc. CPD
was eluted from the affinity column using 2 ml of Tris buffer, pH 8, containing 50 mM NaCl, 25 mM Arg, and 0.01% Triton X-100. Aliquots of the affinity column eluate were analyzed on a
8% denaturing polyacrylamide gel, which was then treated with
Fluoro-hance and exposed to x-ray film (Kodak). Quantitation of
autoradiograms was performed using an image analysis system as
described (28). All autoradiograms used for quantitation were in the
linear range of the system.
For the isolation of CPD and CPE by immunoprecipitation, aliquots of
the extracts described above were used. In some experiments, cells were
extracted directly with 2% SDS using the standard immunoprecipitation protocol (29). Immunoprecipitation was performed using polyclonal antisera against purified rat CPD, purified duck CPD (gp180), the
C-terminal 57 residues of duck CPD, or the C-terminal 9 residues of rat
CPE (11, 27). Antisera-bound CPD was isolated using protein A-Sepharose
4B beads (Sigma) as described (29). CPD was recovered by boiling the
protein A-Sepharose 4B pellet in polyacrylamide gel-loading buffer
containing 1% SDS for 5 min, and the immunoprecipitated material was
analyzed on denaturing 6% polyacrylamide gels as described above. In
some experiments, the immunoprecipitated material was incubated in the
presence of N-glycosidase F (New England Biolabs),
endoglycosidase H (New England Biolabs), or neuraminidase from
Vibrio cholerae (Roche Molecular Biochemicals) for 16 h
at 37 °C. Prior to incubation with the protease-free glycosidases,
the samples were boiled to inactivate any contaminating proteases.
Following digestion, the samples were analyzed on denaturing
polyacrylamide gels as described above.
Permeabilized Cell Preparation and in Vitro Incubations--
The
preparation of permeabilized cells by a swell-scrape method was
described previously (30, 31). Basically, cells were radiolabeled for
10 min with [35S]Met, chased for 120 min (or shorter
times) at 20 °C, and incubated in cold hypotonic buffer (15 mM KCl, 10 mM HEPES, pH 7.2) for 5 min. After
swelling, the cells were scraped off the plate with a rubber policeman
and resuspended in 90 mM KCl, 10 mM HEPES, pH
7.2, and 1 mM MgCl2, as described (30, 31).
Cells used for these analyses included wild type GH3 cells,
AtT-20 cells expressing gp180 (18), and AtT-20 cells expressing
gp180
The assay for nascent secretory vesicle budding was based on
quantitating the release of radiolabeled hormones into a 15,000 × g supernatant following an in vitro incubation
(31, 33). Following the incubation, permeabilized cells were pelleted
and lysed in detergent, and the lysates and supernatants containing nascent secretory vesicles were immunoprecipitated with antisera to
either rat CPD, duck CPD (gp180), CPE, prolactin, or ACTH (a gift of
Dr. Richard Mains, Johns Hopkins University) (this antiserum also
recognizes proopiomelanocortin and intermediate processing forms of
ACTH) as described (31). Immunoreactive material was resolved by
SDS-polyacrylamide gel electrophoresis and detected by fluorography.
Band intensities were quantitated using a Molecular Dynamics model 300A
computing densitometer and Image Quant 3.3 software (Molecular
Dynamics, Sunnyvale, CA).
The biosynthesis of CPD was investigated in AtT-20 cells using
metabolic labeling with [35S]Met and two techniques to
isolate the radiolabeled CPD, substrate affinity chromatography and
immunoprecipitation. After a 15-min pulse with radiolabel followed by
purification of the CPD on a substrate affinity resin, a single 170-kDa
form of CPD was detected (Fig. 1). During
the 30 min chase period, the apparent molecular mass of the
radiolabeled CPD increased to 180 kDa, and the amount of radiolabeled
CPD recovered from the affinity column also increased severalfold (Fig.
1). Quantitation of the results from four separate experiments showed a
reproducible 3-fold increase in the amount of affinity-purified
radiolabeled CPD upon early chase times (Fig. 2). Following this increase, the level of
radiolabeled CPD remained constant for several hours and then decreased
(Figs. 1 and 2). After 20 h, the cellular level of radiolabeled
CPD was approximately 30% of the peak value. No radiolabeled CPD was
detected in the media for any of the time points investigated (Fig.
1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-amidating
monooxygenase (9).
-amidating monooxygenase (22-24). These membrane-bound proteins are
located in the TGN, although they also cycle to the cell surface (25,
26).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C-term, a truncated form of duck CPD that lacks the cytosolic
tail and transmembrane
region.2 The incubation
conditions for these experiments consisted of approximately 5 × 105 permeabilized cells, 20 mM HEPES, pH 7.3, 125 mM KCl, 2.5 mM MgCl2, 1 mM ATP, 200 µM GTP, 10 mM
creatinine phosphate, 160 µg/ml creatinine phosphate kinase
(ATP-regenerating system), 0.5 mM phenylmethylsulfonyl
fluoride, and 5 µg/ml Trasylol. Incubation for 90 min at 37 °C
under these conditions is sufficient to reconstitute both prohormone
processing and nascent secretory vesicle release from the TGN (30,
31).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Pulse-chase analysis of CPD in AtT-20
cells. Cells were pulsed for 15 min, chased for the indicated
period of time, and extracted as described under "Materials and
Methods." Equal proportions of cell extracts and media were subjected
to purification on a p-aminobenzoyl- arginine-Sepharose
6B affinity column, and the column eluates were fractionated on a
denaturing 8% polyacrylamide gel. The positions of prestained size
standards (Bio-Rad) are indicated.
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Fig. 2.
Quantitation of the pulse-chase analysis of
CPD and CPE in AtT-20 cells upon purification by substrate affinity
column. Quantitation was performed using exposures within the
linear range of the film. The levels of each carboxypeptidase are
relative to the maximal level for that protein and are not relative to
each other. Error bars show the standard error of the mean
for n = 4. Data points without error bars
had standard error smaller than the symbol size. CP,
carboxypeptidase.
To compare the relative amount of CPE and CPD produced by the same cells, the affinity columns were first eluted with high pH buffer (to elute CPE) before eluting the CPD with Arg. Analysis of the CPE-containing fractions showed a major band of approximately 55 kDa (not shown). The intensity of this band was substantially greater than that of the radiolabeled CPD. Because the affinity columns appear to bind both proteins quantitatively, the AtT-20 cells synthesize approximately 50-100-fold more CPE than CPD, after adjustment for the number of Met residues in each protein. Quantitation of the radiolabeled CPE from four separate experiments showed a 35% decrease over the first hour followed by a slower decrease over the remainder of the chase period (Fig. 2). As previously reported (34), radiolabeled CPE was detected in the media after 1 h of chase and accumulated with increasing chase time (not shown).
When the radiolabeled CPD in the pulse-chase extracts was isolated by
immunoprecipitation, its apparent molecular mass increased from 170 to
180 kDa during the 1 h chase period, but no increase in the amount
of radiolabeled protein was evident (Fig.
3). Similar results were obtained when
using antiserum raised against either the mature form of rat CPD (Fig.
3) or against its C-terminal region (data not shown). No differences in
gel mobility were detected in comparing the extraction procedures used
for the affinity column procedure (Triton X-100 plus NaCl) with the
procedure used for immunoprecipitation (SDS) (data not shown). Thus,
the increase in the amount of radiolabeled CPD recovered from the
affinity column with time presumably represents an activation step,
possibly protein folding, that is required for binding to the substrate affinity resin. In contrast to the increase in radiolabeled CPD seen
during short chase periods with the affinity purification method, the
decrease in CPD levels upon long chase periods was comparable for both
the affinity purification and immunoprecipitation, indicating that the
reduction is because of loss of CPD protein and not because of less
efficient binding to the affinity column (not shown).
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To investigate whether changes in glycosylation contributed to the increase in apparent molecular mass of CPD, affinity purified material was treated with glycosidases. N-Glycosidase F, which removes all N-linked sugars, reduced the size of the protein isolated immediately after the pulse (i.e. the 170-kDa form) to 147 kDa and the form after chase (i.e. the 180-kDa form) to 150 kDa (Fig. 3). Neuraminidase digestion, which removes terminal sialic acid, decreased the apparent molecular mass of the 180-kDa species to 175 kDa but had no effect on the migration of the 170-kDa form (Fig. 3). This finding indicates that only the 180-kDa form had reached the trans-Golgi (the site of sialyltransferase activity) during 1 h of chase. Endoglycosidase H reduced the apparent molecular mass of the 170-kDa species to 147 kDa but had no effect on the 180-kDa form (Fig. 3). Thus, the 170-kDa species is endoglycosidase H sensitive, although the 180-kDa form is endoglycosidase H resistant, suggesting that the smaller form is predominantly localized to the ER or cis-Golgi, whereas the larger species is present in the medial or trans-Golgi.
To further study the biosynthesis of CPD, the pulse-chase was performed
under several conditions that trap proteins in the ER followed by
isolation using a substrate affinity resin (Fig. 4). To test whether the increase in the
amount of CPD recovered on the affinity resin was because of increased
synthesis of CPD, cycloheximide was used. When cycloheximide was added
to the chase medium to block further protein synthesis, the amount and
size of CPD was identical to that isolated from control untreated cells (Figs. 4 and 5, X). Brefeldin
A added during the chase prevented the increase in apparent molecular
mass but not the amount of CPD recovered from the affinity resin (Figs.
4 and 5, B). Similarly, when the chase was performed at
15 °C, the increase in size was prevented but not the amount of
radiolabeled CPD bound to the affinity resin (Figs. 4 and 5,
15). Incubation at 20 °C, which prevents the exit of
vesicles from the trans-Golgi network (30, 35), partially
blocked the size increase (Figs. 4 and 5, 20). Taken
together, these results suggest that the activation step is an ER event
because treatments that block the exit from the ER do not prevent the
activation.
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To determine if CPD is packaged into nascent secretory vesicles, a
permeabilized cell assay was used. This involves pulse-labeling the
cells with [35S]Met followed by a 2 h chase at
20 °C to trap the radiolabeled proteins in the TGN, followed by
permeabilization of the cells and analysis of the proteins present in
nascent secretory vesicles, which are released from the TGN during
subsequent in vitro incubation at 37 °C (31). For these
experiments, AtT-20 cells were used as well as the rat anterior
pituitary GH3 cell line because preliminary data determined
these cells have relatively high levels of CPD. In addition,
permeabilized GH3 cells have been used extensively to
investigate vesicle budding from the TGN (36) and vesicle fusion with
the plasma membrane (37). In GH3 cells, newly synthesized CPD was approximately 170 kDa (Fig.
6A, lane 1). When
the cells were chased for 2 h at 20 °C, the majority of the
protein remained as the 170-kDa form, although a small amount was
present as a 180-kDa protein (Fig. 6A, lane 2).
Only the larger form of CPD entered nascent secretory vesicles (Fig.
6A, lane 6). Release of the vesicles from the TGN
and the packaging of the 180-kDa CPD was dependent on the presence of
an energy-generating system (compare lanes 6 and
8).
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When wild type AtT-20 cells were used, the level of endogenous CPD was not easily detected in the budded vesicles, and so a cell line overexpressing duck CPD (gp180) was used. In these cells, the processing of CPD from the 170 to the 180-kDa species was much more efficient than in GH3 cells, and only a small fraction of the enzyme remained 170 kDa following a 2 h chase at 20 °C (Fig. 6B, lane 2). For both AtT-20 and GH3 cell lines, the relative amount of CPD that entered into the nascent secretory vesicles represented only a small fraction (12-14%) (Table I) of the total radiolabeled CPD. In contrast, the budding of nascent secretory vesicles containing prolactin (PRL) from the TGN of GH3 cells or of proopiomelanocortin (POMC) from AtT-20 cells was significantly more efficient (36-41%) (Table I) and was similar to results previously obtained (30, 31). As expected the budding of vesicles containing these prohormones was energy-dependent (30, 38) (Fig. 6, C and D). The low apparent budding efficiency of CPD into nascent secretory vesicles was not because of the intrinsic inefficiency of the permeabilized system, because vesicles containing soluble cargo polypeptides (PRL and POMC) were efficiently released from the TGN (Table I).
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To test whether the 170-kDa form of CPD detected after short chase times in AtT-20 cells was unable to enter secretory vesicles as found for the CPD in GH3 cells, the vesicle budding assay was performed on cells that had been chased for only 30 or 60 min at 20 °C prior to generating a permeabilized cell preparation. For these experiments, cells expressing duck CPD (gp180) were used to improve the signal in the budding assay. After 60 min of chase at 20 °C, both 170- and 180-kDa forms of duck CPD were detected (Fig. 6E, lanes 5-8), which is consistent with the results with endogenous CPD in AtT-20 cells (Figs. 1, 3, and 4) and endogenous CPD in GH3 cells (Fig. 6A). The mature 180-kDa form of duck CPD in AtT-20 cells entered the nascent vesicles more efficiently than the immature 170-kDa form (Fig. 6E, lane 8). When the cells were chased for 30 min at 20 °C prior to permeabilization and vesicle budding, the 170-kDa form of CPD was predominant (Fig. 6E, lanes 1-4). Although only small amounts of the 180-kDa form were detected at this early chase time, the relative amount of this form in the nascent vesicles was greater than the 170-kDa form. Very low levels of the 170-kDa form of duck CPD were detected in budded vesicles after 30 min of chase at 20 °C (Fig. 6E, lane 4). It is possible that the vesicles that contain the 170-kDa form at the early chase times were derived in part from the ER or early Golgi compartments.
We hypothesized that the inefficient budding of CPD was a consequence
of its retention in the TGN mediated by its C-terminal region. To test
this idea, permeabilized AtT-20 cells were prepared from a cell line
expressing gp180C-term, a truncated form of CPD, which lacks the
cytoplasmic tail and membrane anchor (Fig. 7). Significantly, the budding of
vesicles containing gp180
C-term was generally similar to that of
POMC and CPE (Table I), suggesting that the C-terminal region
(cytoplasmic tail and/or transmembrane domain) of CPD functions in part
as a retention sequence to decrease the efficiency of CPD packaging
into nascent vesicles.
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DISCUSSION |
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A major finding of the present study is that the turnover of CPD and CPE are substantially different in AtT-20 cells. This difference presumably reflects the intracellular location of each enzyme. CPD is an integral membrane protein that is predominantly localized to the TGN (17), whereas CPE is present in secretory vesicles as soluble and peripheral membrane-associated forms (7, 8). In addition, CPD recycles from the cell surface (17) and is not secreted into the media (Fig. 1), whereas CPE is secreted via the regulated secretory pathway (7, 8). These differences are likely to be responsible for the observed differences in the turn-over rate for the two enzymes.
The large increase in the amount of radiolabeled CPD detected during the 1 h chase period, as determined by binding to a substrate affinity resin (Fig. 1), was not observed when the CPD was isolated by immunoprecipitation (Fig. 3) indicating that CPD requires an activation step for binding to the affinity resin. Although most peptide-processing endopeptidases are initially produced as inactive precursors that require cleavage of a propeptide for activation (39), peptide-processing carboxypeptidases do not typically require proteolytic activation. For example, proCPE and CPE are both enzymatically active (40, 41). It is unlikely that CPD undergoes proteolytic activation because the N terminus of the active form of CPD corresponds to the sequence immediately downstream of the signal peptide cleavage site (19, 20). Also, the activation step does not correlate with the increase in apparent molecular weight that also occurs during the first 30-60 min of synthesis. Furthermore, brefeldin A or incubation at 15 °C blocked the increase in size but had no effect on the amount of CPD bound to the affinity resin (Fig. 4). These observations suggest that the activation step occurs in the ER. Because the activation of CPD does not substantially alter the size of the protein, it is likely that this activation reflects the folding of the CPD into a conformation that is able to bind to the substrate affinity resin.
The increase in the apparent molecular weight of CPD that occurs approximately 30 min after protein synthesis appears to result from changes in glycosylation, because the mobility of CPD on SDS-polyacrylamide gel electrophoresis is increased upon the removal of N-linked carbohydrates (Fig. 3). It is likely that the modification of CPD leading to the shift in apparent molecular mass from 170 to 180 kDa occurs in the TGN because it is blocked by either brefeldin A or incubation at 15 but not 20 °C (Figs. 4 and 6). Brefeldin A causes the collapse of the Golgi apparatus, the redistribution of Golgi proteins into the ER (42), and blocks protein transport to the TGN (43). Incubation at 15 °C also blocks the exit of proteins from the ER, whereas incubation at 20 °C blocks the exit from the TGN (30, 35). The partial effect of the 1-h chase at 20 °C (Fig. 4) on the size shift of CPD in the AtT-20 cells is presumably because of incomplete transport of this protein to the TGN at the reduced temperature; when the chase was performed for 2 h at 20 °C, the majority of the CPD was present in the larger 180-kDa form (Fig. 6). Taken together, it is likely that the increase in size of CPD largely, but not entirely, results from the addition of terminal sialic acid residues, a modification that occurs in the late Golgi and/or TGN. The possibility of other modifications (such as sulfation and palmitoylation) cannot be excluded, although studies using chlorate to block sulfation and chemical treatments to remove palmitate did not provide any evidence for these modifications of CPD (data not shown).
Interestingly, only the larger form of CPD was found to enter nascent secretory vesicles that bud from the TGN (Fig. 6). There are several possible interpretations of this observation; either the lower molecular weight form is prevented from being packaged into nascent secretory vesicles or the enzymes that mediate the size increase are enriched or activated in the budded vesicles so that the processing occurs soon after budding. The former possibility is consistent with the size increase resulting from sialylation because sialyltransferases are present in the TGN. A similar result was observed with furin; only the higher molecular weight sialylated form was found to enter nascent vesicles that bud from the TGN (38).
The efficiency of the budding of CPD is similar in the two cell lines
examined and is substantially lower than that of endogenous hormones.
The difference in efficiency presumably reflects the distribution of
the various proteins; although CPD is enriched in the TGN, peptide
hormones are primarily located in the mature secretory vesicles of
neuroendocrine cells. It is possible that the cytoplasmic tail and/or
transmembrane region of CPD binds to cytoskeletal proteins, thus
preventing the majority of the CPD from entering nascent secretory
vesicles. We speculate that a small fraction of the CPD polypeptides
are modified in the tail domain (possibly by phosphorylation or
dephosphorylation) allowing them to be released and packaged into the
budding vesicles along with the cargo molecules. In this context, it is
noteworthy that the C-terminally truncated gp180C-term CPD, which
lacks both a cytoplasmic tail and transmembrane domain, was sorted into
nascent vesicles with an efficiency similar to that of PRL or POMC
(Fig. 7 and Table I). This finding suggests that the tail and/or
transmembrane domain may play an important role in TGN retention as
previously hypothesized from studies on the distribution of CPD
C-terminal mutants in AtT-20 cells (18). Most significantly, these data demonstrate the specificity of the vesicle budding reaction and strongly suggest that the permeabilized cell system maintains significant selectively with respect to the packaging of cargo molecules into post-Golgi vesicles, which presumably includes both
regulated and constitutive pathway vesicles. If packaging of cargo
proteins were nonselective or vesicle release resulted from the random
fragmentation of the Golgi apparatus/TGN in vitro, then it
would be expected that similar levels of CPD and PRL or POMC would be
present in the vesicle fraction; our data demonstrate that this is not
the case.
The results of the present study, together with previous studies (17,
18), support the proposal that CPD functions in the TGN and/or immature
secretory vesicles to process proteins that transit the secretory
pathway. In this regard, CPD is similar to both furin and
peptidylglycine -amidating monooxygenase. All three enzymes contain
large N-terminal lumenal domains, a transmembrane domain, and a short
C-terminal cytoplasmic tail. Furin is primarily located in the TGN,
although it also cycles to the cell surface and back to the TGN (25,
44). The membrane form of peptidylglycine
-amidating monooxygenase
is also primarily present in the TGN, but smaller soluble forms of this
protein are enriched in mature secretory granules (32). The long
half-life of CPD in the pulse-chase analysis, the lack of detectable
processing of CPD to shorter forms, and the absence of secretion of a
significant portion of the CPD from AtT-20 cells are similar to the
properties of furin. In addition, both furin (38) and CPD (Fig. 6) are
detected in vesicles that bud from the TGN. Taken together, it is
likely that CPD functions following the action of furin or another
related endopeptidase.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledged Dr. Lixin Song for performing some of the preliminary data on the pulse-chase analysis of CPD in the AtT-20 cells.
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FOOTNOTES |
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* This work was primarily supported by National Institutes of Health Grants R01 DK-51271 (to L. D. F.), K02 DA-00194 (to L. D. F.), and R37 DK21860 (to D. S.).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.
2 Varlamov, O., Eng, F. J., Novikova, E. G., and Fricker, L. D. (1999) J. Biol. Chem., in press.
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ABBREVIATIONS |
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The abbreviations used are: PC, prohormone convertase; CPE, carboxypeptidase E; CPD, carboxypeptidase D; TGN, trans-Golgi network; ER, endoplasmic reticulum; PRL, prolactin; POMC, proopiomelanocortin; ACTH, adrenocorticotropic hormone.
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REFERENCES |
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