(Received for publication, August 28, 1995; and in revised form, November 22, 1995)
From the
Carboxypeptidase E (CPE) is involved in the biosynthesis of numerous peptide hormones and neurotransmitters. Previously, the C-terminal region of CPE has been shown to participate in the binding of the protein to membranes and to also contribute to the sorting of CPE into the regulated pathway. In this study, the role of the C-terminal region of CPE was further examined using several approaches. A series of CPE mutants with C-terminal deletions was expressed in the baculovirus system; constructs with a deletion of 14 or 23 residues were expressed at levels comparable to wild-type CPE. In contrast, deletion of 33 or more residues eliminated CPE activity, and the resulting protein was not secreted from the cells. Even though CPE mutants with a deletion of 14 or 23 residues were expressed normally, the resulting protein was mainly soluble, whereas approximately 55% of wild-type CPE was membrane associated. When expressed in AtT-20 cells, CPE with a deletion of 43 C-terminal amino acids was not secreted, whereas CPE with a deletion of 23 residues was secreted via the regulated pathway. Pulse-chase analysis revealed the protein with a deletion of 43 residues to be degraded in a non-acidic intracellular compartment. To investigate whether the C-terminal region of CPE can confer membrane binding and regulated pathway sorting to another protein, portions of the CPE C-terminal region were attached to the C terminus of albumin and the fusion proteins expressed in AtT-20 cells. Of the constructs examined, only the protein containing 51 amino acids of CPE was sorted to the regulated pathway, although with reduced efficiency compared to endogenous CPE. Although the C-terminal 14 amino acids of CPE are sufficient to target albumin to membranes, this fusion protein is not sorted into the regulated pathway. Taken together, these results indicate that the C-terminal 14 amino acids of CPE are important for membrane binding and that membrane binding and sorting require distinct signals.
Carboxypeptidase E (CPE) ()(EC 3.4.17.10), which is
also known as carboxypeptidase H and enkephalin convertase, functions
in the production of a large number of bioactive peptides(1) .
CPE is present in neuroendocrine tissues, and in several tissues this
enzyme has been localized to the peptide-containing secretory
vesicles(2, 3, 4, 5) . Purified CPE
is able to cleave a wide variety of peptides with C-terminal basic
residues, an important step in the production of biologically active
peptides(2, 6, 7, 8) . Further
evidence of the physiological role of CPE comes from the recent finding
that a mouse strain with an inactive form of CPE (due to a point
mutation) does not efficiently convert proinsulin into
insulin(9) .
CPE is present within secretory vesicles in
both soluble and membrane-associated forms(2, 10) .
However, the amino acid sequence (deduced from the DNA sequence) does
not show any potential transmembrane-spanning helical regions,
suggesting that CPE is membrane-bound through another
mechanism(11, 12) . Also, the binding of CPE to
membranes is pH-dependent, with maximal binding occurring at pH
5-6 and minimal binding at pH values greater than 8 (13, 14) . Although CPE has also been found to
aggregate at acidic pH values, this process appears to be distinct from
the membrane-binding process(15) . Several lines of evidence
suggest that the C-terminal region of CPE contributes to the membrane
binding of this protein. First, the C-terminal region of CPE is
predicted to form an amphiphilic -helix(14) . Second,
synthetic peptides that correspond to the C-terminal region of CPE are
able to bind to membranes in a pH-dependent manner(14) . Third,
the only difference found between the soluble and membrane-bound forms
of CPE is within the C-terminal region; antisera directed against the
C-terminal region of CPE show much stronger binding to the membrane
forms of CPE compared to the soluble forms(14, 16) .
However, this point is controversial as it has been reported that the
soluble and membrane forms of CPE show comparable binding of
C-terminally directed antisera(17) . Thus, one focus of the
present study was to further examine the role of the C-terminal region
of CPE in membrane binding.
The pH-dependent membrane binding of CPE has been proposed to be a mechanism for the sorting of CPE(14, 18) . According to this hypothesis, the interaction of CPE with membranes in the trans Golgi network, with an internal pH in the 6-6.5 range, would help drive the sorting of CPE into the regulated pathway. Consistent with this hypothesis was our recent finding that fusion proteins containing 51 amino acids of the C-terminal region of CPE attached to albumin are both membrane bound and partially sorted into the regulated pathway (18) . Another focus of the present study was to investigate whether the sorting and membrane-binding regions within the C-terminal region of CPE were the same or distinct. Several approaches were used to study the C-terminal region of CPE. The first approach used deletion mutations within the C-terminal region to address the role of this domain in protein expression and membrane binding. In the second approach, fusion proteins consisting of albumin with small portions of CPE attached to the C terminus were expressed in AtT-20 cells and examined for sorting and membrane binding. The results of these analyses indicate that the membrane-binding and sorting domains within the C-terminal region are distinct. Our results support the hypothesis that the putative amphiphilic helical region of CPE contributes to the membrane binding but does not support the hypothesis that this region is also involved with intracellular routing of CPE.
Figure 1:
Fusion proteins and deletion mutants. Top, diagram of prepro-CPE and amino acid sequences of
wild-type (wt) CPE and various deletion mutants. The
C-terminal sequence of wild-type CPE is indicated for the region
beginning at Ser and extending to the C terminus of
wild-type CPE (Phe
). The
pro deletion construct
contains the influenza hemagglutinin sequence (HA tag) in place of most
of the pro-region as indicated. Bottom, diagram of
pro-albumin, with portions of CPE attached to the C terminus, and amino
acid sequences of the C-terminal portions of these constructs. Human
albumin ends with the sequence LGL, and for the various fusion proteins
the C-terminal Leu was replaced with the indicated sequence. Dashes indicate gaps in the sequence, and dots indicate
continuing sequence not shown in the
figure.
Human albumin cDNA in
pGEM7zf (Promega) was used to generate albumin/CPE fusion proteins.
Polymerase chain reaction was used to generate fragments of CPE
encoding the various C-terminal regions, with a Bsu36I site on
the 5`-end and a SacI site on the 3`-end. These fragments were
subcloned into the appropriate sites within the albumin/pGEM plasmid;
there is a Bsu36I site at the C terminus of albumin and a SacI site in the pGEM7zf vector. For the expression in AtT-20
cells, the constructs were subcloned into the BamHI and NsiI sites of the eukaryotic expression vector pcDNA/NEO
(Invitrogen). Dideoxynucleotide sequencing was performed to confirm the
sequence of the C-terminal regions of all constructs and the N-terminal
region of the pro and HA-tagged constructs.
A 100-µl aliquot
of the resuspended Golgi/vesicle fraction was centrifuged at 100,000
g for 30 min. The supernatants were removed
(``soluble'' fraction), and the pellets were resuspended and
sonicated in 100 µl of 1 M sodium chloride in 10 mM NaAc buffer following centrifugation again at 100,000
g for 30 min. The second supernatants
(``membrane-1'' extracts) were removed, the pellets were
sonicated in 100 µl of 1 M sodium chloride and 1% Triton
X-100 in the same buffer and centrifuged again, and the supernatants
were removed (``membrane-2'' extracts). Aliquots of each of
the three supernatants were analyzed by Western blotting as described
below.
Figure 2:
Expression of CPE deletion mutants in
baculovirus. Top, Western blot analysis of cells (left) or media (right) after infection for 72 h with
control baculovirus (BV), with baculovirus expressing
wild-type CPE (wt), or with the various deletion mutants. The
Western blot was performed as described under ``Materials and
Methods.'' Bottom, carboxypeptidase activity in the cell
extracts and media was determined using dansyl-Phe-Ala-Arg as described
previously(20) , except that CoCl (which activates
CPE approximately 3-5-fold) was not included. Protein in the cell
extracts was determined using the Bradford assay. Enzyme activity in
both the cell and media samples was normalized to the amount of protein
in the cell extracts, which varied less than 2-fold between samples.
The enzyme determinations were performed with two to three separate
infections, with less than 50% variation in the enzyme measurements
between infections. For each infection, enzyme activity was measured in
duplicate, with less than 10% variation between
replicates.
To
examine whether the various deletions altered the soluble/membrane
distribution of the active forms of CPE, cells expressing the
constructs that showed enzyme activity were sequentially extracted with
NaAc at pH 6.0 (soluble), with 1 M NaCl in NaAc buffer
(membrane-1), and then with a combination of 1% Triton X-100 and 1 M NaCl in NaAc buffer (membrane-2). To ensure that each
extraction was complete, two extractions with each buffer were
performed and assayed separately. Typically, the second extraction with
each buffer contained much less CPE activity than the first extraction (Fig. 3), indicating that the apparent membrane binding is not
due to incomplete tissue disruption or to revesiculation. Approximately
40-50% of the total wild-type CPE and proCPE activity was
detected in the two soluble extracts, 30-40% was detected in the
membrane-1 extracts, and 15-20% was detected in the membrane-2
extracts (Fig. 3). Membranes after the extractions contained
very little CPE activity (not shown). In contrast to the results with
wild-type CPE and
proCPE, the two C-terminal deletion mutants
(CPE
14 and
23) were much more abundant in the soluble
fraction and virtually absent from the membrane-2 fraction (Fig. 3). This finding supports the hypothesis that the
C-terminal region, which is predicted to form an amphiphilic helix, is
important for the NaCl/Triton X-100-dependent binding of CPE to
membranes.
Figure 3: CPE activity in soluble and membrane extracts of baculovirus-infected Sf9 cells. The cells were infected for 72 h and then extracted and assayed as described under ``Materials and Methods.'' The results are from a single experiment assayed in duplicate. Error bars indicate the variation between replicates; values without error bars had negligible variations.
Removal of the C-terminal 14 amino acids has a minor
effect on the net charge, with the elimination of two acidic groups and
one basic group (Fig. 1). In contrast, removal of the C-terminal
23 residues has a larger effect on the net charge. To test whether the
decrease in membrane binding of CPE23 is due to an alteration in
the net charge, which affects the ability of the protein to aggregate,
we examined the pH-dependent aggregation of wild-type CPE and
CPE
23. CPE was purified from baculovirus-infected Sf9 cells and
tested for aggregation as described previously(15) . Wild-type
CPE and CPE
23 showed substantial aggregation at pH 5.0, less
aggregation at pH 5.5, and virtually no aggregation at pH 6.0 or higher
(data not shown). The similar pH-dependent aggregation of the two forms
of CPE implies that this aggregation is not responsible for the
membrane-binding properties of CPE, which show substantial differences
between wild-type CPE and CPE
23 (Fig. 3).
To test
whether the C-terminal 23 residues of CPE contributes to the efficient
sorting of this protein into the regulated pathway, the HA-epitope tag
sequence was introduced in the N-terminal region (to distinguish the
mutant CPE from endogenous CPE) and the protein expressed in AtT-20
cells. The N-terminal HA sequence does not interfere with the enzymatic
properties of CPE or the sorting of this protein into the regulated
pathway (data not shown). AtT-20 cells expressing HA-tagged CPE23
were stimulated with secretagogues for 30 min, and the amount or
protein in the media was analyzed by Western blot analysis. The
monoclonal antisera that recognizes the HA sequence binds to a single
protein in the media of transfected AtT-20 cells (Fig. 4);
untransfected cells do not show any signal with this antisera (data not
shown). Treatment of the cells with either forskolin or
tetradecanoylphorbol-13-acetate stimulates the levels of HA-tagged
CPE
23 in the media approximately 2-3-fold (Fig. 4).
The same media samples were also analyzed for endogenous CPE, which was
detected using an antisera directed against the C-terminal region (i.e. the region missing from the CPE
23 construct). The
endogenous CPE that reacts with the C-terminally directed antisera is
stimulated by the two secretagogues to the same extent as the
CPE
23 (Fig. 4), indicating that the C-terminal region is
not necessary for the efficient sorting of CPE into the regulated
pathway.
Figure 4:
Effects of secretagogues on the secretion
of CPE23 and endogenous CPE from AtT-20 cells. AtT-20 cells
expressing HA-tagged CPE
23 were treated with control media (C) or media containing 10 µM forskolin (F) or 0.1 µg/ml tetradecanoylphorbol-13-acetate (T) for 30 min. The media were analyzed on two separate
Western blots. To detect CPE
23, one blot was probed with the 12CA5
monoclonal antiserum, which recognizes the HA tag. To detect endogenous
wild-type CPE, the other blot was probed with the CPE C-terminally
directed antisera, as described under ``Materials and
Methods.'' Left, representative blots for the two
antisera. Right, quantification of the results from multiple
analyses. Error bars show standard error of the mean (n = 6). * indicates p < 0.05 using
Student's t test.
CPE with a deletion of 43 C-terminal amino acids was
expressed in AtT-20 cells to examine whether the C-terminal region is
required for secretion in a mammalian cell line that normally expresses
CPE. Immunoprecipitation of the [S]Met-labeled
extract from cells expressing CPE
43 showed a major band of
approximately 44 kDa and a minor band of 50 kDa, in addition to the
endogenous proCPE (56 kDa) and various minor bands that were also
detected with wild-type AtT-20 cells (Fig. 5). Analysis of 5
separate clones by [
S]Met labeling and
immunoprecipitation and 30 separate clones by Western blot analysis
showed a similar presence of two additional proteins of approximately
44 and 50 kDa. Both forms of CPE
43 do not change during the 30-min
chase but then largely disappear from the cells after 180 min of chase (Fig. 5). Neither form of CPE
43 appears in the media,
indicating that the mutant is degraded during the 180-min chase. In
contrast, the endogenous CPE becomes slightly smaller and shows a
weaker signal after 30 min of chase. This is consistent with the
removal of Met-containing N-terminal (propeptide) and C-terminal
sequences from a portion of the molecules; altogether, 4 out of the 9
total Met residues in mouse proCPE are located within 13 residues of
the N and C termini (GenBank accession no. X61232). After 180 min of
chase, the endogenous CPE is detected in the media in amounts equal to
those in the cells (Fig. 5).
Figure 5:
Pulse-chase analysis of wild-type CPE and
CPE43 in AtT-20 cells (C) and media (M). The
chase time (in minutes) is indicated. Immunoprecipitation was performed
with an antisera raised against the N-terminal region of CPE and
analyzed on a denaturing polyacrylamide gel, as described under
``Materials and Methods.'' The mobilities and apparent
molecular weights of prestained standards (Bio-Rad) are indicated.
Similar results were obtained with 5 distinct CPE
43-expressing
AtT-20 cell lines.
To further investigate the fate
of CPE43 in the AtT-20 cells, the cells were labeled with
[
S]Met and then either analyzed directly or
chased under a variety of conditions that block various cellular
processes. After 3 h of chase, both the endogenous CPE and the two
bands of CPE
43 were substantially decreased relative to the levels
before the chase (Fig. 6). Analysis of the media showed the CPE,
but not the CPE
43, to be secreted (not shown) as found for Fig. 5. The degradation of CPE
43 that occurs during the 3-h
chase is blocked when the chase is performed at either 15 or 20 °C (Fig. 6). Brefeldin A, which blocks transport between the
endoplasmic reticulum and Golgi, does not block the degradation of
CPE
43, although this does prevent the secretion of CPE (Fig. 6). Neither chloroquine nor ammonium chloride, which block
the acidification of vesicles, prevents the degradation of CPE
43 (Fig. 6). Taken together, these results suggest that CPE
43
is degraded by a temperature-sensitive pre-Golgi enzyme.
Figure 6:
Pulse-chase analysis of
CPE43-expressing AtT-20 cells using various conditions that block
intracellular trafficking. Cells were labeled for 30 min and then
chased for either 0 or 3 h under control conditions at 37
°C(-), at 15 or 20 °C, or at 37 °C with 5 µg/ml
brefeldin A (B), with 100 µM chloroquine (C), or with 10 mM NH
Cl (N) as
indicated. Data shown are representative of four separate
determinations using two different CPE
43-expressing cell lines.
The mobilities and apparent molecular weights of prestained standards
(Bio-Rad) are indicated.
Figure 7:
Western blot analysis of AtT-20 cells
expressing albumin/CPE fusion proteins. Either whole cell homogenates (center panel) or an enriched vesicle/microsomal fraction of
the cells (left and right panels) were separated into
soluble extracts (S), NaCl membrane extracts (M), or NaCl/Triton X-100 membrane
extracts (M
), as described under
``Materials and Methods.'' The mobilities and apparent
molecular weights of prestained standards (Bio-Rad) are indicated.
Similar results were obtained in three separate experiments using two
distinct clones for each construct.
Sorting of the albumin/CPE constructs into the regulated
pathway was investigated by stimulating the cells with forskolin and
then measuring either albumin or CPE immunoreactivity using Western
blot analysis. For all cell lines, the secretion of endogenous CPE in
the presence of forskolin was 200-300% of the secretion of CPE
from unstimulated cells (Fig. 8). This result indicates that the
cell lines expressing the various albumin/CPE constructs possess a
functional regulated secretory pathway. As previously reported, the
forskolin-induced secretion of immunoreactive albumin from the
Alb+51-expressing cells is 135% of the control level of secretion (Fig. 8). In contrast, forskolin does not cause a significant
change in the secretion of immunoreactive albumin from any of the other
cell lines (Fig. 8). This result indicates that the
membrane-binding domain (i.e. the C-terminal 14 residues) is
not sufficient for sorting into the regulated pathway. Also, the
Alb+23 and the Alb+5114 constructs contained overlapping
segments of the C-terminal region of CPE, and the lack of correct
sorting of either construct suggests that the signal for sorting is not
a short linear sequence.
Figure 8:
Relative amounts of albumin or CPE
immunoreactivity secreted from fusion protein-expressing AtT-20 cells.
Cells were stimulated with forskolin for 30 min, and the media were
analyzed on Western blots using albumin- and CPE N-terminal-directed
antisera as described under ``Materials and Methods.'' For
each antisera and for each construct, the data are shown relative to
the level of immunoreactive protein in the control media. Error
bars show standard error of the mean for the cell lines expressing
Alb+51 (n = 7), Alb+23 (n =
6), Alb+14 (n = 6), Alb+5114 (n = 16), and Alb (n = 5). *, differs from
control (p < 0.05) using Student's t test.
The present study used two separate approaches: deletion mutation and fusion protein analysis. These different approaches provide complementary information. The fusion protein analysis reveals whether a region can confer a particular targeting pattern to another protein. A positive result is strong evidence that the particular region performs the targeting function, although it does not reveal whether additional regions participate. However, a negative result does not rule out the possibility that a region is involved with the function since the structure of this region within the fusion protein may not resemble the structure of this region in the native protein. The deletion approach provides information as to the consequences of the elimination of a particular region. A positive result is good evidence for a functional role, although it is hard to rule out a nonspecific change in structure, caused by the deletion, which causes the observed effect. Also, a negative result does not necessarily indicate the lack of a particular function since two or more regions could contribute, and deletion of a single region may not cause a dramatic effect. For these reasons, the combined approach of fusion proteins and deletion analysis provides stronger results than either approach alone.
A major finding of the present study is that the C-terminal 14 residues of CPE are both necessary (from the deletion mutant analysis) and sufficient (from the fusion protein analysis) for the NaCl-independent membrane binding at pH 5.5. This conclusion fits the hypothesis that the C-terminal 14 residues form an amphiphilic helix, which helps anchor the protein to membranes. Adjacent to this putative amphiphilic helix is a highly charged region that was previously proposed to contribute to the salt-dependent membrane binding (i.e. the membrane-1 form)(29) . However, our finding that the mutant with a deletion of 23 C-terminal residues is still able to bind to membranes in a salt-dependent manner argues against this charged region being solely responsible for the ionic binding of CPE to membranes. In addition to CPE, many other proteins within secretory vesicles have been reported to exist in membrane-associated forms even though the protein does not contain a predicted transmembrane-spanning helix(30, 31, 32, 33, 34) . Some of these proteins (such as prohormone convertases 1 and 2) also contain a predicted C-terminal amphiphilic helix, although there is no direct sequence similarity between CPE and these other proteins.
The
C-terminal region of CPE has been highly conserved among species. The
last exon of CPE, which encodes the C-terminal 32 amino acids, is 100%
identical in human, rat, mouse, and bovine CPE and contains only four
conservative substitutions in Anglerfish CPE (Refs. 11, 12, 35, and 36
and GenBank accession no. X61232). Except for gp180(37) , a
duck protein which may be the homologue of the recently discovered
bovine carboxypeptidase D(38) , none of the other members of
the metallocarboxypeptidase gene family have significant homology to
this region of
CPE(39, 40, 41, 42, 43, 44, 45) .
Furthermore, several family members (such as pancreatic
carboxypeptidase A and B) are approximately 130 amino acids shorter
than CPE, with most of the difference in size due to the length of the
C-terminal region(39, 40) . Based on these sequence
comparisons, it was expected that all of the C-terminal deletion
mutants would be enzymatically active. Our finding that CPE lacking 33
or more C-terminal residues is inactive implies that the region between
23 and 33 residues from the C terminus of CPE is necessary for the
production of enzymatically active CPE. Since CPE33 and CPE
43
are unable to bind to a substrate affinity column, it is likely that
they are not correctly folded, although a direct role for the
C-terminal region in substrate binding is also possible. Our finding
that CPE
23 is active and secreted from Sf9 cells is consistent
with a study by Manser et al.(46) , who found that CPE
with a deletion of 26 C-terminal residues was both active and secreted
from C6 cells.
The finding that CPE43 is degraded and is not
secreted from AtT-20 cells is consistent with the possibility that the
C-terminal region is important for proper folding of the protein. The
effect of brefeldin A and other compounds on the degradation of
CPE
43 is similar to the effect of these compounds on the
degradation of other proteins that have been expressed in mammalian
cell lines. Examples include T-cell receptor subunits expressed in
fibroblasts(47) , apolipoprotein B100 in HepG2
cells(48) , the immunoglobulin
chain in CH12
lymphoma-derived cells(49) , and the heavy chain of class 1
major histocompatibility complex in human embryonic lung
fibroblasts(50) . As found for CPE
43, the degradation of
these other proteins is insensitive to lysosomotrophic agents
(chloroquine, weak bases) and to blockers of transport from endoplasmic
reticulum to Golgi (such as brefeldin A), but their degradation is
prevented by incubation at low temperatures.
Protein sorting into the regulated pathway is thought to involve one of two mechanisms. One mechanism requires that the regulated pathway proteins contain specific sequences that are recognized by a ``sortase'' protein that binds to and directs the sorting of these proteins(51) . However, except for recent studies on the sorting of proopiomelanocortin(52) , there is no evidence for a specific sorting signal on regulated pathway proteins, and a previous report describing a ``sortase'' (53) does not appear to be correct(54) . The other potential mechanism involves the aggregation of regulated pathway proteins; these aggregates would then be sorted into the regulated pathway either by a size selection (i.e. if the aggregates are simply too large to fit into constitutive vesicles) or by some other mechanism(28, 55, 56, 57) . The pH-dependent membrane binding of CPE was previously proposed to be a mechanism for aggregates of CPE (and possible co-aggregates of CPE and other proteins) to bind to secretory vesicle membranes, thus driving the sorting process(14) . An important finding of the present study is that the membrane-binding domain of CPE is not sufficient for sorting of a fusion protein (Fig. 8) or necessary for the sorting of CPE (Fig. 4) into the regulated pathway. These results argue against our previous prediction that the membrane binding of CPE would contribute to the sorting of this protein (14, 18) .
In summary, there appear to be three separate functions within the C-terminal region of CPE. The 51 C-terminal amino acids direct the sorting of albumin into the regulated pathway, although with a lower efficiency compared to that of CPE. Since smaller portions of this C-terminal region are not effective in directing the sorting of albumin, this ``sorting'' domain is distinct from the other two regions identified in the present study. Another important region is located 23-33 amino acids from the C terminus; this region may be required for the proper folding of CPE since protein lacking this region is neither active nor secreted from cells. The third domain, which is located within the predicted amphiphilic helix of the C-terminal 14 residues, is involved with the salt-independent binding of CPE to membranes. The presence of multiple domains within the C-terminal region of CPE could account for the high degree of conservation of this region among CPE from different species.