(Received for publication, August 14, 1995)
From the
A major unresolved issue in the field of secretory granule
biogenesis is the extent to which the aggregation of granule content
proteins is responsible for the sorting of regulated from
constitutively secreted proteins. The aggregation process is postulated
to take place in the trans-Golgi network and immature
secretory granules as the proteins encounter mildly acidic pH and high
calcium concentrations. We have developed in vitro assays that
reconstitute the precipitation out of solution of secretory granule
content proteins of anterior pituitary gland and adrenal medulla. In
the assays, all of the major granule content polypeptides form a
precipitate as the pH is titrated below 6.5, and this precipitate can
be recovered in the pellet fraction after centrifugation. Addition of
calcium is required for the aggregation of chromaffin granule content.
In contrast to the proteins secreted by the regulated pathway, the
constitutively secreted proteins IgG, albumin, and angiotensinogen,
when added to the assays, remain predominantly in the supernatant.
Among the individual proteins tested, prolactin is found to aggregate
homophilically under these conditions and can drive the co-aggregation
of other proteins, such as the chromogranins. Soluble forms of granule
membrane proteins, including dopamine -hydroxylase and peptidyl
glycine
-amidating enzyme also co-aggregated with granule content
proteins. The results are consistent with the idea that spontaneous
aggregation of proteins occurring under ionic conditions similar to
those at the sites of granule formation is a property restricted to
those proteins packaged in secretory granules. In addition, the
association of luminal domains of membrane proteins with content
proteins in vitro raises the possibility that analogous
interactions between membrane-bound and content proteins also occur
during granule formation in intact cells.
A hallmark of secretory granules is the presence within them of
condensed cores containing aggregated content proteins. Morphological
studies have indicated that the formation of these cores begins in
dilated extensions of the trans-Golgi network (TGN) ()(1, 2) and continues in the immature
secretory granules/condensing vacuoles (ISG) that are intermediates in
the granule formation process(3) . Progressive condensation of
the content proteins occurs during maturation of the ISGs into mature
secretory granules.
During the process of secretory granule formation, the granule content and membrane proteins are segregated from molecules constitutively transported to the plasma membrane. This segregation is thought to occur both in the TGN and in the ISGs(4, 5, 6) . A major unresolved issue is what role spontaneous protein aggregation has in this sequestration of the granule-specific proteins, particularly for the content proteins. Most of the available information concerning the aggregative properties of granule content proteins has derived from the observed in vitro behavior of the chromo/secretogranins (Cg), which can aggregate at mildly acidic pH in the presence of calcium(4, 7, 8) , conditions thought to resemble those existing in the TGN(9) .
If aggregation mediated sorting is to be a general mechanism for the segregation of regulated from constitutively secretory proteins then several criteria should be met. First, granule content proteins should aggregate under the ionic and pH conditions thought to be present in the TGN even in cells where Cgs are minor components or not present. Second, constitutively secreted proteins should not be able to co-aggregate with the granule content proteins. In previous studies where the aggregation of Cgs was examined either in vitro or in detergent-treated cell extracts, the behavior of the other granule content proteins or well characterized constitutively secreted proteins was not examined systematically(4, 7, 8) .
To test whether aggregation of content proteins can potentially mediate the targeting of proteins to secretory granules, we have developed assays to measure protein aggregation in vitro. In these assays, granule content proteins undergo pH-dependent aggregation in a process that excludes constitutively secreted proteins. Some of the content proteins aggregate at low pH when assayed individually in the absence of the other proteins, while others require an aggregating partner to precipitate. Soluble forms of membrane-associated proteins also undergo co-aggregation with the content proteins under conditions where they do not self-associate. The results are consistent with a potential role for protein aggregation in the segregation of both secretory and membrane proteins to storage granules.
Figure 1:
Pituitary granule content proteins
aggregate when the pH is reduced. Secretory granule content (2 mg/ml)
was prepared from bovine pituitary gland and equilibrated with 5 mM HEPES, 10 mM MES, pH 7.5. The pH was reduced as indicated
and the pellet and supernatant fractions recovered as described under
``Materials and Methods.'' The protein in both fractions was
measured and is plotted as the mean and standard error of the protein
in the pellet as a percentage of the total (pellet + supernatant).
150 mM KCl and 10 mM CaCl were added
where indicated. In both cases, aggregation began by
pH 6.5 and
increased as the pH was reduced further.
When
analyzed by SDS-PAGE, all of the major proteins in the granule content
were observed to precipitate as the pH was reduced (Fig. 2).
This effect was also reversed when the pH was retitrated back to 7.5 (Fig. 2, right panel). The two most prominent proteins
were identified as Prl and GH based on the migration positions of the
purified proteins and immunoblotting using specific antibodies (data
not shown). Thus, these granules, as is typical for purified pituitary
granules(14) , consist primarily of those from somatotrophs and
mammotrophs, the most abundant cells in the pituitary. However, other
known pituitary proteins could be identified by immunoblot analysis
using specific antibodies. A pH-dependent aggregation was observed for
LH, FSH, and chromogranin A (CgA) (Fig. 3). In addition, a
37-kDa protein reacting with anti-ACTH antibodies underwent
pH-dependent aggregation as well (Fig. 3). This protein
co-migrated on polyacrylamide gels with authentic porcine POMC. In
general, all of these pituitary proteins aggregated as well as did GH
and Prl in the same experiments.
Figure 2: The major pituitary secretory granule content proteins aggregate at mildly acidic pH. Granule content from bovine pituitary gland was utilized in the standard assay (see Fig. 1). Left panel, the pH was reduced to 6.2 or 5.5. The entire pellet (P) (lanes c, f, and i), 20% of the initial sample (I) (lanes a, d, and g), and 20% of the remaining supernatant (S) after centrifugation (lanes b, e, and h) were then subjected to SDS-PAGE and the gel was stained with Coomassie Blue. The migration positions of the indicated proteins, prolactin (Prl) and growth hormone (GH), were determined from those of the corresponding purified proteins and confirmed by immunoblotting using appropriate antibodies. Note the greatly increased appearance of the major proteins in the pellets after pH reduction (lanes f and i). For Prl, the percent aggregation at pH 7.5, 6.2, and 5.5 was 4, 14, and 21%, respectively, and the corresponding values for GH were 7, 35 and 44%. The migration positions of prestained marker proteins are indicated at the right margin. Presented is one of three similar experiments. Right panel, the reversibility of the pH-dependent aggregation was measured after reduction of the pH to 5.5, incubation for 30 min, and retitration back to pH 7.5. Control samples were maintained for 30 min at pH 7.5 and 5.5, respectively. The amounts of Prl and GH in the pellets and supernatants were measured by densitometric scanning of digitalized images of the Coomassie-stained gels and are presented as the overall mean and standard error of three separate experiments.
Figure 3:
The major pituitary hormones precipitate
at mildly acidic pH. Granule content (3 mg/ml) prepared from bovine
pituitary glands was subjected to the aggregation assay at either pH
7.5 (lanes a and b) or 5.6-5.8 (lanes
c and d). Pellet (P; lanes a and c) and
30% of the supernatant (S; lanes b and d) fractions
were subjected to SDS-PAGE and transfer to nitrocellulose. The
indicated proteins were identified by immunoblotting using specific
antibodies followed by the ECL procedure (in the case of POMC) or by
I-protein A and autoradiography. All of the content
proteins underwent pH-dependent aggregation, including FSH (4% at pH
7.5 versus 50% at pH 5.6), LH (3% at pH 7.5 versus 24% at pH 5.6), and chromogranin A (CgA; 2% at pH 7.5 versus 48% at pH 5.6). As expected, GH, included as a control, was found
predominantly in the pellet fraction at low pH (6% at pH 7.5 versus 42% at 5.6).
37-kDa POMC, from a separate experiment, also
underwent pH-dependent aggregation (6% at pH 7.5 versus 39% at
pH 5.6 after normalization for differences in Prl/GH aggregation). In
each case, the results represent one of two similar
experiments.
To extend these results to other
types of secretory granule content, the same assay was conducted with
purified adrenal chromaffin granule content proteins, consisting almost
entirely (>90%) of the Cgs. Unlike what was observed using the
pituitary granule content, the chromaffin granule proteins when used at
2 mg/ml did not undergo pH-dependent aggregation (Fig. 4).
Aggregation did occur, however, when calcium was added (Fig. 4)
and this effect was greater as the pH was reduced. The aggregation was
also reversible. In three separate experiments where the pH was lowered
to 5.6 in the presence of 20 mM CaCl
and, after a
30-min incubation, titrated back to pH 7.5, the recovery of CgA in the
pellets was reduced to the level of the pH 7.5 control (data not
shown). The concentration of calcium required for maximal aggregation
was
20 mM (not shown), higher than the 10 mM believed to be present in the TGN (4) but lower than the
40 mM present in mature chromaffin granules(15) . The
high calcium requirement is likely to be due to the relatively low
concentrations of protein employed, far lower than that of mature
granules (>100 mg/ml). It has been already established that
aggregation of CgA in vitro requires less calcium as the
protein concentration is increased(8) . Thus, the overall
aggregation of adrenal content is consistent with that expected for the
chromogranins themselves.
Figure 4:
Adrenal chromaffin granule content
proteins aggregate in the presence of calcium. Granule content (2
mg/ml) was obtained from bovine adrenal medulla as described under
``Materials and Methods.'' This content was subjected to the
standard aggregation assay in the presence or absence of 40 mM CaCl. Total protein in the pellets was measured and is
plotted as the mean ± S.E. Little precipitation of the
chromaffin granule content was observed in the absence of
CaCl
, or in the presence of 150 mM KCl and 10
mM CaCl
(not shown). However, when high
concentrations of CaCl
were used, aggregation did occur,
with increased aggregation at lower pH.
A similar set of experiments was conducted using chromaffin granule content proteins induced to aggregate in the presence of calcium. As shown in Fig. 5, none of the three constitutive markers, IgG, angiotensinogen, or BSA, co-aggregated effectively (<5%) with the adrenal content proteins, whereas CgA, the major content protein, did precipitate well (30-39% at pH 5.6). Taken together, these data show that protein-protein interactions occurring during protein aggregation are specific and that constitutively secreted proteins aggregate less efficiently than granule content proteins, at least in vitro.
Figure 5:
Constitutively secreted proteins do not
co-aggregate with adrenal chromaffin granule content proteins. Granule
content (2 mg/ml) from adrenal medulla was subjected to the aggregation
assay at the indicated pH values in the presence of 25 mM CaCl after addition of
I-labeled
angiotensinogen (Ang) and unlabeled rabbit IgG (panel
A) or
I-labeled BSA (panel B). Pellets (P; lanes a and c) and 20% of the supernatants (S; lanes b and d) were analyzed after SDS-PAGE (no
reducing agent was added) by autoradiography on a PhosphorImager (panels A and B, top) or by Coomassie Blue
staining of the gel (panels A and B, bottom). These markers of the constitutive pathway did not
co-precipitate significantly with granule content proteins in the
presence of CaCl
. At pH 5.6, 5% of angiotensinogen, 4% of
IgG, and 0% of BSA were detected in the pellet fractions as compared to
30% (panel A) and 39% (panel B) for CgA. For each
constitutive protein, the results represent one of two similar
experiments.
Figure 6:
IGF-1 also co-aggregates with granule
content proteins. I-Labeled IGF-1 (10,000 cpm) was added
to the aggregation assays together with content proteins from pituitary (panel A) or adrenal medulla (heat-treated; panel B).
The assays were conducted as described in the legends to Fig. 1and Fig. 4for pituitary and adrenal, respectively,
at the pH values indicated with 40 mM calcium in the adrenal
samples at pH 6.4 and 5.9. Total protein in the pellet and supernatant
fractions was measured using a BCA assay while
I was
determined by counting on a
-counter. The mean and standard error
of triplicate samples are presented after correcting for free
I (10%). IGF-1 co-aggregated with both the pituitary and
adrenal content proteins at low pH.
Figure 7:
Prolactin, but not other pituitary granule
content proteins, homotypically self-aggregates at reduced pH. Bovine
Prl, human GH, and ovine LH were subjected to the standard aggregation
assay (see Fig. 1). 150 mM KCl and 10 mM CaCl was included in one set of the samples as
indicated. Total protein in the supernatants and pellets was measured
in triplicate samples and is plotted as the mean ± S.E., which
in most cases was too small to be represented on the graph. Prolactin
underwent a strong self-aggregation as the pH was reduced <6.5. This
aggregation was not affected by the addition of salt. GH did not
aggregate well, although some pH-dependent aggregation was detected in
the absence of salt (up to 12% in some experiments). By contrast, LH
and the constitutively secreted proteins, IgG and BSA, did not
self-aggregate detectably under these
conditions.
CgA, GH, and LH, which did not self-associate under these conditions, did undergo substantial aggregation in the pituitary content itself (Fig. 2), implying that their aggregation, at least in vitro, is dependent on their interaction with other proteins in the mixture, perhaps self-aggregating proteins like prolactin. To investigate these types of protein-protein interactions, Cgs, in the form of adrenal content, and purified LH were added to Prl and the pH reduced so as to initiate Prl aggregation. Prl itself was capable of driving the aggregation of the major chromaffin granule proteins, including CgA (data not shown), suggesting that these proteins could potentially interact during granule formation. LH, on the other hand, remained mostly if not entirely soluble when mixed with Prl alone in the assay (Fig. 8A), illustrating the specificity of the protein-protein interactions that occur at low pH. The aggregation of LH could be induced, however, by including LH together with adrenal content and Prl. In other words, the aggregation of LH was dependent upon its interaction with proteins in the adrenal content, most likely chromogranins, which in turn can associate with the aggregating Prl. Addition of the same amount of LH to assays conducted with pituitary extracts led to a similar level of aggregation of LH (not shown). To analyze directly the potential interaction of LH with adrenal content proteins, this hormone was added to adrenal content and Cg aggregation was induced in the presence of calcium. As depicted in Fig. 8B, LH was also induced to co-precipitate together with the Cgs in the presence of calcium, whereas neither LH nor the Cgs aggregated in its absence.
Figure 8:
Co-aggregation of luteinizing hormone with
adrenal components occurs in the presence of prolactin or calcium. Panel A, aggregation assays were performed as described in the
legend to Fig. 2at pH 5.8 using 2 mg/ml LH alone (lanes a and b), Prl alone (lanes c and d), Prl
together with LH (lanes e and f), Prl, LH together
with adrenal content (lanes g and h), and LH together
with pituitary content (lanes i and j). Pellet (P; lanes a, c, e, g, and i) and 20% of the
supernatant (S; lanes b, d, f, h, and j) fractions
were subjected to SDS-PAGE in the absence of reducing agent (lanes
g and h are from a separate gel). Prl by itself failed to
induce the aggregation of LH (lanes e and f).
However, LH did sediment when added together to adrenal content in the
presence of Prl (lanes g and h). CgA also sedimented
when Prl was mixed with adrenal content under these conditions whether
LH was present (lanes g and h) or not (not shown). Panel B, the aggregation assays were performed as in Fig. 5. LH (lanes e and f), adrenal content (lanes c and d, g and h), and a mixture of
both (lanes c and d, i and j) were incubated
in the absence (lanes a-d) or presence (lanes e-j) of
25 mM CaCl. In the absence of CaCl
both LH and CgA failed to aggregate. In the presence of calcium,
however, LH co-precipitated with the aggregating adrenal proteins,
including CgA. The percent aggregation (% Aggreg.) obtained
for each of the indicated proteins is listed at the bottom. The results
indicate that LH can interact directly with chromaffin content proteins
at low pH. It will sediment when the aggregation of Cgs is induced
either by calcium or by Prl. Presented is one of two similar
experiments.
These results are in agreement with observations made in vivo concerning the packaging of these proteins in secretory granules. Cgs and Prl are packaged together in the granules of pituitary-derived cell lines in culture and in rat mammotrophs, while LH and chromogranins appear in the same granules in pituitary gonadotrophs(20, 21, 22) .
As can
be seen in Fig. 9, the endogenous soluble form of DBH in adrenal
extracts aggregated only modestly when the chromaffin granule content
was titrated to pH 6.0 in the absence of calcium. When adrenal
content was added to pituitary granule extracts prior to the assay, the
major content proteins, including CgA, underwent aggregation at low pH
in the absence of calcium (Fig. 9A, lanes k and l). In this case, DBH was also prominent in the pellet
fractions. This result indicates that under conditions where DBH does
not self-aggregate well, it can interact with other granule content
proteins. When adrenal content aggregation was induced by addition of
calcium to the assay, DBH sedimented (Fig. 9B).
Although homotypic self-aggregation of DBH in the presence of calcium
cannot be ruled out, the data suggest that DBH is in fact
co-aggregating with the Cgs in the chromaffin extracts.
Figure 9:
The soluble form of the chromaffin granule
membrane protein dopamine--hydroxylase aggregates together with
pituitary and adrenal content proteins. Panel A, aggregation
assays were performed exactly as described in the legend to Fig. 1at the indicated pH values using either 2 mg/ml adrenal
content alone (lanes a-d), 2 mg/ml pituitary content (lanes e-h), or 0.5 mg/ml adrenal mixed with 2 mg/ml
pituitary content protein (lanes i-l). Pellets (P;
lanes a, c, e, g, i, and k) and 30% of the supernatant (S; lanes b, d, f, h, j, and l) fractions were
subjected to SDS-PAGE in the absence of reducing agent. The samples
were transfered to nitrocellulose and stained with Ponceau S (bottom). The nitrocellulose was probed with rabbit antibodies
to bovine DBH and
I-protein A and the radioactive bands
detected on a PhosphorImager (top). The adrenal content
proteins (including CgA) do not aggregate at low pH in the absence of
calcium (lanes c and d) but do aggregate when mixed
with the pituitary extract (lanes k and l). Top, the 150-kDa adrenal DBH dimer precipitated in the
pituitary extract in a pH-dependent fashion but only modestly in the
adrenal content itself (compare top, lanes c and d, k, and l). The migration positions of molecular mass standards are
noted at the right. Panel B, adrenal chromaffin
granule content proteins were subjected to the aggregation assay at pH
7.5 (lanes a and b) or 5.8 (lanes c and d) in the presence of 25 mM CaCl
as
described in the legend to Fig. 6. Samples were analyzed as in panel A but using the ECL immunoblotting procedure. DBH
aggregated at low pH in the presence of calcium as did CgA. Thus, DBH
can co-aggregate with the pituitary content at low pH under conditions
where it self-aggregates poorly. It also aggregates in the adrenal
extracts in the presence of calcium, conditions that promote
self-aggregation of the chromogranins. The percentage of the indicated
proteins in the pellet fractions (% Aggreg.) is listed at the bottom of each panel. Presented is one of two similar
experiments.
PAM, in
contrast to DBH, could undergo a vigorous pH-dependent aggregation in
the absence of other granule components. A significant fraction of
purified PAM3 (a soluble isoform consisting of virtually the entire
luminal domain) was observed to sediment after reduction of the pH to
5.8 regardless of whether the assay was conducted in the presence of
pituitary or adrenal granule content, or nonaggregating proteins such
as hemoglobin (Fig. 10) or BSA (not shown). However, PAM
self-aggregation was inhibited by CaCl (Fig. 10, lanes e and f). This inhibition was specific for
calcium and not observed in the presence of the same molar equivalents
of potassium ion (data not shown). When added to the pituitary or
adrenal assays in the presence of calcium, PAM did sediment effectively (Fig. 10, lanes k and l), indicating that it
can interact with other granule content proteins.
Figure 10:
A soluble form of peptidyl glycine
-amidating monooxygenase aggregates at low pH both with itself and
with other granule content proteins. Panel A, purified PAM3
(1-2 µg/ml), a soluble form of the protein (see text), was
added to either 1.5 mg/ml hemoglobin (globin; lanes a-f) or
pituitary content (lanes g-l) and the standard aggregation
assays conducted at pH 7.5 (lanes a and b, g and h) or 5.8 (lanes c-f and i-l) as described
under ``Materials and Methods.'' 25 mM CaCl
was included in one set of samples in each case (lanes e and f, k and l). Pellet (P; lanes a, c, e,
g, i, and k) and 20% of the supernatant (S; lanes b,
d, f, h, j, and l) samples were analyzed by SDS-PAGE on a
12% gel followed by transfer to nitrocellulose and staining with
Ponceau S (bottom) and immunoblotting using the ECL procedure (top). At low pH, PAM underwent a strong homophilic
aggregation (35% at pH 5.8) in the presence of globin, which did not
itself precipitate (3%, lanes c and d). In comparison
to the samples containing globin, PAM aggregated only slightly better
when incubated with pituitary content proteins at pH 5.8 (41%, lanes i and j). However, in the presence of calcium,
the self-aggregation of PAM was reduced (13%, lanes e and f) and under these conditions PAM still precipitated strongly
in the pituitary samples (57%, lanes k and l). By
comparison, the aggregation of Prl and GH together at pH 5.8 was 19% in
the absence and 17% in the presence of calcium. Panel B,
purified PAM3 was added to 1.5 mg/ml adrenal content proteins or globin
and the assay was performed as described in the legend to Fig. 9. Samples were analyzed as described in panel A using a 10% polyacrylamide gel. In the absence of calcium at pH
5.8, 23% of PAM aggregated in the globin sample and 32% was found in
the pellets of the adrenal samples. In the presence of calcium, PAM
aggregated much better when mixed with adrenal content (55%, lanes
k and l) than with globin (3%, lanes e and f). CgA recovered in the pellet was only 2% in the absence of
calcium but 33% in its presence. Endogenous PAM did not contribute to
the results as it was not readily detectable in immunoblots of
pituitary and adrenal content under these conditions (not shown). Thus,
PAM is capable of associating with granule content proteins at low pH.
The results represent one of two similar
experiments.
Proteins traversing the TGN and ISG, the sites of sorting of constitutive from regulated proteins, are exposed to mildly acidic pH and high concentrations of calcium ions(9, 28, 29, 30, 31) . The maintenance of an acidic pH in the TGN and ISG is known to be important for granule biogenesis. Treatment of secretory cells with lysosomotropic amines to dissipate pH gradients blocks maturation of secretory granules, as has been shown in both pancreatic acinar cells (32, 33) and islet cells (34) . Moreover, incubation of AtT20 cells with high concentrations of chloroquine promotes the diversion of POMC from the regulated to the constitutive secretory pathway(35) . The results presented here showing spontaneous aggregation of granule proteins in vitro support the idea that the ionic milieu of the TGN and ISG are prime contributors to the selective targeting of proteins to granules and to the condensation of secretory material that is observed to occur by electron microscopy(1, 2) .
The conditions promoting the precipitation are somewhat different in each type of granule content. In pituitary extracts, calcium ion is not required. All of the major pituitary content proteins tested (CgA, prolactin, growth hormone, POMC, FSH, and LH) precipitate. An important finding was that the constitutively secreted proteins IgG, angiotensinogen, and BSA do not co-precipitate with the pituitary content proteins. Moreover, as demonstrated previously(12) , a secretory form of the pancreatic membrane protein GP2, which is not packaged in granules in endocrine cells, does not co-aggregate with pituitary content proteins. Thus, the packaging of content proteins in storage granules correlates well with the behavior of the regulated and constitutive secretory proteins in the aggregation assays in vitro.
Adrenal content proteins, consisting primarily of the
Cgs(15) , do not precipitate under the same ionic conditions as
those of the anterior pituitary gland. Their precipitation requires
calcium, as does the major constituents of these granules, the
chromogranins. Further evidence that it is Cg aggregation being
measured in our assay comes from experiments conducted using a
preparation that consists almost entirely of soluble Cgs, obtained
after chromaffin granule extracts were heated to 100 °C and
centrifuged(37) . The same level of
Ca-dependent aggregation of CgA was observed (e.g.Fig. 6). However, a recent study has provided
evidence that Ca
is not required for the
incorporation of proteins into ISGs(38) . The transport of
secretogranin II (CgC) from the TGN to ISGs in permeabilized PC12 cells
was found to be insensitive to chelation of cytosolic Ca
and to the addition of the Ca
-H
ionophore A23187. This suggests that either some protein sorting
in the TGN can occur without aggregation of the Cgs or that sorting in
this system largely occurs later, after formation of ISGs themselves,
where Ca
could still play a role. In favor of the
latter explanation is the appearance in the ISGs, with a sorting index
comparable to that of secretogranin II, of the constitutively secreted
protein, heparan sulfate proteoglycan in this study(38) . It is
also possible, however, that interaction of the granule content
proteins with membrane proteins, a process that our data using DBH and
PAM3 suggest may occur at mildly acidic pH even in the absence of
calcium, is sufficient to insure some preferential sorting of these
proteins in the TGN. In favor of this view, CgA and CgB have been
reported to bind to chromaffin granule membranes at low pH in the
absence of Ca
(39, 40) .
Further
support for the notion that interactions between granule content
proteins themselves are involved in selective packaging of proteins in
granules has been inferred from the properties of the Cgs. Cgs have
been shown to self-aggregate when calcium is
present(7, 8) . Calcium and low pH can also stabilize
the Cg-containing aggregates formed in PC12 and GH4 cells(4) .
At neutral pH in the absence of Ca, the
S O
-labeled Cgs, while still in the TGN, were
extracted from detergent-permeabilized cells but at pH 6.4 in the
presence of 10 mM Ca
, they remained
sedimentable. The behavior of content proteins other than Cgs was not
reported(4) . More importantly, the use of BiP/GRP78, protein
disulfide isomerase, and free glycosaminoglycans as markers of the
constitutive secretory pathway that did not efficiently co-aggregate
with Cgs is problematic. BiP and protein disulfide isomerase are
normally resident endoplasmic reticulum proteins and have not been
shown to exit endocrine secretory cells via the constitutive pathway.
Glycosaminoglycans, in contrast to bona fide constitutively secreted
proteins, are exported from the cell by both constitutive and regulated
pathways(41) . Similarly, Gorr et al.(7) reported that ovalbumin did not co-aggregate with CgA.
However, ovalbumin has not been expressed in endocrine or
neuroendocrine secretory cells; hence it is not known whether it is a
regulated or constitutive secretory protein. Thus it is difficult to
directly relate aggregation experiments using ovalbumin, BiP, protein
disulfide isomerase, or glycosaminoglycan chains to the general problem
of protein sorting to secretory granules. It should be noted that the
aggregation of pancreatic zymogen granule content proteins in vitro at low pH has been previously reported(42) . IgG added to
the assay was also shown not to co-aggregate with amylase and other
pancreatic zymogens.