1 Department of Biological and Biophysical Sciences, University of Louisville Health Sciences Center, Louisville, Kentucky 40292; and 2 Division of Endocrinology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461
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ABSTRACT |
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For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH4C1 cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, in GH4C1 cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH4C1 cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.
proinsulin; insulin; chromogranin
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INTRODUCTION |
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CONCEPTS CONTINUE TO EVOLVE concerning the storage of secretory granule proteins in the regulated exocytotic pathway of endocrine cells. A decade or more ago, the sole view was that regulated secretory proteins required specific sorting signals for entry into forming storage granules (i.e., active sorting), whereas soluble secretory proteins lacking this specific sorting information entered the constitutive secretory pathway by default (10, 50, 51). More recently, however, a sorting-by-retention model has been suggested in which several soluble proteins that are not stored in secretory granules can nevertheless enter immature granules, only to be later sorted out during the granule maturation process (13, 39, 43). Thus, whereas regulated secretory proteins tend to be retained (i.e., stored via passive sorting) during granule maturation (41), some fraction of nonretained proteins may be released via the constitutive-like secretory pathway (3).
In both models of protein sorting (which should not be viewed as mutually exclusive), regulated secretory proteins contain specific domains that are required for intragranular storage. Thus regulated secretory proteins may be removed from the soluble phase by binding to specific proteins or lipids in the forming granule membrane (21, 49), by condensation with each other (14, 23, 29, 31), or both (17, 25, 45, 55, 56, 72). These reactions are thought to depend on the acidic pH of the trans-Golgi network (TGN) and immature secretory granules (69), and condensation is additionally thought to be promoted by millimolar concentrations of calcium in these compartments (11, 30).
In recent years, several groups have argued that specific disulfide loop structures, which overlap with a postulated common structural motif involved in sorting (28, 65), may play a role in the efficient granule storage of a subset of regulated secretory proteins. In particular, methods leading to disruption of a disulfide in proopiomelanocortin (POMC) (19) or the disulfide in chromogranin B (CgB) (16) [which may play a role in chromogranin homodimerization (67)] have been linked to an increase in unstimulated (constitutive) secretion of these (newly synthesized) proteins. In the case of POMC, sorting has been proposed to be accomplished by binding of the sorting motif to a membrane-associated form of carboxypeptidase E (CPE), acting as a sorting receptor (21). Initial evidence also suggested a role for CPE in the sorting of chromogranin A (CgA) (21); however, very recent evidence has led to a revision of this view (20, 52, 53).
Although many secretory proteins contain disulfide bonds, the evidence
for a role of disulfide bonds in Golgi/post-Golgi sorting remains
limited, and the requirement for a disulfide loop in the sorting of
regulated secretory proteins (25, 40), separate from a role in overall
protein folding, has been questioned (2, 58). To further examine the
potential role of disulfide bonds in the sorting of regulated secretory
proteins, we set out to investigate the events that follow
dithiothreitol (DTT)-mediated reduction of proinsulin (which contains
three disulfide bonds) in -cells of live pancreatic islets and
compared them to the effects of DTT treatment on CgB (which contains
one disulfide bond) in live PC-12 and
GH4C1
cells. Interestingly, on treatment with DTT, neither reduced proinsulin
nor insulin exhibits increased release via constitutive or
constitutive-like secretory pathways. By contrast, in PC-12 cells,
unstimulated secretion of newly synthesized CgB is significantly
increased by DTT-mediated reduction. However, in
GH4C1
cells, the unstimulated secretion of newly made CgB is not
significantly increased by DTT-mediated reduction, and, in transiently
transfected
GH4C1
cells, neither the absence nor presence of the conserved disulfide
affects the calcium/low-pH-induced multimeric aggregation of CgA or the
stimulus-dependent secretion of this granule protein. Evidently, the
effects of disulfide integrity on secretory protein trafficking are
protein and cell-type dependent, indicating the potential for numerous
possible protein interactions that may influence the final outcome of
protein targeting in the distal secretory pathway.
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METHODS |
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Materials. Collagenase was from Worthington (Freehold, NJ); Hypaque (sodium diatrizoate), human serum albumin, BSA, soybean trypsin inhibitor, DTT, iodoacetamide (IAA), and phorbol 12-myristate 13-acetate (PMA) were from Sigma (St. Louis, MO); BAY K 8644 was from Calbiochem-Novabiochem (La Jolla, CA); calf serum was from GIBCO (Long Island, NY); gelding equine serum and FCS were from Hyclone (Logan, UT); antibiotic-antimycotic solutions were from either GIBCO or Sigma; [35S]methionine/cysteine (Express) was from NEN (New Bedford, MA); [3H]leucine was from ICN; [14C]leucine was from Amersham; DMEM, both deficient and complete, were either from Sigma or from Life Technologies (Grand Island, NY), while mouse epidermal growth factor (EGF) and Lipofectamine were from Life Technologies. Rabbit antisera to the amino terminus and carboxy terminus of CPE were graciously provided by Dr. L. Fricker (Albert Einstein College of Medicine, Bronx, NY), while the antiserum to bovine CgA was generously provided by Dr. D. V. Cohn (Univ. of Louisville, Louisville, KY).
Isolation and pulse-chase studies of mouse pancreatic islets. Islets from CD-1 mice were isolated by pancreatic ductal perfusion with collagenase, flotation on a Hypaque gradient, picking of individual islets, and recovery overnight in DMEM containing 10% calf serum plus 1% penicillin-streptomycin. Islets were washed twice with methionine-free, cysteine-free DMEM, before pulse labeling, for times ranging up to 12 min at 37°C in the same deficient medium that contained ~300 µCi of [35S]methionine plus cysteine. Labeling of islets in batch was performed as described in Ref. 34. When appropriate, labeled proinsulin accumulation in the TGN was accomplished with chase incubation of 2 h at 19.5°C. When appropriate, labeled proinsulin accumulation in the immature secretory granules was accomplished by a 105-min chase at 37°C. Appropriately chased islets were divided into equal numbers for different treatment conditions. Preliminary experiments confirmed that, with the numbers of islets used (800 per experiment), equally divided aliquots contained roughly equal amounts of total protein. The islet aliquots were then either mock treated or incubated in chase medium containing DTT at a final concentration of 20 mM. Unlike lower concentrations of DTT necessary for protein reduction in the endoplasmic reticulum, this concentration is required to achieve adequate reduction of proinsulin in the TGN of pancreatic islets (35). For islets in which labeled proinsulin was accumulated in the TGN, the DTT treatment was performed first for 10 min at 19.5°C, and the islets were then further warmed for 10 min to 37°C before continuation of the experiment (the warm-up was included because intracellular DTT-mediated reduction is less effective at 19.5°C). For islets in which labeled proinsulin chased to immature secretory granules, the DTT treatment was simply performed for 10 min at 37°C.
After the transient treatment, washout of DTT and subsequent chase were
performed as described in the figure legends. All labeling and chase
media also contained 0.5 mg/ml human serum albumin as well as 0.005%
soybean trypsin inhibitor. For the purposes of this report, chase
medium collected under "unstimulated" conditions was simply
complete DMEM, which contains 5.5 mM glucose. In some cases, samples
were stimulated with a -cell secretagogue cocktail that included 22 mM glucose, 1 mM tolbutamide, 1 mM IBMX, and 100 nM PMA.
Stimulated secretion for 1 h from isolated mouse islets generally achieves 15-20% of total insulin measured by RIA.
At the conclusion of all chase incubations, labeled islets were rapidly washed in ice-cold Tris-buffered saline containing 50 mM IAA and were lysed by bath sonication in the same buffer including 1% Triton X-100, 25 mM IAA, and an anti-protease cocktail of aprotinin (1 mU/ml), leupeptin (0.1 mM), pepstatin (10 mM), EDTA (5 mM), and diisopropyl fluorophosphate (1 mM). The islet lysate was spun briefly in a microcentrifuge to remove debris, and proportionate volumes of the clarified lysate and secretions were analyzed by SDS-PAGE.
PC-12 cell culture and pulse-chase experiments. PC-12 cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% FCS and penicillin/streptomycin in a humidified atmosphere at 37°C and 5% CO2. For each experiment one-half of the cells from near-confluent 75-cm2 flasks were plated in six-well cluster plates. The cells were maintained in culture for an additional 1-2 days before experiments.
For secretion experiments, PC-12 cells were preincubated for at least 45 min with labeling medium (0.3 mM CaCl2, 3.82 mM KCl, 0.62 mM MgCl2, 127 mM NaCl, 1.1 g/l glucose, 1.0 mM sodium pyruvate, 2.05 mM glutamine, 15 mM HEPES, pH 7.4). The preincubation medium was discarded and the cells were labeled with 1 ml per well of labeling medium containing 100 µCi [3H]leucine or 8 or 50 µCi of [14C]leucine for 5 min at 37°C. The cells were briefly rinsed and then chased for 1 h at 37°C in complete DMEM to which had been added 5 mM DTT from a freshly prepared 20× stock solution or an equivalent volume of water.
At the conclusion of the chase, 10 mM N-ethylmaleimide (NEM) was added to the collected chase media, which were then spun briefly in a microcentrifuge to remove debris. The cells were briefly rinsed in a buffer containing 20 mM NEM and were lysed by freeze-thawing in solubilization buffer (20 mM Tris · HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.3% Tween 20, 1 mM phenylmethylsulfonyl fluoride) to which 10 mM NEM was then added; these extracts were then spun briefly in a microcentrifuge to remove cellular debris. In some experiments, to make conditions for protein recovery identical for all samples, radiolabeled secretion was subsequently mixed with unlabeled cell extracts from a control plate that had been incubated in parallel, while radiolabeled cell extracts were mixed with unlabeled medium from the control plate. In experiments in which the mixing was not performed, the media samples were adjusted to 5 mM EDTA and 0.3% Tween 20 before further analysis. At this time, radiolabeled cell extracts and medium samples were heated for 30 min at 100°C, followed by centrifugation for 20 min at 16,000 g. The heat-stable supernatant fractions were mixed with TCA (10% final concentration), incubated 20 min on ice, and spun for 20 min at 16,000 g. In all cases, visible pellets were obtained without the addition of carrier protein. The TCA precipitates were finally washed with 80% acetone before resuspension in solubilization buffer.
GH4C1 cell culture and pulse-chase experiments. GH4C1 cell culture and hormone treatment (1 nM estradiol, 300 nM insulin, and 10 nM EGF) were performed as previously described (26).
For secretion experiments, the cells were plated at a concentration of 106 cells/well in six-well cluster plates and were hormone treated for 4 days. The cells were rinsed with Krebs-Ringer-HEPES (KRH) buffer and preincubated in fresh KRH buffer for 75 min at 37°C. The cells were then labeled for 5 min in 1 ml/well of KRH containing 100 µCi [3H]leucine (or, in some cases, 250 µCi [3H]proline). The labeling media were discarded and the cells were briefly rinsed and then chased for 1 h at 37°C in KRH containing 2 mM leucine to which had been added 5 mM DTT from a freshly prepared 20× stock solution or an equivalent volume of water. For analysis of stimulated secretion following the first 60-min chase period (see Fig. 8B), the cells were rinsed with KRH containing 2 mM leucine and then chased for 30 min in fresh KRH with leucine plus either 50 mM NaCl, 100 nM 4Transient expression of CgA in GH4C1 or PC-12 cells. The wild-type CgA (wt-CgA) cDNA (1) was subcloned into pcDNA3 (Invitrogen, San Diego CA). A mutant form of CgA ("CgA-CC") lacking the amino-terminal disulfide bond was prepared by PCR-based mutagenesis, using separate primers (5'-catggtacccttggagagtgtgtcagagatgacctcgacgatagacttcat-3' and 5'-tacggtacccccatgccagtcagcaaggagtcttttgag-3') designed to mutate the cysteine residues at positions 17 and 38, respectively, to serine residues. Primers for the 5'- and 3'-untranslated regions were also designed. A new Kpn I restriction site was incorporated in the coding sequence between the two mutation sites. The amino- and carboxy-terminal PCR products were cloned into pcDNA3 and joined in frame at the new Kpn I site. Incorporation of the Kpn I site necessitated two conservative changes at Pro29Gly and Ser30Thr. The identity of the mutated CgA was confirmed by DNA sequencing.
GH4C1 or PC-12 cells were plated at a density of 5 × 104 cells/cm2 in six-well plates in complete medium (described above). Two days later, the cells were transfected using 8 µl of Lipofectamine with 1 µg DNA/well (GH4C1 cells) or 8 µl of PerFect Lipid (InVitrogen) with 2 µg DNA/well (PC-12 cells). The medium was changed the following day, and stimulated secretion (in response to a secretagogue cocktail described above for GH4C1 cells or 2 mM BaCl2 for PC-12 cells) was assayed on the second day after transfection. The medium containing secreted CgA was heated at 100°C for 10 min and centrifuged to remove denatured proteins. For GH4C1 cells, 200 µl of the heat-stable supernatant (containing CgA) were then assayed quantitatively by dot immunoblotting essentially as previously described for secretogranin II (SgII) and prolactin (26). Control samples consisted of the heat-stable supernatant from media bathing cells that had been transfected with pcDNA3 not containing the CgA insert. The samples were applied to nitrocellulose membranes in a 96-well dot-blot apparatus (Bio-Rad Laboratories, Hercules CA). These membranes were washed in 20 mM Tris · HCl, pH 7.4, 500 mM NaCl, 0.05% Tween 20 (TTBS). For PC-12 cells, the medium was precipitated with TCA and resolved by SDS-PAGE followed by transfer to polyvinylidene difluoride membranes. For both dot blotting and Western blotting, the blots were then blocked for 5 min with 2% Tween 20 in TTBS before incubating overnight with antiserum to bovine CgA (a kind gift from Dr. David V. Cohn, Univ. of Louisville) diluted 1:10,000 in TTBS containing 10 mg/ml BSA. The blots were washed with TTBS and then incubated for 2 h with goat anti-rabbit IgG conjugated to horseradish peroxidase (1:2,000). The blots were developed with chemiluminescent substrate and recorded on Biomax film. Exposure times were selected to ensure linearity of the assay. The images were quantitated by densitometric scanning using a BioImage image analyzer (BioImage, Ann Arbor, MI) as previously described (26). In some experiments the control samples yielded a modest background signal that was subtracted to obtain the final measured values. In vitro multimeric aggregation assay for CgA. Media containing secreted wt-CgA or CgA-CC were heated and centrifuged to remove denatured proteins. The heat-stable supernatants (containing granins) were adjusted to pH 7.4 with 10 mM HEPES and concentrated fivefold by centrifugation through a Centricon-30 filter unit (Amicon). Each sample (100 µl) was incubated for 1 h at room temperature with 100 µl of 20 mM NaCl plus 100 mM HEPES, pH 7.4, or 20 mM CaCl2 plus 100 mM MES, pH 6.0, followed by centrifugation for 20 min at 16,000 g. Pellets and supernatants were analyzed by SDS-PAGE and immunoblotting with anti-CgA. The in vitro aggregation of CgA shown in Fig. 8 actually represents the difference value between the percent sedimentation at pH 6.0 minus background values at pH 7.4. SDS-PAGE and fluorography. Islet samples were analyzed by 15% acrylamide SDS-PAGE plus urea using a Tricine buffer system (61). Reduced and oxidized mouse proinsulins were run as standards on all gels. Insulin gels were fixed initially in 20% TCA without alcohol, then in 12.5% TCA plus 50% methanol, then incubated briefly with water, and finally incubated with 1 M sodium salicylate for 20 min. Dried gels were exposed to XAR film at ![]() |
RESULTS |
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Effects of in vivo disulfide reduction of proinsulin
and insulin in islet -cells. The conserved disulfide
loop in CgB (which behaves as a regulated secretory protein) has been
proposed to function as a sorting signal at the TGN for entry into
forming secretory granules (25, 40). The amino-terminal disulfide bond
was originally found to be an essential part of this signal, since
disulfide disruption using DTT treatment in live PC-12 cells causes
newly synthesized CgB to exhibit enhanced unstimulated release rather
than intracellular storage, suggesting its missorting from the
regulated to the constitutive secretory pathway (16). Disulfide
disruption of a similar amino-terminal disulfide-bonded loop in POMC
reportedly produces an analogous phenotype in Neuro-2a cells (19); thus
we have been interested in exploiting disulfide disruption to explore
the general significance of disulfide bond integrity in regulated
secretory protein sorting.
As proinsulin advances along the secretory pathway in pancreatic
-cells, hexamerization causes proinsulin disulfide bonds to be
increasingly buried and less accessible to membrane-permeant reducing
agents, so that disulfide disruption in vivo can no longer be obtained
with monothiol reducing agents like 2-mercaptoethanol (70) or with DTT
at doses
5 mM (35). Nevertheless, brief exposure to DTT at 20 mM can
still result in significant intracellular reduction of luminal protein
disulfides within the TGN or secretory granules of pancreatic islets
(35). To determine whether DTT treatment also affects proinsulin
trafficking, we first pulse labeled isolated mouse pancreatic islets
with 35S-labeled amino acids for
10 min followed by accumulation of labeled proinsulin in the TGN at
20°C and a brief treatment with 20 mM DTT (or mock treatment, see
METHODS). At this time, most labeled proinsulin in control islets has not yet exited the TGN (41). As shown
in Fig.
1A,
lane 1, little insulin had yet been
formed. However, analysis by nonreducing SDS-PAGE confirmed that, under these conditions, a partial but significant cystine reduction in
labeled intracellular proinsulin had occurred in live islets treated
with DTT (compare A and B of Fig.
1).1
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When analyzed for secretion, the medium collected during the brief DTT treatment did not contain augmented secretion of proinsulin (Fig. 1C). After washout of DTT, subsequent unstimulated and stimulated chase periods were collected. Interestingly, the unstimulated chase period contained only modest labeled proinsulin secretion that was clearly not stimulated by prior DTT treatment (Fig. 1D). Furthermore, stimulated secretion of labeled prohormone and/or hormone from islets briefly treated with DTT was also less than that observed from control islets (Fig. 1E). Accompanying the decrease in labeled proinsulin and/or insulin secretion from DTT-treated islets was an increase in the labeled proinsulin that remained intracellular at the end of the experiment (Fig. 1F). Importantly, analysis of media by nonreducing SDS-PAGE (not shown) revealed that only the residual oxidized forms of proinsulin and insulin were secreted from islets treated with DTT, suggesting that proinsulin molecules with disrupted disulfide bonds could not advance from the TGN into any secretory pathway. In addition, DTT treatment led to obvious inhibition of endoproteolytic conversion to labeled insulin (Fig. 1, E and F).
To exclude a general toxic effect of the brief islet exposure to 20 mM
DTT, it was important to examine proinsulin and insulin that had
already entered immature granules of the regulated secretory pathway.
For this purpose, we modified our protocol such that pancreatic islets
were chased to enrich for labeling of immature -granules (see
METHODS), the compartment in which
endoproteolytic conversion of proinsulin to insulin takes place (34,
41). The islets were then briefly exposed to DTT or were mock treated (see METHODS). It was clear that DTT
penetrated the granules and structurally altered proinsulin as well as
insulin, because there was a dramatic decrease in the recovery of both
labeled intracellular proteins on nonreducing SDS-PAGE (Fig.
2A).1
This evidently reflected a structural perturbation rather than proteolysis, because, in separate experiments, DTT-treated islets boiled in the presence of 2% SDS and 25 mM DTT yielded recovery of
reduced labeled proinsulin and insulin that was comparable with control
samples (Fig. 2B). Thus DTT does not
alter the amount of insulin or proinsulin in the immature granules but
instead alters the structure of these proteins so as to affect their
mobility on nonreducing gels. [This kind of structural
perturbation induced by in vivo reduction, detected by differential
underrecovery on nonreducing SDS-PAGE, has been reported previously for
other proteins (22, 36, 38, 54, 59).] Importantly, however, after
in vivo reduction of labeled proinsulin and insulin within immature granules, the unstimulated and stimulated secretions did not differ from those of control (mock-treated) islets (Fig.
3). Essentially identical results were
obtained on reduction of insulin after chase to mature secretory
granules (chase time = 5 h at 37°C); indeed, by reducing SDS-PAGE
followed by fluorography/scanning densitometry, stimulated insulin
secretion from islets that had been briefly treated with 20 mM DTT
averaged 18.3%, whereas stimulated insulin secretion from control
islets averaged 18.4% (n = 4 experiments). Together, these data suggest that the effects of DTT on
labeled proinsulin secretion were not a general effect of the DTT
treatment on the viability or responsiveness of the islets. Rather,
disruption of proinsulin disulfides in the TGN resulted in structural
alterations that inhibited its advance into the post-Golgi secretory
compartments comprising the constitutive, constitutive-like, and
stimulated secretion pathways. Although there were also major
structural sequelae for disruption of proinsulin or insulin disulfides
within secretory granules (Fig. 2), because these proteins were already contained within the regulated secretory pathway, there was no demonstrable diminution of the fraction of labeled intracellular hormone that underwent stimulus-dependent exocytosis (Fig. 3).
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In vivo disruption of CgB disulfide in PC-12 and
GH4C1 cells.
Because newly synthesized proinsulin did not exhibit increased
unstimulated release in response to DTT treatment (Fig. 1), we decided
to reexamine the effect of DTT-mediated disulfide reduction on the
intracellular routing of newly synthesized CgB in endocrine cells. For
this, we deferred DTT treatment or mock treatment of newly synthesized
proteins until immediately after radiolabeled amino acid incorporation,
because DTT inclusion during the pulse labeling can substantially
inhibit the synthesis of CgB and other secretory proteins (16). After a
5-min pulse labeling of PC-12 cells with radioactive leucine, the cells
were chased for an additional 60 min in the presence or absence of 5 mM
DTT. The heat-stable radioactive proteins recovered from media or cell
lysates (containing CgB) were analyzed by reducing SDS-PAGE and
fluorography (14, 16, 24). The identity of CgB was independently
confirmed by immunoprecipitation (not shown) with a specific antiserum
(kindly provided by Dr. J. Scammell, Univ. of South Alabama). In
agreement with earlier results (16), the presence of DTT during the
chase resulted in enhanced unstimulated release of newly synthesized CgB from PC-12 cells, which was increased approximately sixfold over
that released from control PC-12 cells (Fig.
4,
left).
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Disulfide disruption does not affect the targeting of CgA in
GH4C1 cells.
We next wished to test the role of the amino-terminal disulfide bond in
chromogranin targeting using a means independent of DTT treatment. For
this, we chose CgA, which also is targeted to secretory granules and
exhibits homotypic calcium/low-pH-dependent assembly properties (31,
73) and contains the highly conserved disulfide-bonded loop structure
(6) that has been described as essential for sorting to secretory
granules in PC-12 cells (67). We transiently expressed wt-CgA or CgA in
which both cysteine-17 and cysteine-38 (which form the amino-terminal
disulfide loop) were converted to serine by site-directed mutagenesis
(CgA-CC; see METHODS), thereby
abolishing the sole granin disulfide bond. It is important to note that
the expression of a disulfide-deficient mutant of CgB has recently been
shown to be targeted normally to secretory granules in PC-12 cells,
presumably because the disulfide-deficient CgB can homotypically
associate with wild-type endogenous CgB (40). In addition, PC-12 cells
express endogenous CgA. Indeed, when we overexpressed CgA-CC in PC-12
cells, it also exhibited a clear stimulus-dependent secretion (similar
to that observed for expressed wt-CgA, Fig.
7), quite possibly because the
disulfide-deficient CgA-CC mutant can homotypically associate with
endogenous wt-CgA. However,
GH4C1
cells do not express endogenous CgA, effectively eliminating homotypic
interactions between transfected and endogenous CgA molecules.
[Note also that, while CgA may interact with CgB under highly
acidic conditions not comparable with those found in the TGN (73, 74),
our previously published (and unpublished) results have suggested that
heterotypic CgA-CgB interactions are in fact quite limited (31).]
We therefore compared the degree of stimulation of wt-CgA or CgA-CC
secretion induced by secretagogue treatment of
GH4C1
cells at steady state (a measure of granule targeting and storage
efficiency). The data in Fig.
8A clearly demonstrate that the ability of secretagogue addition to elicit a
stimulated secretion of CgA from
GH4C1
cells is independent of the presence of its amino-terminal disulfide
bond.
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CPE levels are not very different in PC-12 and
GH4C1 cells.
One possible difference in chromogranin routing in PC-12 vs.
GH4C1
cells could be in the binding of chromogranins to a putative sorting
receptor that could interact with the amino-terminal disulfide bond.
Thus far, the only common component that has been postulated to
function as a neuroendocrine-specific receptor for the entry of
secretory proteins into forming granules (21) or in secretory protein
retention within maturing granules (57) [including in cells
synthesizing growth hormone or prolactin (63)] is CPE. It
therefore was of interest to determine whether there might be important
differences in the levels of CPE between
GH4C1
and PC-12 cells. As measured by immunoblotting (see
METHODS), total immunoreactive CPE
appeared comparably abundant in these cells (Fig.
9). These data are consistent with the idea
that CPE per se does not directly relate to the mechanism of
chromogranin sorting to the regulated secretory pathway (20, 52, 53)
and cannot explain differences in sorting between PC-12 and
GH4C1
cells after disruption of the chromogranin disulfide bond.
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DISCUSSION |
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We have endeavored to explore the general significance of disulfide disruption on the fate of luminal proteins in the distal secretory pathway of several regulated secretory cell types. Although there are several distinct models of protein sorting to secretory granules (3, 66), one idea common to all models is that sorting depends on conformational features in the secretory proteins. In particular, recent evidence has implicated disulfide-enclosed loop structures in the sorting of several regulated secretory proteins (15, 16, 25, 28, 40, 65, 67).
The results presented in this report raise new issues that must be
considered when trying to interpret how disulfide disruption in newly
synthesized secretory proteins may affect their targeting. Proinsulin
contains three highly conserved disulfide bonds, including the A6-A11
bond that is known to be highly inaccessible to solvent, based on the
insulin crystal structure (5, 7). Thus global structural rearrangement
is expected to accompany reduction of the three disulfide bonds in
proinsulin or insulin. Remarkably, our results in pancreatic -cells
seem to suggest that DTT treatment does not alter the fraction of
labeled insulin that undergoes regulated exocytosis, because that
fraction of molecules has already gained entrance to the regulated
secretory pathway before exposure to the reducing agent (Fig. 3).
(These findings also eliminate the idea that transient DTT exposure
produces a toxic effect that blocks subsequent regulated exocytosis.)
By contrast, when proinsulin is accumulated in the TGN of islet
-cells (Fig. 1) or shortly after prolactin synthesis in
GH4C1
cells (Fig. 5), disulfide disruption renders these molecules unable to
advance into either unstimulated or stimulated secretory pathways.
Thiol-dependent conformational perturbation of secretory proteins
within the endoplasmic reticulum is well established (8, 9, 36-38,
47, 48, 59); however, to our knowledge, such an effect has not been
previously reported for labeled proteins that have already reached the
TGN (Fig. 1). Indeed, these observations contrast with a previous
conclusion that protein retention mediated by disulfide reduction (to
free thiols) does not take place in or beyond the Golgi (70). Thus the
mechanism of thiol-mediated retention of proteins in the Golgi complex
appears to be protein dependent and quite possibly does not involve
endoplasmic reticulum molecular chaperones (37). Moreover, recent
studies have established that luminal proteins may be retained within
the Golgi complex, even in the absence of disulfide disruption (32, 46,
62).
Unlike proinsulin or prolactin, reduction of the single conserved disulfide bond does not impair chromogranin transport through the endoplasmic reticulum and Golgi complex (Fig. 6A). Indeed, we have confirmed that, in PC-12 cells, unstimulated secretion of newly synthesized CgB is greatly augmented as a result of DTT treatment (Fig. 4). In GH4C1 cells, dramatic perturbation of newly synthesized prolactin clearly indicated DTT penetration and disulfide disruption within the secretory pathway (Fig. 5); however, DTT treatment did not lead to any significant rerouting of CgB in these cells (Figs. 4 and 6B). Consistent with these findings, CgA mutated to lack the homologous amino-terminal disulfide bond also exhibits normal stimulated secretion when transiently expressed in GH4C1 cells (Fig. 8). Importantly, although CgA-CC targeting to secretory granules in PC-12 cells (Fig. 7) can be explained by homotypic interactions with endogenous CgA that "rescue" the mutant [analogous to that reported for the rescue of disulfide-deficient CgB by endogenous CgB (40)], the same result in GH4C1 cells cannot be explained by interactions with endogenous CgA (because it is not expressed) but can be explained by homotypic multimeric aggregation of the transfected mutant CgA molecules (Fig. 8 and see below). We have used two entirely independent approaches, and neither has provided any evidence to support the idea that chromogranin sorting to the regulated secretory pathway in GH4C1 cells requires the presence of its amino-terminal disulfide bond. These findings are not only consistent with a recent conclusion that manipulation of disulfide bonds differentially affects the intracellular transport, sorting, and processing of various neuroendocrine secretory proteins (71) but also suggest the complex notion (which is gaining increasing acceptance) that cell-type-specific protein interactions play important roles in the targeting of regulated secretory proteins (13, 18, 64). However, interactions with CPE are unlikely to account for chromogranin sorting or for differences in sorting between PC-12 and GH4C1 cells [and this agrees with recent reports (20, 52, 53)], as levels of CPE by immunoblotting were comparable between the two cell types (Fig. 9).
There is a growing consensus that multimeric aggregation in the luminal
ionic environment of the TGN and immature granules is a critical step
in the sorting of numerous regulated secretory proteins (3, 68). For
example, this appears to be of major importance not only for
intragranular insulin storage in the maturing granules of pancreatic
-cells (12, 42) but also after proinsulin expression in
GH4C1
cells (R. Kuliawat and P. Arvan, unpublished observations). Moreover,
we note that SgII, a homolog of CgB, lacks the amino-terminal disulfide
bond and yet is targeted normally to secretory granules in both PC-12
and
GH4C1
cells. Importantly, the targeting of SgII to secretory granules cannot
be explained by rescue interactions with CgB, since these two granins
apparently do not form heterotypic complexes (15, 16), although it is known that SgII independently exhibits calcium/low-pH-induced multimeric aggregation properties (24). Thus, in both PC-12 and
GH4C1
cells, molecular interactions employing structural features over and
above the disulfide-bonded loop are likely to be involved in granin
targeting to secretory granules.
With these ideas in mind, it seemed reasonable to examine the multimeric aggregation properties of disulfide-deficient granins (which might potentially serve as a starting point for further investigations of granin sorting differences in PC-12 vs. GH4C1 cells). Notably, reduction of the disulfide bond in CgB does not affect its properties of calcium/low-pH-induced multimeric aggregation (15). Also, we do not find a significant difference in the calcium/low-pH-induced multimeric aggregation of CgA containing or lacking the disulfide bond (Fig. 8). We therefore hypothesize that multimeric aggregation behavior is likely to account largely for the disulfide-independent granin targeting to secretory granules in GH4C1 cells (Figs. 4, 6, 8). We intend to test this hypothesis by expressing in PC-12 and GH4C1 cells a truncated, nonaggregating form of CgA (31) that, nevertheless, contains the amino-terminal disulfide-bonded loop.
In conclusion, protein trafficking in regulated secretory cells appears to be a complex process involving multiple potential interactions among different secretory proteins, including potential interactions between secretory proteins and the surrounding membranes that form both the constitutive and the regulated secretory pathways (3). Thus, even with experiments that may demonstrate that a given region of a model protein is necessary and sufficient for trafficking to granules within the context of a single cell type, no one model protein or cell line can be trusted to faithfully replicate and to be fully representative of all aspects of post-Golgi trafficking among the great variety of neuroendocrine secretory cells.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Jonathan G. Scammell (Univ. of South Alabama) for providing antiserum to CgB, Dr. Lloyd Fricker (Albert Einstein College of Medicine) for antisera to CPE, and Dr. David V. Cohn (Univ. of Louisville) for antiserum to bovine CgA. Dr. Douglas S. Darling is thanked for assistance with cloning techniques and Yancy R. Moore is thanked for expert technical assistance and for maintaining PC-12 and GH4C1 cell cultures.
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FOOTNOTES |
---|
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-48280 (to P. Arvan) and DK-53367 (to S.-U. Gorr) and by grants from the Kentucky Affiliate of the American Heart Association, Jewish Hospital Research Foundation (Louisville, KY).
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. §1734 solely to indicate this fact.
1 We have previously reported that, because a fresh isolation of pancreatic islets is performed for each experiment, there is variability in the degree of reduction of the contents of post-Golgi compartments in different preparations of islets, which are clusters of 1,000-5,000 cells. Incomplete reduction is commonly observed in preparations enriched in larger islets, which may diminish the degree of DTT penetration during incubation with the reducing agent (35).
Address for reprint requests and other correspondence: P. Arvan, Div. of Endocrinology and Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Golding Bldg., Rm 501, 1300 Morris Park Ave., Bronx, NY 10461 (E-mail: arvan{at}aecom.yu.edu).
Received 15 March 1999; accepted in final form 28 April 1999.
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