(Received for publication, April 4, 1996, and in revised form, October 16, 1996)
From the Groupe de Biologie de la Cellule Neuroendocrine, CNRS URA 1115 and INSERM U 36, Collège de France, 11, Place Marcellin Berthelot, 75231 Paris Cedex 05 France
Secretogranin II (SgII) is a protein specific to the matrix of the secretory granules in neurons and neuroendocrine cells. We have already demonstrated the precursor-product relationship between sulfated SgII and four N-terminal derived peptides in GH3B6 prolactin cells. In this study, we have investigated the subcellular compartment in which the cleavage of SgII is initiated by taking advantage of its tyrosine sulfation in the trans-Golgi network (TGN). In order to prevent export of radiosulfated SgII from the TGN, we used brefeldin A (BFA) as well as incubation at 20 °C. BFA completely inhibited the cleavage of SgII when added immediately post-pulse. BFA added a few minutes post-pulse or after a 20 °C incubation, however, permitted the cleavage of SgII in the presence of the drug. These SgII-derived peptides generated in the presence of BFA could not be released upon stimulation of the cells by either thyroliberin, a physiological secretagogue, or KCl. These results demonstrate that SgII can be cleaved in the TGN. They also evidence that the cleavage occurs in a distal compartment of the TGN different from the sulfation site. The transfer of SgII from the sulfation site to this distal compartment of the TGN involves BFA-sensitive membrane dynamics.
Bioactive peptides and hormones secreted by neurons and neuroendocrine cells are synthesized as precursors that must undergo post-translational processing before their release. The different steps of this processing occur during the vectorial transport of the proteins from the rough endoplasmic reticulum, where they are synthesized, to the secretory granules, in which they are stored. Proteolytic processing of the precursors is one of the last of these post-translational modifications. Although it occurs mainly in the secretory granules, it can be initiated in the trans-Golgi network (TGN)1 (reviewed in Ref. 1). Indeed, immunocytochemical (2-5) and biochemical studies (6-9) have evidenced the cleavage of precursors in the TGN as well as in the secretory granules. In this study, we have investigated the intracellular cleavage site of secretogranin II (SgII) in GH3B6 prolactin cells.
SgII is a member of the granin family (reviewed in Ref. 10). These
proteins are specific to the dense core secretory granules of
neuroendocrine cells and neurons (reviewed in Ref. 11-13). They are
considered as precursors of secreted peptides (reviewed in Ref. 14).
SgII is the precursor of secretoneurin (15), a peptide whose activity
has recently been proposed (16). SgII and chromogranin B are expressed
in normal anterior pituitary prolactin cells and in GH3B6 prolactin
cells (17). Both are present in the same secretory granules with
prolactin but display different intragranular localizations (18). We
have demonstrated the precursor-product relationship between SgII and
four sulfated fragments in prolactin cells (see Fig. 1) (19). The
terminal sulfated fragment is a 21-kDa protein that is accumulated in
the secretory granules and released upon stimulation by thyroliberin
(TRH), a prolactin secretagogue, or KCl. The processing of SgII is
fast, and more than 70% of mature SgII is cleaved 30 min after a 5-min
pulse with [35S]sulfate (19).
We took advantage of the tyrosine sulfation of SgII to study the
intracellular site of its proteolytic processing. Indeed, sulfation is
a post-translational modification specific to the TGN (20-22).
Pulse-labeling PC12 cells with [35S]sulfate has already
allowed the use of sulfated SgII as a marker of intracellular transport
between the TGN and the plasma membrane via the dense core secretory
granules (23-25). PC12 cells, however, lack the prohormone convertases
PC1 and PC2, which are involved in the processing of SgII (26, 27). Our
experiments were performed in GH3B6 cells, a subclone of GH3 cells,
which express PC2 but not PC1 (28). Cultivating these cells in the
presence of insulin, 17-estradiol, and epidermal growth factor
increases the number of secretory granules (29), the expression of PC2
(28) and the storage of SgII-derived peptides in the granules (19).
GH3B6 cells therefore provide an ideal model for studying the
proteolytic processing of SgII in the TGN and secretory granules.
We used brefeldin A (BFA) and 20 °C incubation to block anterograde transport of sulfated proteins in the TGN. We have already described the effect of these treatments on the distribution and secretion of prolactin in GH3B6 cells (30). BFA is a fungal metabolite that blocks anterograde transport of proteins at different steps of the secretory pathway (reviewed in Ref. 31). It inhibits the formation of secretory vesicles and granules from the TGN, without preventing the exocytosis of the secretory granules stored in the cytoplasm (8, 32, 33). Incubating the cells at 20 °C accumulates secretory products in the TGN (34, 35) and thus permits the study of post-translational modifications at the exit of the Golgi apparatus (7-9, 20, 36, 37). The combination of the sulfate labeling with the BFA and 20 °C transport blocks allowed an accurate study of the steps of transport of SgII in the TGN. Our results indicate that the cleavage of SgII may occur in the TGN in a compartment distal from the sulfation site but before the packaging of SgII in the secretory granules.
GH3B6 cells were cultured in Ham's F-12
medium supplemented with 15% horse serum (PAA Labor) and 2.5% fetal
calf serum (Life Technologies, Inc.) at 37 °C in 5%
CO2. For experiments, they were grown in 35-mm dishes for 6 days in the presence of 180 nM insulin
(Sigma), 10 nM epidermal growth factor
(Becton Dickinson Labware), and 1 nM 17-estradiol
(Sigma).
Before metabolic labeling, cells were incubated in sulfate-free Ham's F-12 medium for 30 min. They were then pulse-labeled for 5 or 10 min at 37 °C in the presence of 200-250 µCi/dish carrier free [35S]sulfate (ICN Biomedicals, Inc.). Cells were washed three times with Ham's F-12 on ice before further chase. The 20 °C incubations were performed by transferring the cells from ice to a 19-20 °C water bath. BFA (Epicentre Technologies) was always used at 10 µg/ml. When added after a 20 °C incubation, BFA was always added at 20 °C, 3-5 min before the temperature shift to a 37 °C water bath. Chloroquine (Sigma) was always used at 40 µM. In some experiments, the release of SgII was stimulated with either 30 nM TRH (Calbiochem) or 30 mM KCl. All the experiments were performed with duplicate culture dishes and reproduced three times.
Extraction of Secretogranins and SDS-PAGECells were
scraped on ice in 20 mM Tris, pH 7.4, 150 mM
NaCl, 5 mM EDTA, 0.3% Tween 20 containing 200 µM phenylmethylsulfonyl fluoride, 1 µM
leupeptin, and 1 µM pepstatin. Lysates were boiled for 3 min, cooled on ice, and centrifuged 10 min at 23,000 × g. Supernatants were collected and acetone precipitated
overnight at 20 °C. Media were collected and centrifuged to pellet
any detached cell before trichloroacetic acid precipitation of the proteins overnight at 4 °C. Proteins were separated by SDS-PAGE on
acrylamide gradients (5-20%) mini-gels (Hoeffer). Gels were stained
with Coomassie Blue R-250 either before direct autoradiography or
treatment for fluorography with Amplify (Amersham Corp.). Gels were
exposed to either Bio-Max films (Kodak) or
-Max films (Amersham Corp.). Quantitation of autoradiograms was performed by densitometric analysis with Agfa Arcus Plus and NIH Image.
To investigate the intracellular site of sulfation, we
pulse-labeled GH3B6 cells with [35S]sulfate for 5 min in
both the presence and the absence of 10 µg/ml BFA. In control cells,
[35S]sulfate was incorporated in heparan sulfate
proteoglycans, chromogranin B, and SgII (see Fig. 2A).
Sulfation was completely abolished after a 30-min pretreatment of the
cells with BFA (see Fig. 2A). Longer pretreatment of 120 min
provided the same results (not shown). If BFA was added at the
beginning of the pulse without pretreatment, sulfation was strongly
inhibited and could not be increased by extending the pulse up to 15 min in the presence of the drug (see Fig. 2A).
We also investigated the effect of BFA on the cleavage of sulfated SgII by adding the drug immediately post-pulse. In control cells, sulfated SgII is sequentially cleaved from the C terminus to the N terminus (Fig. 1). The cleavage products are accumulated in the cells as 27- and 21-kDa fragments (Fig. 2B) (19). BFA completely prevented this proteolytic processing. It also inhibited the secretion of sulfated SgII (not shown), as already described in PC12 cells (32, 33).
BFA Blocks the Transport of Sulfated SgII to the Cleavage CompartmentBFA blocks anterograde transport by inhibiting the
association of the ADP-ribosylation factor and the coat proteins with
the membranes of the organelles involved in the secretory process (38,
39). The coat proteins associated with the Golgi apparatus (-COP)
and the TGN (
-adaptin) are dispersed in the cytoplasm within 1 min
in the presence of BFA (40, 41). We have taken advantage of the
rapidity of the dispersion to study the kinetics of the inhibition of
SgII proteolytic processing by BFA. Chase media were replaced by BFA
containing media either 10, 20, or 30 min after a 5-min pulse (Fig.
3). Whereas BFA completely inhibited the processing of
SgII when added immediately post-pulse (Fig. 3, lane 5), it
had no effect 20 min or more after the pulse (Fig. 3, lane 7 and 8). If BFA was added 10 min after the pulse (Fig. 3,
lane 6), two pools of sulfated SgII were present in the cell extracts: one was BFA-sensitive and not cleaved, the other was BFA-resistant and cleaved. 10 min after the pulse, the processing of
SgII had not begun (Fig. 3, lane 1), and the fragments of
SgII present when BFA was added at 10 min chase time were thus
generated in the presence of the drug. It is worth noting that this
cleaved SgII was not accumulated as 80- or 40-kDa intermediate
fragments but rather as 27- and 21-kDa terminal fragments. This
demonstrated that BFA inhibited the cleavage of SgII through a block of
transport from the sulfation site to the cleavage compartment and not
through the inhibition of the proteolytic activity itself.
The SgII-derived Peptides Generated in the Presence of BFA Cannot Be Released by Secretagogues
The transport step inhibited by BFA
could correspond to the formation of secretory granules as evidenced by
several groups (32, 33) or could occur within the TGN itself. In order
to distinguish between these two possibilities, we studied the release of the proteolytic products generated in the presence of BFA by stimulating the cells with 30 nM TRH. Cells were
pulse-labeled with [35S]sulfate for 5 min and chased in
control medium for 5 min before the addition of BFA for 55 min. This
provided the same pattern of cleavage as on Fig. 3 (lane 6):
one pool of BFA-sensitive uncleaved SgII and another of BFA-resistant
SgII, which was present as 27- and 21-kDa fragments. Cells were then
stimulated with TRH for 30 min (Fig. 4). This
secretagogue released the SgII maturation products from control cells.
BFA did not inhibit this release if it was present only during the
second chase with TRH. When cells were incubated in the presence of BFA
during both chases, the maturation products that had been generated in
the presence of BFA could not be released by TRH (Fig. 4). Experiments
performed with 30 mM KCl provided the same results (not
shown). Some SgII fragments could therefore be generated in the
presence of BFA but not released.
The Proteolytic Processing of SgII Is Strongly Slowed Down at 20 °C
Incubating cells at 20 °C accumulates proteins in the
TGN and blocks the formation of secretory vesicles (34, 35). SgII was
sulfated in GH3B6 cells incubated at 20 °C (not shown). When the
cells were pulse-labeled at 37 °C and chased at 20 °C, SgII could
be processed to the same peptides, but the kinetics of the cleavage was
dramatically slowed down: the first fragments appeared between 30 and
60 min (Fig. 5). In order to determine if this inhibition resulted from the transport block or from the inhibition of
the proteolytic activity, we took advantage of the results obtained
with BFA, i.e. the absence of effect of the transport block
on the cleavage of SgII 20 min after the pulse. Cells were first chased
at 37 °C for 30 min in order to allow transport of SgII to the
cleavage compartment. They were then incubated at 20 °C for 30 min
(Fig. 5). The cleavage pattern observed at the end of this second chase
was similar to that observed after the first 30 min chase at 37 °C.
This demonstrated that SgII was not further processed during the second
chase at 20 °C. The inhibition of proteolytic processing at 20 °C
was thus caused by the inhibition of the proteolytic activity rather
than by the transport block alone. This could possibly be related to
the inhibition of PC2 at 20 °C already evidenced in vitro
(42).
A 20 °C Preincubation Lowers the Effect of BFA on the Cleavage of SgII but Not on Its Release
We investigated the effect of BFA
after an incubation at 20 °C. Cells were thus chased at 20 °C for
30 min, after a 5-min pulse with [35S]sulfate and prior
to a 60-min incubation at 37 °C in the presence of BFA. This
protocol provided the same proteolytic pattern of SgII as that already
observed when BFA was added 10 min after the pulse: one pool of
sulfated SgII was BFA-sensitive and uncleaved, whereas the other was
BFA-resistant and accumulated as 27- and 21-kDa fragments (Fig.
6A). We also investigated the release of the
fragments generated in the presence of BFA by stimulating the cells
with 30 nM TRH (Fig. 6B). TRH could release the
fragments generated under control conditions, even if it was added
together with BFA. The fragments generated in the presence of BFA
could, however, not be released upon stimulation of the cells. The same results were obtained after stimulation of secretion with 30 mM KCl (not shown). These experiments demonstrate that the
transport block induced by BFA, which results in the inhibition of the
cleavage of SgII, was upstream from the 20 °C accumulation site,
unlike the other block, which results in the inhibition of the release of the fragments of SgII.
Acidification of Luminal pH Is Necessary for the Cleavage of SgII
The acidification of luminal pH in the TGN and the secretory
granules is necessary for the proteolytic processing of some peptide
precursors and pro-hormones (reviewed in Refs. 1 and 43). We
investigated the involvement of the acidification of luminal pH in the
processing of SgII with 40 µM chloroquine, a lysosomotropic agent that blocks intra-organelle acidification. Chloroquine had no effect on the sulfation of SgII, but it completely inhibited the proteolytic processing of sulfated SgII (Fig.
7A). We also investigated the secretion of
sulfated SgII in the presence of chloroquine. Uncleaved sulfated SgII
could be released in the medium, and this release could be stimulated
by TRH (Fig. 7B). This indicates that the inhibition of SgII
cleavage induced by chloroquine was not due to the derouting of SgII
from its regulated secretory pathway.
In this study we investigated the proteolytic processing of endogenous SgII in GH3B6 prolactin cells. Recent biochemical studies have provided evidences for the cleavage of precursors in the TGN as well as in the secretory granules (7-9). Using BFA and 20 °C incubations, we were able to demonstrate that sulfated SgII is cleaved in the TGN of GH3B6 cells. Sulfation is a post-translational modification specific to the TGN (20-22), and sulfated SgII has been used as a marker for the transport between the TGN and the plasma membrane in PC12 cells (23-25). Unlike GH3B6 cells, this cell line does not process SgII to smaller peptides. Sulfated SgII in GH3B6 cells therefore provides a unique model for studying in detail the proteolytic processing in the TGN. Our experiments indeed permitted to evidence two BFA-sensitive steps in the delivery of SgII from its sulfation site in the TGN to the secretory granules: sulfated SgII is first transported to a distal compartment of the TGN where its cleavage is initiated and then packaged in the secretory granules.
Biochemical studies and subcellular fractionation have demonstrated that sulfation is a post-translational modification specific to the TGN (20, 23). BFA has also been used to demonstrate that sulfation occurred in the TGN of epithelial (21) and neuroendocrine cells (22, 33). Our experiments are in agreement with these studies: pretreatment of the cells with BFA completely abolished sulfation, and treatment at the beginning of the pulse also strongly inhibited sulfation. These results are relevant of the absence of redistribution of the sulfotransferase to the rough endoplasmic reticulum together with the cis-, medial, and trans-Golgi (reviewed in Ref. 31). In such a hypothesis, the inhibition of sulfation observed results from the lack of delivery of substrate to the TGN, as already proposed by others (21, 22), even though we cannot exclude that the sulfotransferase activity itself is inhibited by the BFA treatment.
We used BFA to study the site of the initiation of the proteolytic
processing of SgII. BFA completely abolished the cleavage of SgII in
GH3B6 cells when it was added immediately post-pulse. This demonstrates
that the cleavage compartment of SgII is different from the sulfation
compartment. This observation is in agreement with the lack of direct
sulfation of any SgII proteolytic fragment that we have already
described (19). When BFA was added a few minutes after the pulse, the
fragments of 27 and 21 kDa were, however, generated. This demonstrates
that the cleavage of SgII may occur in the presence of BFA and that BFA
blocks the delivery of sulfated SgII to the cleavage site without
inhibiting the proteolytic activity itself. Two lines of evidence
indicate that this transport block occurs before the formation of the
secretory granules. First, incubating the cells at 20 °C as briefly
as 30 min permitted the cleavage of sulfated SgII in the presence of
BFA. Second, the 27- and 21-kDa sulfated fragments generated in the
presence of BFA could not be released in the medium when the cells were
stimulated by 30 nM TRH or 30 mM KCl, even
though BFA had no effect on the release of the stored granules. This
indicates that the SgII fragments generated in the presence of BFA were
not present in secretory granules, not even in immature secretory
granules, which have the same exocytotic potency as the mature granules
(44, 45). This is in contrast to results obtained in -cells in which
BFA inhibited the cleavage of proinsulin accumulated in the TGN at 20 °C (8). This difference could be relevant of the cell types. Indeed, immunocytochemical studies have also demonstrated that the
cleavage of proinsulin occurred only in the secretory granules of
-cells (2), whereas that of pro-opiomelanocortin took place in the
TGN of pituitary corticotropes (4).
The use of sulfate labeling in the TGN together with the BFA and
20 °C transport blocks has therefore permitted demonstration of the
cleavage of endogenous SgII in the TGN and definition of the two steps
in the transport of SgII from its sulfation site to the secretory
granules (Fig. 8). The first step occurs in the TGN
itself and corresponds to the delivery of sulfated SgII to the cleavage
site, which is a distal compartment of the TGN. The second step
corresponds to the packaging of SgII in the secretory granules. The
distinction between these two steps was only possible under conditions
that accumulate the proteins at the exit of the TGN, i.e. in
the presence of BFA or at 20 °C. The cleavage of SgII in the TGN may
therefore result from an increase in the residence time of SgII in this
organelle. Such a hypothesis is in agreement with the increased
sialylation of chromogranin B in the TGN of PC12 cells in the presence
of BFA (33). Our results do not exclude that under normal conditions of
transport, SgII is mainly cleaved in secretory granules, as suggested
by the stimulation of the secretion of uncleaved SgII (19). They
demonstrate that the conditions necessary for the cleavage of SgII are
present in the TGN.
The cleavage site that we have defined as a distal compartment of the TGN could possibly correspond to the dilated zones of the TGN where regulated secretory products aggregate in neuroendocrine cells. The acidification of luminal pH in the TGN to 6-6.5 triggers the aggregation of SgII (46) and prolactin (47). Such a pH was also necessary for the cleavage of prosomatostatin in infected GH3 cells (42). Our own experiments with chloroquine demonstrate that the pH acidification is also necessary for the cleavage of SgII. The acidification of pH may thus be responsible for both the aggregation and the cleavage of SgII in the TGN. Ultrastructural studies have evidenced the aggregation of proteins in specific regions of the more distal part of the TGN in endocrine cells (48), including prolactin cells (49, 50). The TGN is a very dynamic structure whose size and shape vary greatly according to the cell type and activity (51, 52). Three-dimensional morphological studies of prolactin cells lead to the hypothesis that secretory granules were formed concomittantly with the network and that this process itself consumed the membrane by fragmentation of the network (50). The same was described concerning the formation of secretory vesicles in NRK cells (53). Such a dynamic model could well be conciliated with our results (Fig. 8). The first step of transport of sulfated SgII in the TGN might correspond to the formation of the network concomittantly with the combination of the factors necessary for the cleavage. The second step might correspond to the fragmentation of the network during the formation of the secretory granules.
We gratefully acknowledge Eric Etienne for the photographic work. We thank Dr. Denis Hervé (INSERM U 114) for help in the densitometric analysis of the autoradiograms and Richard Killion for help in preparing the manuscript.