(Received for publication, July 12, 1995; and in revised form, October 26, 1995)
From the
Our laboratory has been using a permeabilized cell system
derived from rat anterior pituitary GH cells expressing
prosomatostatin (pro-SRIF) to study prohormone processing and nascent
secretory vesicle formation in vitro. Because calcium is
necessary for prohormone processing enzyme activity, secretory granule
fusion with the plasma membrane, and possibly sorting to the regulated
pathway, we treated permeabilized cells with the calcium ionophore
A23187 to determine the role of calcium in pro-SRIF cleavage and
nascent vesicle formation from the trans-Golgi network (TGN).
Here we demonstrate that pro-SRIF cleavage was markedly inhibited when
lumenal free calcium was chelated with EGTA in the presence of A23187.
Surprisingly, submillimolar free calcium (
15 µM) was
sufficient to maintain prohormone cleavage efficiency, a value far
lower than that estimated for total calcium levels in the TGN and
secretory granules. Experiments using both A23187 and the protonophore
CCCP revealed that free calcium is absolutely required for efficient
pro-SRIF cleavage, even at the optimal pH of 6.1. Secretory vesicle
formation by contrast was not inhibited by calcium chelation but rather
by millimolar extralumenal free calcium. Together, these observations
demonstrate that pro-SRIF processing and budding of nascent secretory
vesicles from the TGN can be uncoupled and therefore have distinct
biochemical requirements. Interestingly, our data using intact GH
cells demonstrate that basal secretion of SRIF-related material
is largely calcium-dependent and therefore cannot be equated with
constitutive pathway secretion. These results underscore the importance
of determining calcium requirements before assigning a secretion event
to either the constitutive or regulated secretory pathway.
The secretory pathway in eukaryotes consists of a series of
topologically distinct intracellular membrane-enclosed compartments
that are specialized in composition and function. Vesicular transport
permits sorting without compromising compartmental identity. In
polypeptide hormone-producing cells, the trans-Golgi network
(TGN) ()has been implicated in specific protein trafficking
and endoproteolytic maturation events in the distal secretory pathway
(for review see Shields and Danoff (1993)). These cells can store and
concentrate hormones in dense core secretory granules that deliver
their contents to the cell surface by calcium-dependent fusion with the
plasma membrane after an appropriate extracellular stimulus. In
addition, neuroendocrine cells possess the ubiquitous constitutive
pathway, whereby secretory proteins are continually delivered to the
cell surface in a calcium-independent manner (Turner et al.,
1992; Miller and Moore, 1991). Morphological evidence indicates that
sorting of nascent secretory proteins into the regulated versus constitutive pathway occurs in the TGN (Sossin et al.,
1990; Orci et al., 1987), although recent studies suggest that
in pancreatic cells sorting may also occur after TGN exit in immature
secretory granules through a pathway called ``constitutive-like
secretion'' (Kuliawat and Arvan, 1994; Arvan and Castle, 1992).
Biochemical evidence supports a model whereby the low pH and high
calcium in the TGN lumen triggers selective aggregation and hence
sorting of proteins destined for secretory granules (Song and Fricker,
1995; Colomer et al., 1994; Chanat and Huttner, 1991), whereas
constitutively secreted proteins are excluded from such aggregates.
Lumenal ionic conditions also appear to play an important role in hormone activation. Many peptide hormones are initially synthesized as larger inactive polyprotein precursors or prohormones that undergo endoproteolytic maturation during intracellular transport. Prohormone cleavage occurs within the TGN and secretory granules at a defined set of basic residues by the recently discovered prohormone convertase enzymes (PCs) (for reviews see Seidah et al.(1993) and Steiner et al.(1992)). PC1/3 and PC2, which are the predominant PCs expressed in neuroendocrine tissue, are calcium-dependent endoproteases that require an acidic pH for maximal activity (Shennan et al., 1995; Rhodes et al., 1993; Davidson et al., 1988).
To investigate prohormone sorting, endoproteolytic
maturation, and secretory vesicle formation in vitro, we have
developed a permeabilized cell system (Xu and Shields, 1994, 1993)
derived from growth hormone and prolactin secreting rat anterior
pituitary GH cells stably expressing human or anglerfish
prosomatostatin (pro-SRIF) (Elgort and Shields, 1994; Stoller and
Shields, 1988). These cells possess significant levels of PC2 and
cleave pro-SRIF with
70% efficiency to generate the amino-terminal
75-80 amino acid propeptide and the carboxyl-terminal 14-amino
acid mature SRIF (Elgort and Shields, 1994; Stoller and Shields, 1988).
Pro-SRIF cleavage in permeabilized GH
cells requires ATP to
generate an acidic TGN lumenal pH (Xu and Shields, 1994). For
concomitant formation of nascent secretory vesicles, both ATP and GTP
are required (Xu and Shields, 1993).
We have employed the permeabilized cell system in conjunction with the calcium ionophore A23187 to examine the role of free calcium in prohormone processing and vesicle formation from the TGN. Here, we demonstrate that lumenal free calcium is absolutely required for efficient pro-SRIF cleavage but not for secretory vesicle formation. We also show that lumenal ionic conditions permissive for pro-SRIF cleavage in the TGN are nonpermissive extralumenally for vesicle formation.
Figure 1:
Calcium
requirement for pro-SRIF cleavage in vitro. A, cells
were pulse-labeled with [S]methionine for 12 min
at 37 °C, chased for 2 h at 19 °C, and permeabilized.
Permeabilized cells were then preincubated with or without 1 µM calcium ionophore A23187 in the presence of 2 mM EGTA or
0.015 (standard condition), 0.5, 2.0, 5.0, or 10.0 mM additional calcium chloride for 10 min at 4 °C, followed by a
further incubation with ATP and GTP for 2 h at 37 °C. Samples were
then separated into pellet (P) and supernatant (S)
fractions to monitor vesicle formation (see Fig. 4) by
centrifugation at 15,000
g for 10 s and
immunoprecipitated with rabbit anti-SRIF-propeptide or anti-SRIF (RSS1)
serum (see ``Experimental Procedures''). Immunoreactive
peptides were resolved by SDS-polyacrylamide gel electrophoresis (20%
acrylamide) and detected by fluorography. B, cleavage of
pro-SRIF in permeabilized cells requires submillimolar lumenal free
calcium. Quantitation by densitometry of fluorograms was as in A. Pro-SRIF cleavage efficiency = ([sum of the
propeptide band intensities from the pellet and supernatant]
[sum of the pro-SRIF and propeptide band intensities from
the pellet and supernatant])
100. The values represent
means ± S.E. from at least three experiments. Incubations with
up to 10 mM EGTA resulted in similar cleavage efficiencies to
those with 2 mM EGTA (not shown).
Figure 4:
Nascent secretory vesicle formation does
not require free calcium and is inhibited by millimolar extralumenal
free calcium. The pellet and supernatant (nascent vesicle) fractions
from Fig. 1were treated sequentially with anti-SRIF-propeptide,
anti-prolactin, and growth hormone antisera and the immunoprecipitated
material quantitated by densitometry of fluorograms. Budding efficiency
= ([sum of the hormone band intensities from the
supernatant] [sum of the hormone band intensities
from the pellet and supernatant])
100. The values
represent the means ± S.E. from at least three experiments.
Budding efficiency in the absence of A23187 is shown and was not
significantly affected by inclusion of 1 µM A23187 (Fig. 1A and data not
shown).
To confirm the lumenal calcium requirement for pro-SRIF cleavage in vivo, intact cells were incubated at 37 °C with or without free calcium in the presence of A23187 following a 2 h chase at 19 °C. Consistent with the in vitro results, chelation of cellular free calcium with EGTA in the presence of A23187 inhibited pro-SRIF cleavage (Fig. 2). Pro-SRIF cleavage efficiency in the presence of A23187 was significantly higher in samples containing trace (10-50 µM) or 10 mM added calcium, although for unknown reasons, processing was not completely restored to control levels. Thus lumenal free calcium in the low micromolar range was also sufficient for endoproteolytic maturation of pro-SRIF in intact cells.
Figure 2:
Cleavage of pro-SRIF in intact cells
requires submillimolar calcium. Cells were pulse-labeled with
[S]methionine for 12 min at 37 °C, chased
for 2 h at 19 °C, and then preincubated with or without 5
µM A23187 in the presence of 10 mM EGTA,
0.03 mM (con), or 10.0 mM calcium
chloride for 10 min at 4 °C, followed by a further incubation for 2
h at 37 °C. Pro-SRIF cleavage efficiency was determined by
immunoprecipitation from cell lysates and media followed by
densitometric quantitation of the fluorograms. The values represent the
means ± S.E. from at least three
experiments.
Our earlier studies demonstrated that a
pH of 6.0-6.2 is necessary for pro-SRIF cleavage in the TGN (Xu
and Shields, 1994), a value that corresponds to estimates of the
endogenous lumenal pH in the TGN (Seksek et al., 1995; Yilla et al., 1993; Anderson and Orci, 1988). To determine if the
lumenal free calcium requirement for pro-SRIF cleavage is independent
of the pH requirement, permeabilized cells were incubated under varied
calcium and pH conditions in the presence of both A23187 and the
protonophore CCCP simultaneously (Fig. 3, black bars).
When lumenal calcium was chelated with EGTA at the suboptimal pH of 7.3
(double nonpermissive condition), cleavage was inhibited. In contrast,
when millimolar free calcium was present at the optimal pH of 6.1
(double permissive condition), pro-SRIF cleavage was 45-50%
efficient. Chelation of lumenal free calcium with EGTA at the optimal
pH of 6.1 (single nonpermissive condition) only partially inhibited
pro-SRIF cleavage because the affinity of EGTA for calcium is strongly
pH-dependent and greatly diminished at low pH (Bers et al.,
1994). The calcium chelator BAPTA exhibits a much lower pH sensitivity
and consequently is more effective at pH 6.1 (Bers et al.,
1994). When permeabilized cells were treated with BAPTA at the optimal
pH of 6.1, pro-SRIF maturation was potently inhibited (<10%
cleavage). As observed for the single ionophore experiments, trace
micromolar free calcium maintained efficient pro-SRIF cleavage at pH
6.1 in the presence of A23187 and CCCP (data not shown). Thus even at
the optimal pH of 6.1 lumenal free calcium (no more than 15
µM) is absolutely required for efficient endoproteolytic
maturation of pro-SRIF. In contrast to our previous single ionophore
experiments with CCCP at pH 7.3 (Xu and Shields, 1994), inhibition of
cleavage at this pH was not complete in the presence of both ionophores
and 10 mM calcium (data not shown) for reasons that are
unclear at present. Nevertheless, our data here demonstrate that
although acidic lumenal pH is important, lumenal free calcium is
absolutely required for efficient pro-SRIF cleavage. Cleavage
efficiency was much less sensitive to calcium and pH manipulations when
ionophores were omitted from the incubations (Fig. 3, hatched bars), consistent with the ability of the TGN and
nascent secretory vesicles of the permeabilized cells to maintain a
relatively stable lumenal environment in the presence of variations in
the extralumenal environment.
Figure 3: Lumenal free calcium and pH requirements for pro-SRIF cleavage in vitro. Cleavage of pro-SRIF in permeabilized cells at optimal pH requires calcium. Permeabilized cells were preincubated with or without 1 µM A23187 and 30 µM CCCP at pH 7.35 or 6.1 in the presence of either 2 mM EGTA, 2 mM BAPTA, or 10.0 mM calcium chloride for 10 min at 4 °C, followed by a further incubation with ATP and GTP for 2 h at 37 °C. Pro-SRIF cleavage efficiency was determined by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis and densitometry. The values represent the means ± S.E. from at least three experiments.
Figure 5:
Prolactin is packaged into sedimentable
and membrane-enclosed secretory vesicles. Permeabilized cells were
incubated as in Fig. 1with 1 µM A23187 in the
presence of 2 mM EGTA. One sample (CON) was separated
into 15,000 g pellet (lane 1) and supernatant (lane 2) fractions; a second sample (SED) separated
into 15,000
g pellet (lane 3) and supernatant
fractions. This supernatant was then centrifuged at 200,000
g for 20 min to generate a second pellet (lane 4)
(nascent vesicle) and supernatant (lane 5) fractions. Samples PtK and PtK/Det were incubated with 25 µg/ml
proteinase K in the absence or the presence of 1% Triton X-100,
respectively, for 15 min at 4 °C, quenched with 1 mM
phenylmethylsulfonyl fluoride, and separated into 15,000
g pellet (lanes 6 and 8) and supernatant (lanes 7 and 9) fractions. Budding efficiency was
determined by immunoprecipitation using rabbit anti-prolactin serum.
For the SED sample, budding efficiency is defined as
([prolactin band intensity from the SP fraction]
[sum of the prolactin band intensities from the P, SP, and SS fractions]). Similar
results were obtained for budding of growth hormone and SRIF propeptide
+ pro-SRIF (not shown).
Figure 6:
Basal secretion of pro-SRIF and prolactin
from intact cells requires calcium. Cells were pulse-labeled with
[S]methionine for 12 min at 37 °C, chased
for 2 h at 19 °C, and then preincubated with or without 5
µM A23187 in the presence of 10 mM EGTA,
0.03 mM (con) or 10.0 mM calcium
chloride for 10 min at 4 °C, followed by a further incubation for 2
h at 37 °C. Secretion efficiency was determined by
immunoprecipitation from cell lysates and media followed by
densitometric quantitation of the fluorograms and is defined as
([hormone band intensity from the medium fraction]
[sum of the hormone band intensities from the medium and cell
lysate fractions]). The values represent the means ± S.E.
from at least three experiments.
An advantage of using a permeabilized cell system to study
organelle function is that cytosolic conditions can be directly
manipulated without necessarily changing conditions within the lumen of
the organelle. Our permeabilized cell system allows us to examine an
important event in the lumen of the TGN and immature secretory
granules: the endoproteolytic maturation of a prohormone. In addition,
this system permits examination of an isolated step in the transport of
hormones from the TGN to the cell surface: the formation of nascent
secretory vesicles. In this study, we exploited the permeabilized cell
system to (a) determine the lumenal free calcium requirement
for endoproteolytic maturation of pro-SRIF, (b) compare the
requirements for low pH versus calcium for pro-SRIF
processing, and (c) determine the effect of perturbed calcium
concentration and pH on the formation of vesicles from the TGN. Our
data demonstrate that low levels (15 µM) of lumenal
free calcium are sufficient for efficient endoproteolytic maturation of
pro-SRIF in permeabilized GH
cells (Fig. 1, A and B). This is independent of the requirement for low
pH, because inefficient cleavage occurred at the optimal pH of 6.1 when
lumenal free calcium was chelated with BAPTA (Fig. 3). The
requirement for submillimolar free calcium could also be demonstrated
in intact cells (Fig. 2).
In light of the high levels of
intralumenal calcium (estimated to be 10 mM or higher) in the
TGN and secretory granules (Chanat and Huttner, 1991), it was
surprising that such low concentrations of free calcium were sufficient
to support pro-SRIF cleavage. However, much of this calcium may in fact
be bound to high capacity, low affinity calcium-binding proteins such
as the chromogranins (Rosa et al., 1992; Huttner et
al., 1991; Reiffen and Gratzl, 1986). Indeed, studies using a
calcium-selective electrode suggest that in chromaffin granules, only
24 µM calcium exists in the free unbound state
(Bulenda and Gratzl, 1985). Thus our present observations showing a low
micromolar free calcium requirement for pro-SRIF cleavage is consistent
with earlier measurements of endogenously available lumenal free
calcium in regulated secretory granules. We do not know if the exchange
between free and protein-bound pools of calcium is sufficiently rapid
to permit depletion of the protein-bound pool when free calcium is
chelated during our incubations with A23187, although laser scanning
confocal and ion microscopy studies revealed significant (70-90%)
depletion of total lumenal calcium in the Golgi of porcine kidney
LLC-PK
, mouse fibroblast 3T3, and rat myoblast L5 cells by
5 min using EGTA in the presence of A23187 (Chandra et al.,
1991). Thus the free calcium requirement for pro-SRIF cleavage in our
system could be indirect through maintenance of the protein-bound
lumenal pool.
A direct requirement for submillimolar free calcium is
consistent with the observed in vitro properties of PC2, the
enzyme likely responsible for pro-SRIF cleavage in our system (Brakch et al., 1995; Seidah et al., 1994; Xu and Shields,
1994; Mackin et al., 1991). Using PC2 purified from rat
insulinoma granules, Hutton and colleagues observed low micromolar
calcium optima for cleavage of purified chromogranin A (K
11 µM) (Arden et
al., 1994), proopiomelanocortin (K
=
5-80 µM) (Rhodes et al., 1993), and
proinsulin (K
=
100 µM)
(Bennett et al., 1992). The calcium optimum for cleavage by
PC2 synthesized in vitro using Xenopus egg extracts,
however, was higher (K
=
1-4
mM) (Shennan et al., 1995). In contrast to PC2
purified from granules, PC1 displayed a much higher calcium optimum in vitro (K
=
2.5 mM for proinsulin cleavage; Davidson et al.(1988) and Zhou
and Lindberg(1993)).
Our data also demonstrate that free calcium is
not required for vesicle formation from the TGN of permeabilized cells (Fig. 1A and 4). A similar observation was made
previously using permeabilized MDCK cells (Bennett et al.,
1988). Interestingly, a recent study investigating the sorting of
secretogranin II from the TGN of permeabilized PC12 cells (Carnell and
Moore, 1994) suggests that lumenal free calcium may not be required for
sorting. In our system, formation of vesicles from the TGN was strongly
inhibited by extralumenal free calcium roughly 5 orders of magnitude
greater than physiological cytoplasmic conditions (Fig. 1A and Fig. 4). We were unable to demonstrate a calcium block
on TGN exit in intact cells (Fig. 6) and presume that in
permeabilized cells, millimolar calcium nonspecifically disrupts the
TGN budding machinery. Interestingly, elevated cytosolic calcium levels
(100 µM) in permeabilized cells can cause
mistargeting of plasma membrane clathrin-coated vesicle adaptor
complexes (Seaman et al., 1993). We also found that an acidic
extralumenal pH of 6.1 strongly inhibits both budding in permeabilized
cells and secretion in intact cells (not shown). Inhibition of
clathrin-mediated transport events from the TGN and plasma membrane by
cytosol acidification is well documented (Hansen et al., 1993;
Cosson et al., 1989; Heuser and Anderson, 1989; Heuser, 1989;
Sandvig et al., 1987) and appears to involve reversible
structural alterations in the clathrin cages.
Significantly, our results reveal that pro-SRIF endoproteolytic maturation and entry into nascent secretory vesicles are not obligatorily coupled events (compare Fig. 1B and Fig. 4). Pro-SRIF cleavage was inhibited by chelation of lumenal free calcium, whereas formation of nascent vesicles was unimpaired. Conversely, vesicle formation but not pro-SRIF cleavage was inhibited by raising extralumenal free calcium to millimolar levels or by lowering the pH, which reinforces our previous finding that pro-SRIF endoproteolytic maturation can proceed in the TGN if exit is prevented (Xu and Shields, 1993). These observations contrast with recent studies using pancreatic islets (Huang and Arvan, 1994), which suggested that proinsulin endoproteolytic maturation cannot proceed if TGN exit is blocked. It is not clear if this discrepancy is due to tissue- or substrate-specific differences or methodological differences. It is noteworthy that proinsulin processing requires both the activities of PC1 and PC2, the former having significantly higher calcium and lower pH requirements than the latter (Zhou and Lindberg, 1993; Rhodes et al., 1993; Davidson et al., 1988). Overall, our data demonstrate that by using the permeabilized cell system, prohormone endoproteolytic maturation can be dissociated from entry into secretory vesicles.
Inhibition of
pro-SRIF basal secretion from intact cells by chelation of free
calcium, and to a lesser extent that of prolactin and growth hormone,
reveals a calcium-dependent transport step distal to TGN exit (Fig. 6). An important distinction must be made between basal
secretion and constitutive pathway secretion. Distal steps in
constitutive secretion do not require calcium (Turner et al.,
1992; Miller and Moore, 1991). In the regulated pathway by contrast,
calcium-dependent fusion of secretory vesicles with the plasma membrane
has been well documented (Turner et al.(1992) and Martin and
Walent(1989); for review see Kelly (1993)). We propose that
calcium-dependent basal secretion is in fact secretion by the regulated
pathway rather than the constitutive pathway. It is conceivable that
the threshold for activation of the regulated pathway has been
diminished in immortalized cell lines such as GH,
permitting some secretion by this pathway even in the absence of
secretagogues. Consistent with this hypothesis, two-thirds of basally
secreted SRIF-related material was released in a calcium-dependent
manner (Fig. 6). If this material were largely derived from the
regulated pathway, it should have undergone significant processing.
Indeed, we found that two-thirds of basally released SRIF-related
material appeared as free propeptide (67 ± 9%, data not shown)
and mature SRIF (
60%, Stoller and Shields, 1988). Accordingly, we
feel that calcium dependence more precisely defines regulated secretion
than secretagogue stimulation above basal levels.