(Received for publication, June 9, 1995; and in revised form, October 16, 1995)
From the
Chromogranins A and B (CGA and CGB), the major proteins of the
secretory vesicles of the regulated secretory pathway, have been shown
to aggregate in a low pH and high calcium environment, the condition
found in the trans-Golgi network where secretory vesicles are
formed. Moreover, CGA and CGB, as well as several other secretory
vesicle matrix proteins, have recently been shown to bind to the
vesicle membrane at the intravesicular pH of 5.5 and to be released
from it at a near physiological pH of 7.5. The pH- and
Ca-dependent aggregation and interaction of
chromogranins, as well as several other matrix proteins, with the
vesicle membrane are considered essential in vesicle biogenesis.
Therefore, to gain further insight into how vesicle matrix proteins
find their way into the secretory vesicles, the pH- and
Ca
-dependent aggregation and vesicle membrane binding
properties of the vesicle matrix proteins were studied, and it was
found that most of the vesicle matrix proteins aggregated in the
presence of Ca
at the intravesicular pH of 5.5.
Furthermore, most of the vesicle matrix proteins bound not only to the
vesicle membrane but also to CGA at pH 5.5, with the exception of a few
matrix proteins that appeared to bind only to CGA or to vesicle
membrane. Purified CGB was also shown to interact with CGA at pH 5.5.
The extent and Ca
-sensitivity of the aggregation of
vesicle matrix proteins lay between those of purified CGB and CGA, CGB
aggregation showing the highest degree of aggregation and being the
most Ca
sensitive at a given protein concentration.
Hence, in view of the abundance of chromogranins in secretory vesicles
and their low pH- and high calcium-dependent aggregation property,
combined with their ability to interact with both the vesicle matrix
proteins and the vesicle membrane, CGA and CGB are proposed to play
essential roles in the selective aggregation and sorting of potential
vesicle matrix proteins to the immature secretory vesicles of the
regulated secretory pathway.
One of the fundamental interests in secretory vesicle biogenesis
is to understand how the regulated secretory proteins find their way
into the secretory vesicles of the regulated secretory pathway. Unlike
many other cellular organelles, secretory vesicles contain high
concentrations of Ca and H
, bringing
their intravesicular Ca
concentration to a millimolar
range (Winkler and Westhead, 1980; Bulenda and Gratzl, 1985; Hutton,
1989) and the pH to 5.5 (Johnson and Scarpa, 1976; Casey et
al., 1977). The high calcium and low pH environment exists in the trans-Golgi network (TGN) (
)where secretory
vesicles are formed due to the presence of Ca
and
H
pumps (Roos, 1988; Hutton, 1989; Mata and Fink,
1989; Orci et al., 1986). This acidic pH and high
Ca
milieu appears to be critical for secretory
vesicle formation in the TGN. Interestingly, the major constituents of
secretory vesicles, the chromogranins, were shown to aggregate in an
acidic pH and high calcium environment (Gerdes et al., 1989;
Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner,
1991; Yoo, 1995a), the condition found in the TGN. Based on this low
pH- and high Ca
-induced aggregation property,
combined with their abundance in secretory vesicles, chromogranins A
and B were suggested to play key roles in secretory vesicle biogenesis
(Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and
Huttner, 1991; Pimplikar and Huttner, 1992; Yoo, 1995a). Nevertheless,
there was a marked difference in Ca
sensitivity
between CGA and CGB despite the similarity in amino acid sequences and
the high acidic nature of both proteins (Benedum et al., 1986;
Iacangelo et al., 1986; Bauer and Fischer-Colbrie, 1991).
Chromogranin B exhibited a Ca
sensitivity at least
two orders of magnitude greater than that of chromogranin A (Yoo,
1995a); CGB aggregation required less than 1/100th the Ca
concentration required for CGA aggregation at pH 5.5 (Yoo,
1995a), suggesting that CGB will begin to aggregate in the TGN long
before CGA aggregation occurs.
We have shown previously that
chromogranin A interacts with the vesicle membrane at the
intravesicular pH of 5.5 and is released from it at a near
physiological pH of 7.5 (Yoo, 1993a). Subsequent studies indicated that
chromogranin A interacts with several integral membrane proteins of
secretory vesicles, including a 260-kDa protein reactive to inositol
1,4,5-trisphosphate receptor antibody (Yoo, 1994). Similarly,
chromogranin B has also been shown recently to interact with the
secretory vesicle membrane at pH 5.5 but not at pH 7.5 (Yoo, 1995b),
and its interaction with the vesicle membrane was much stronger than
that of CGA (Yoo, 1995b), suggesting the interaction of CGB with the
vesicle membrane long before the interaction of other vesicle matrix
proteins, including CGA, with the vesicle membrane. In addition,
chromogranins A and B bind Ca (Bulenda and Gratzl,
1985; Reiffen and Gratzl, 1986) and have been shown to undergo pH- and
Ca
-dependent conformational changes (Yoo and
Albanesi, 1990, 1991; Yoo, 1995a). Moreover, the Ca
binding is known not only to cause conformational changes both at
pH 5.5 and 7.5 but also to expose previously buried hydrophobic regions
of CGA (Yoo and Albanesi, 1990). Although the pH-dependent
conformational changes were proposed to be responsible for the dimeric
existence of CGA at pH 7.5 and the tetrameric existence of CGA at pH
5.5 (Yoo and Lewis, 1992), Ca
was instrumental in
changing the tetramerization reaction of CGA from an enthalpically
driven to an entropically driven reaction (Yoo and Lewis, 1992), thus
clearly underscoring the role Ca
plays in the
self-association of CGA. Furthermore, Ca
stabilized
the interaction between tetrameric CGA and four molecules of the
intraluminal loop peptide of inositol 1,4,5-trisphosphate
receptor/Ca
channel (Yoo and Lewis, 1995).
In view
of the need for the vesicular matrix proteins of the regulated
secretory pathway to be segregated from other proteins and to associate
with the potential vesicle membrane during vesicle biogenesis in the
TGN (Huttner et al., 1988, 1991a; Yoo, 1993b), elucidation of
the sorting process of the vesicle matrix proteins is essential for a
better understanding of vesicle biogenesis. Hence, in the present
study, the pH- and Ca-induced aggregation property of
the vesicular matrix proteins has been studied along with the
interactions between CGA and CGB and between CGA and the vesicle matrix
proteins. The results are discussed in the context of the proposed
roles of chromogranins A and B in vesicle biogenesis.
In light of the pH- and Ca-induced
aggregation of chromogranins (Gerdes et al., 1989; Gorr et
al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991; Yoo,
1995a), the pH- and Ca
-dependent aggregation of the
secretory vesicle matrix proteins was also studied by measuring
Ca
-induced turbidity changes (Fig. 1). As
shown in Fig. 1A, Ca
induced
aggregation of the vesicle matrix proteins, and the aggregation pattern
was dependent upon the protein concentration; a lower Ca
concentration was needed to induce the aggregation as the protein
concentration was increased. Moreover, the extent of aggregation was
also dependent on Ca
concentration at each protein
concentration.
Figure 1:
Aggregation of secretory vesicle lysate
as a function of Ca concentration. A,
secretory vesicle lysate concentrations are 0.8 mg/ml (
), and 0.4
mg/ml (
). The A
values shown are those
taken 2 min after the addition of Ca
. The dashed portion of each curve indicates that the aggregation
peaked and the aggregated protein started to precipitate out of the
solution. B, at three different levels of aggregation as
indicated by numbers 1-3 above the upper aggregation
curve (0.8 mg/ml) in A, 100 µl from each level of
aggregation were pelleted by centrifugation at 200,000
g for 1 h. After removal of the supernatant, the pellet was
resuspended in 60 µl of water. 20-µl aliquots from each
supernatant and pellet suspension were then separated on a 7%
SDS-polyacrylamide gel. Lane a, vesicle lysate (10 µg); lanes b-d, pellet (P) 1-3 (the numbers 1, 2, and 3 indicate the protein aggregates
1, 2, and 3 as shown in the upper aggregation curve of A); lanes e-g, supernatant (S)
1-3. Intact CGB and CGB fragments are indicated by closed arrows, and CGA by an open
arrow.
In order to determine what kind of matrix proteins
are aggregated in the presence of increasing concentration of
Ca at pH 5.5, the matrix protein aggregates at three
different levels of aggregation were pelleted by centrifugation. The
pelleted proteins were then separated on a 7% SDS-polyacrylamide gel.
As shown in Fig. 1B, most of the matrix proteins were
present in the pellet obtained from the highest level of aggregation
(see lane d), clearly demonstrating the fact that most of the
matrix proteins eventually aggregated in the presence of Ca
at pH 5.5.
In light of the fact that CGA and CGB aggregation
is highly dependent on pH, i.e. the extent and Ca sensitivity of the CGA and CGB aggregation at pH 5.5 are at least
2 times higher and more sensitive than those at pH 7.5 (Yoo and
Albanesi, 1990; Yoo, 1995a), the effect of pH on the aggregation of
vesicle matrix proteins was also studied (Fig. 2). However,
unlike the CGA and CGB aggregation in which the extent of CGA and CGB
aggregation at pH 7.5 was approximately one-third (Yoo and Albanesi,
1990) and one-half (Yoo, 1995a) that at pH 5.5, respectively, there was
very little aggregation of the vesicle matrix proteins at pH 7.5.
Figure 2:
Effect of pH on secretory vesicle lysate
aggregation. Aggregation of vesicle lysate proteins (0.4 mg/ml) at pH
5.5 () and 7.5 (
) was measured as a function of
Ca
concentration. The buffers were either 20 mM sodium acetate, pH 5.5, or 20 mM MOPS, pH 7.5. The A
values are those taken 2 min after the
addition of Ca
.
In
view of the observation that most of the vesicle matrix proteins
aggregated in the presence of Ca at pH 5.5 (Fig. 1), it was of interest to determine whether constitutive
secretory proteins can co-aggregate with the vesicle matrix proteins of
the regulated secretory pathway. To address this question, a
constitutive secretory protein, immunoglobulin G (IgG), was mixed with
the vesicle matrix proteins and the Ca
-induced
aggregation at pH 5.5 was studied (Fig. 3). Although IgG itself
failed to aggregate in the presence of Ca
at pH 5.5
(not shown), the mixture of vesicle matrix proteins and IgG aggregated
in a Ca
- and protein concentration-dependent manner (Fig. 3A). To analyze the aggregated proteins, the
protein aggregates were pelleted by centrifugation, and the proteins
were separated on a SDS-polyacrylamide gel in nonreducing condition. As
shown in Fig. 3B, IgG appeared primarily in the
supernatant. Densitometric quantitation of the relative amount of IgG
over the reference protein CGA in each sample showed that in the
original mixture (lane d) the ratio of IgG to CGA was 0.17 and
the IgG/CGA ratio in the supernatant (lane a) was 0.33,
whereas that in the pellet (lane b) was 0.05, clearly
indicating that IgG failed to aggregate under the condition that caused
the pH- and Ca
-induced aggregation of the vesicle
matrix proteins. This result underscores the specific nature of the pH-
and Ca
-induced aggregation of the vesicle matrix
proteins of the regulated secretory pathway.
Figure 3:
Aggregation of secretory vesicle lysate
and immunoglobulin G mixture as a function of Ca
concentration. A, aggregation of the mixture of vesicle lysate
and immunoglobulin G in 15 mM sodium acetate, pH 5.5, was
measured at two different concentrations of the mixture as a function
of Ca
concentration. The A
values are those taken 2 min after the addition of
Ca
. B, at the highest level of aggregation
as indicated by an arrow above the upper aggregation curve (0.8 mg of vesicle lysate/ml + 0.04 mg of IgG/ml), 100 µl
of the aggregate were pelleted by centrifugation at 200,000
g for 1 h. After removal of the supernatant, the pellet was
resuspended in water. 5 µg of proteins each from the supernatant
and the pellet suspension were then separated on a 7%
SDS-polyacrylamide gel in nonreducing condition. Lane a,
supernatant (S) (5 µg); lane b, pellet (P) (5 µg); lane c, immunoglobulin G (0.3
µg); lane d, vesicle lysate (7 µg) + IgG (0.35
µg); lane e, vesicle lysate (7
µg).
In order to delineate
the relative contribution of each of CGA and CGB aggregation to the
overall aggregation of the vesicle matrix proteins, the extent and
Ca sensitivity of the aggregation of CGA, CGB, and
the vesicle matrix proteins were compared. As shown in Fig. 4,
CGB exhibited the highest degree of aggregation and was the most
sensitive to Ca
, whereas CGA lagged behind the matrix
proteins both in Ca
sensitivity and in the extent of
aggregation.
Figure 4:
Comparison of vesicle lysate aggregation
with those of chromogranin A and chromogranin B as a function of
Ca concentration. Protein concentration was 0.4 mg/ml
in 15 mM sodium acetate, pH 5.5. The A
values for CGB (
), vesicle lysate (
), and CGA (
)
are those taken 2 min after the addition of
Ca
.
Although most of the vesicle matrix proteins aggregated
in the presence of Ca at pH 5.5, it was still not
clear how these proteins find their way into the secretory vesicles.
Given the fact that they do end up in the vesicle, it appeared that
these matrix proteins have to either bind to the vesicle membrane
directly or interact with CGA or CGB, which is known to bind to the
vesicle membrane (Yoo, 1993a, 1995b), for their entry into the vesicle.
In fact, it has been shown previously that several vesicle matrix
proteins bind to the vesicle membrane at pH 5.5 and are released from
it at pH 7.5 (Yoo, 1993b). However, since CGA has been known to bind to
the vesicle membrane at pH 5.5, it was not clear what portion of the
vesicle membrane-bound matrix proteins interacted with the vesicle
membrane, not through their own interaction with the vesicle membrane,
but by virtue of their binding to CGA which in turn bound to the
vesicle membrane.
Therefore, in order to determine whether any of the vesicle matrix proteins binds to CGA at pH 5.5, the pH of the vesicle lysates was adjusted to 5.5, and the lysates were loaded onto a CGA-coupled Sepharose 4B column equilibrated with a pH 5.5 buffer (Fig. 5). After thorough washing, the column was eluted with a 1 M KCl buffer. As shown in Fig. 5, a small amount of protein was eluted in the high salt elution, suggesting the possibility that a large amount of protein is retained in the column. Further elution with a pH 7.5 buffer released a large amount of protein from the column, thus suggesting that a significant amount of vesicle matrix proteins was bound to CGA at pH 5.5 and released from it when the pH was raised to 7.5.
Figure 5: Chromogranin A-coupled Sepharose 4B chromatography of the matrix proteins of the secretory vesicle. 1 mg of the vesicle matrix proteins in 1 ml of buffer A (20 mM sodium acetate, pH 5.5, 0.1 M KCl) was loaded onto a CGA-coupled Sepharose 4B column (0.8-ml volume) equilibrated with buffer A. The column was washed with 10 ml each of buffer A, followed by buffer A containing 0.15 M KCl, and again with buffer A. The protein then was eluted with 1 M KCl in the same acetate buffer. After this high salt elution, the pH of the elution buffer was changed to 7.5 (buffer B: 20 mM Tris-HCl, pH 7.5, 1 M KCl), and the elution was continued. The fraction size was 1 ml/fraction for the washes (Fraction I) and 0.5 ml/fraction for the elutions (Fraction II), and the chromatography was carried out at room temperature.
To determine whether the change of pH from 5.5 to
7.5 was sufficient to free the bound matrix proteins from CGA, the
column was loaded with the lysate proteins, washed thoroughly, and
directly eluted with a pH 7.5 buffer. As shown in Fig. 6A, elution of the column with a pH 7.5 buffer
(buffer B: 20 mM Tris-HCl, pH 7.5, 1.0 M KCl)
released a large amount of protein from the column, comparable in
quantity to that released by the high salt elution plus the pH 7.5
elution shown in Fig. 5, indicating that the change of pH to 7.5
was sufficient to free most of the bound vesicle matrix proteins. Since
the vesicle lysate solution used in the present study already contained
approximately 0.5-1.0 mM Ca, due to
approximately 40 mM intravesicular Ca
already present inside the secretory vesicles, this concentration
of Ca
appeared sufficient for the matrix proteins to
bind CGA at pH 5.5. Further addition of more Ca
(up
to 4 mM) did not affect the results appreciably, although
removal of Ca
from the vesicle lysate by Chelex 100
treatment and addition of 2 mM EGTA significantly reduced the
amount of vesicle lysate proteins bound to CGA (Fig. 6B).
Figure 6:
Chromogranin A-coupled Sepharose 4B
chromatography of the matrix proteins of the secretory vesicle. The
chromatography was carried out as described in Fig. 5except
that two different conditions were used. A, after the washing
step (-
) the elution was carried out with a pH
7.5 buffer (buffer B) (
-
). B, after
the loading and washing step in the absence of Ca
(
-
) the elution was carried out with the
same pH 7.5 buffer (buffer B) containing 2 mM EGTA
(
-
). Other conditions are the same as in Fig. 5.
The pH-dependent binding of matrix proteins appeared to be a specific interaction between the matrix proteins and chromogranin A, for the CNBr-activated Sepharose 4B resin that had gone through all the coupling routine but without CGA was not able to bind to any of the matrix proteins. The CGA-coupled column used in the present experiment, which had 0.8 ml (wet volume) of resin with 1.08 mg of coupled CGA, was able to bind and elute approximately 85-90 µg of matrix proteins. Although a saturating amount (1 mg) of matrix proteins was loaded onto the column in the present experiment, a similar amount of matrix proteins was bound and eluted from the column when as little as 450 µg of matrix proteins were loaded. However, loadings of less than 450 µg of matrix proteins began to lower the amount of matrix proteins bound and eluted. When 300 µg of matrix proteins were loaded, approximately 55-60 µg of matrix proteins was eluted from the same column. This result indicated that 1.08 mg of immobilized CGA can bind approximately 85-90 µg of matrix proteins, and the amount of matrix proteins bound appeared to reflect an upper limit of binding by the column from the fact that a proportionately larger amount of proteins was bound and eluted from columns with a larger volume of the CGA-coupled resin (not shown).
The interaction between the vesicle
matrix proteins and CGA was manifested only at the intravesicular pH of
5.5, since all of the matrix proteins appeared to pass right through
the CGA-coupled column at pH 7.5 without any sign of interaction.
Moreover, the presence or absence of Ca did not
affect the result at pH 7.5, strongly demonstrating the importance of
the acidic pH.
To analyze the CGA-interacting matrix proteins, the eluted proteins were separated on a 10% SDS-polyacrylamide gel. As shown in Fig. 7, several vesicle matrix proteins bound to CGA at pH 5.5 and were released at pH 7.5. In particular, the four proteins larger than CGA, along with several smaller proteins, have been shown to interact with CGA at pH 5.5.
Figure 7: SDS-PAGE of the vesicle lysate proteins and the eluted proteins. The eluted proteins from the CGA-coupled Sepharose 4B column containing 1 M KCl were diluted 5-fold with 20 mM Tris-HCl, pH 7.5, to reduce the salt concentration. The diluted proteins were then concentrated 25-fold, and 20-µl aliquots are analyzed on a 10% SDS-polyacrylamide gel and visualized by Coomassie staining. Lane a, preloading vesicle lysate proteins (12 µg); lane b, the flow-through (10 µg) from the underloaded column (350 µg loaded); lanes c, d, and e, fractions 14, 15, and 16 of the pH 7.5, 1 M KCl elution of Fig. 5. Intact CGB and CGB fragments are indicated by closed arrows and CGA by an open arrow.
Moreover, in order to determine whether the pH-dependent matrix protein binding to CGA can also be demonstrated with other proteins, bovine serum albumin was coupled to CNBr-activated Sepharose 4B for a pH-dependent matrix protein binding study. Bovine serum albumin is an acidic protein like chromogranin A, and has a molar mass of 67 kDa. Although the bovine serum albumin-coupled Sepharose 4B column appeared to bind and release, on a weight to weight basis of the proteins coupled, approximately 10-15% of the matrix proteins that can be bound and released by a comparable CGA-coupled column, analysis of these eluates by a 10% SDS-polyacrylamide gel and Coomassie staining as has been done in Fig. 7showed no discernible protein bands (not shown), clearly indicating that other proteins cannot substitute for CGA in exerting pH-dependent matrix protein binding activity.
In view of the fact that many vesicle matrix proteins also bind to the vesicle membrane at pH 5.5 and are released from it at pH 7.5 (Yoo, 1993b), it was of interest to compare the vesicle matrix proteins that bind to CGA and to the vesicle membrane. Therefore, the eluted proteins from both the vesicle membrane-coupled and the CGA-coupled columns were separated by SDS-PAGE. As shown in Fig. 8, some vesicle matrix proteins were eluted only from the CGA-coupled column, while others were eluted only from the vesicle membrane-coupled column, although some of them were eluted from both columns. These results indicated that several vesicle matrix proteins are capable of interacting with both CGA and vesicle membrane in the vesicle, while some appeared to interact with either the vesicle membrane or CGA exclusively.
Figure 8: SDS-PAGE of the eluted vesicle lysate proteins from the CGA-coupled and the vesicle membrane-coupled Sepharose 4B columns. The vesicle lysate proteins (3.5 µg each) eluted from the CGA-coupled (lane a) and the vesicle membrane-coupled (lane b) columns were separated on a 10% SDS gel and visualized by Coomassie staining. Intact CGB and CGB fragments are indicated by closed arrows and CGA by an open arrow.
In order to identify some of the eluted proteins, the vesicle matrix proteins were separated on a two-dimensional SDS-polyacrylamide gel (Fig. 9), and four proteins which are larger than CGA (marked with arrows) and had been shown to interact with both CGA and the vesicle membrane at pH 5.5 were isolated and sequenced as described under ``Experimental Procedures.'' The top three proteins (marked with open arrows from the left side) had the N-terminal sequences of MPVDIR(NH), identical to that of bovine chromogranin B (Bauer and Fischer-Colbrie, 1991), while a tryptic fragment of the fourth protein (marked with an arrow from the right side) had a sequence of LVNXA, corresponding to residues 289-(292)293 of bovine secretogranin II (also called chromogranin C) (Fischer-Colbrie et al., 1990). This result indicated that the top three proteins were chromogranin B, the smaller CGB fragments being CGB with proteolyzed C-terminal ends, whereas the fourth protein was secretogranin II.
Figure 9: Two-dimensional electrophoresis of the vesicle lysate proteins. The secretory vesicle lysate proteins (26 µg) from bovine adrenal medulla were subjected to two-dimensional electrophoresis. Four proteins that are larger than CGA were isolated and sequenced as indicated by arrows. The determined amino acid sequences (in the single-letter code) are shown along with the corresponding sequences of CGB and secretogranin II (SgII). The numbers above the amino acids indicate the position of each amino acid residue in the protein.
To further determine whether CGB interacts with CGA directly, purified CGB (the mixture of intact CGB and two CGB fragments as indicated by open arrows in Fig. 9) was loaded onto a CGA-coupled Sepharose 4B column, washed thoroughly, and eluted with a pH 7.5 buffer (buffer B). As shown in Fig. 10, chromogranin B was eluted from the CGA-coupled column, indicating that CGB bound to CGA at pH 5.5 and was released at pH 7.5. In the present experiment where a mixture of intact CGB and two CGB fragments without the C-terminal regions was loaded, the eluted proteins consisted of the intact CGB plus CGA released from the column (see Yoo(1994)) and a varying amount of CGB fragments. The CGA-coupled Sepharose 4B column used in the present experiments has been extensively washed before loading the sample so that no CGA was eluted by eluting with the buffers. However, when the column was loaded with the CGA-interacting proteins, elution caused a release of some CGA from the CGA-Sepharose column (Yoo, 1994). This is probably due to the fact that CGA molecules exist in a dimeric or tetrameric state (Yoo and Lewis, 1992) and that some CGA which are not covalently bound to the resin still remain in the column owing to their interaction with other CGA molecules that bound to the resin covalently. When these noncovalently bound CGA begin to interact with the newly loaded CGA-interacting proteins, this new interaction may weaken the existing CGA-CGA interaction in the column, thus resulting in the release of some of these CGA when other CGA-interacting proteins are eluted from the column. Moreover, in spite of the observation that CGA appeared to interact primarily with intact CGB at pH 5.5 (Fig. 10), a significant amount of the CGB fragments without the C-terminal regions was also eluted from the column although the amount varied from experiment to experiment, suggesting that these CGB fragments also interact with CGA to a certain extent.
Figure 10: Chromogranin A-coupled Sepharose 4B chromatography of purified chromogranin B and SDS-PAGE of the eluted proteins. A, 160 µg of purified bovine chromogranin B (a mixture of intact CGB and two CGB fragments) in 1.2 ml of buffer A (20 mM sodium acetate, pH 5.5, 0.1 M KCl) were loaded onto a CGA-coupled Sepharose 4B column (0.8-ml volume) equilibrated with buffer A, washed thoroughly as in Fig. 5, and eluted with a pH 7.5 buffer (buffer B: 20 mM Tris-HCl, pH 7.5, 1 M KCl). The fraction size was 1 ml/fraction for the washes and 0.5 ml/fraction for the elutions, and chromatography was carried out at room temperature. B, the eluted proteins from the CGA-coupled Sepharose 4B column containing 1 M KCl were diluted 5-fold with 20 mM Tris-HCl, pH 7.5, to reduce the salt concentration. The diluted proteins were then concentrated 25-fold, and 20-µl aliquots were analyzed on a 10% SDS-polyacrylamide gel and visualized by Coomassie staining. Lane a, vesicle lysate proteins (8 µg); lane b, the preloading purified CGB (1 µg); lanes c, d, and e, fractions 3, 4, and 5 of the pH 7.5-1 M KCl elution (Fraction II) of A. Intact CGB and CGB fragments are indicated by closed arrows, and CGA by an open arrow.
The present results demonstrate that most of the vesicle
matrix proteins aggregate in the presence of Ca at
the intravesicular pH of 5.5. The aggregation was specifically
demonstrated with the vesicle matrix proteins of regulated secretory
pathway and was not shown with a constitutive secretory protein like
immunoglobulin G (Fig. 3), which is consistent with the previous
result that showed a selective aggregation of secretogranin II, at the
exclusion of immunoglobulin G, in the presence of 10 mM Ca
at pH 5.2 (Gerdes et al., 1989).
Recently, another vesicle matrix protein carboxypeptidase E was also
shown to aggregate in a pH- and Ca
-dependent manner
(Song and Fricker, 1995). Taken together, this low pH- and high
calcium-dependent aggregation property seems to be the hallmark of
vesicle matrix proteins of the regulated secretory pathway,
distinguishing the regulated secretory proteins from others in the TGN.
Unlike the aggregation of chromogranins A and B, which showed
significant aggregation at pH 7.5, amounting to as much as one-third
(Yoo and Albanesi, 1990) to one-half (Yoo, 1995a) that at pH 5.5,
respectively, the aggregation of the whole vesicle matrix proteins at a
near physiological pH of 7.5 was less than 1/14th that at pH 5.5 even
at high Ca concentrations (Fig. 2). This
aggregation pattern suggested that the vesicle matrix proteins, each
with different pH and Ca
sensitivity, interact with
each other at pH 5.5, thereby influencing the aggregation pattern of
individual matrix proteins. Indeed, many vesicle matrix proteins were
shown to interact with CGA at the intravesicular pH ( Fig. 5and Fig. 7). Nevertheless, it is not clear what fraction of the
matrix proteins interact with CGA directly, although it appears
reasonable to assume that some matrix proteins bind to CGA directly
while other proteins interact with CGA indirectly by virtue of their
pH- and Ca
-dependent interactions with the CGA-bound
proteins. Moreover, the fact that very little aggregation of the
vesicle matrix proteins occurred at pH 7.5 (Fig. 2) suggested
that there may be virtually no aggregation of the matrix proteins at
physiological pH levels and the matrix proteins in the TGN will not
begin to aggregate unless the pH starts to drop. Given that the
internal pH of the TGN is acidic with high concentration of
Ca
(Roos, 1988; Hutton, 1989; Mata and Fink, 1989;
Anderson and Pathak, 1985; Orci et al., 1986), the present
results suggest that the potential vesicle matrix proteins in the TGN
will start to aggregate upon binding Ca
while
excluding constitutive secretory proteins. Further acidification and
increase in Ca
concentration of the immature
secretory vesicles after budding from the TGN will further increase not
only the aggregation of the vesicle matrix proteins but also the
interactions between the vesicle matrix proteins and between the
vesicle matrix proteins and the vesicle membrane.
In view of the
fact that CGB exhibits both a high Ca sensitivity and
a high degree of aggregation at the acidic pH (Fig. 4) (Yoo,
1995a), it is likely that CGB might be the first protein to undergo pH-
and Ca
-induced aggregation in the TGN. This initial
aggregation of CGB might serve as a facilitator of ensuing aggregation
of other matrix proteins. In this regard, the pH- and
Ca
-induced aggregation of CGB appears likely to serve
as the initiation force for the selective aggregation of the vesicle
matrix proteins in the TGN. In addition, CGB has recently been shown to
interact with the vesicle membrane much more tightly than CGA at pH 5.5
(Yoo, 1995b). Therefore, it is not difficult to envision the
interaction of the matrix protein aggregate with the vesicle membrane,
through the initial aggregation and tight interaction of CGB with the
potential vesicle membrane, in the early stage of vesicle biogenesis in
the TGN. In this regard, the tightly membrane-associated form of CGB
reported by Pimplikar and Huttner(1992) may be the soluble CGB that
remained bound to the fused vesicle membrane due to their high affinity
for the secretory vesicle membrane, possibly reflecting the essential
role of CGB in secretory vesicle biogenesis. Although it is not known
whether other vesicle matrix proteins interact with CGB directly, the
interaction of CGB with CGA (Fig. 10) and the interaction of CGA
with other potential vesicle matrix proteins ( Fig. 5and Fig. 7) will ensure the transmission of the effect of CGB
aggregation to the rest of the potential vesicle matrix proteins in the
TGN.
The pH- and Ca-induced aggregation and
binding of the vesicle matrix proteins to the vesicle membrane appear
to be due to the pH- and Ca
-induced conformational
changes of the matrix proteins. In particular, chromogranins A and B
are known to undergo pH- and Ca
-dependent
conformational changes (Yoo and Albanesi, 1990, 1991; Yoo, 1995a), and
CGA has previously been shown to expose hydrophobic sites upon binding
Ca
(Yoo and Albanesi, 1990). In a study carried out
with a hydrophobic fluorescent probe, bis-ANS, which is known to bind
to hydrophobic regions of protein and fluoresce upon binding, the
addition of Ca
to CGA in the presence of saturating
amount of bis-ANS caused an increase of fluorescence emission by
bis-ANS (Yoo and Albanesi, 1990). This result indicated that
hydrophobic regions of CGA have been newly exposed upon binding
Ca
. It was further shown that CGA exists not only in
a more extended conformation at pH 5.5 than at pH 7.5 (Yoo and
Ferretti, 1993) but also in a dimeric state at pH 7.5 and in a
tetrameric state at pH 5.5 (Yoo and Lewis, 1992). Therefore, although
it is not known at present whether binding of Ca
to
CGB exposes additional hydrophobic regions in CGB, it is conceivable
that the Ca
-exposed hydrophobic regions of CGA and
CGB may participate in the interactions between CGB and CGA and between
CGA and other matrix proteins.
Considering that the interaction
between the vesicle matrix proteins or between the vesicle matrix
proteins and the vesicle membrane either does not occur, or is
substantially reduced, at pH 7.5 regardless of the presence of
Ca despite the fact that Ca
can
cause CGA to bind to calmodulin even at pH 7.5 (Yoo and Albanesi,
1990), the present results highlight the importance of the pH-induced
conformational changes of the matrix proteins for interaction.
Nonetheless, the pH-induced conformational change alone was not
sufficient to cause the selective aggregation of the matrix proteins,
underscoring the importance of Ca
as well.
Interestingly, Ca
has previously been demonstrated to
stabilize the pH-dependent interaction between the tetrameric CGA and
the intraluminal loop region of inositol 1,4,5-trisphosphate receptor
(Yoo and Lewis, 1995), one of the integral membrane proteins of
secretory vesicles that had been shown to interact with CGA (Yoo,
1994), and to lead the interacting molecules to more ordered structures
(Yoo and Lewis, 1995). Therefore, another important role of
Ca
in the acidic environment appears to be the
stabilization of the interaction of chromogranins or vesicle matrix
proteins with the vesicle membrane.
From these results, it seems
evident that the acidic pH and high Ca closely work
together to bring about 1) the selective aggregation, 2) the needed
interaction, and 3) the stablilization of the intravesicular structures
of the matrix proteins during vesicle biogenesis. Furthermore, in light
of the fact that chromogranins are the predominant proteins in the
secretory vesicles of virtually all neuroendocrine cells
(O'Connor and Frigon, 1984; Simon and Aunis, 1989; Huttner et
al., 1991b; Winkler and Fischer-Colbrie, 1992), it is highly
likely that pH- and Ca
-induced chromogranin
aggregation and vesicle membrane binding will be the dominating
factors, which dictate the fate of chromogranins and the
chromogranin-associating vesicle matrix proteins, in the selective
aggregation and sorting of the vesicle matrix proteins to the secretory
vesicles. Any matrix proteins that do not associate with chromogranins
may still ensure their entry into the secretory vesicles through their
direct binding to the vesicle membrane.
In the past, a few models
for vesicle biogenesis have been proposed, attributing essential roles
to the pH- and Ca-induced chromogranin aggregation
(Huttner et al., 1988, 1991a; Yoo, 1993b). In line with these
models, the present results appear to explain the selective segregation
and sorting of vesicle matrix proteins in the TGN and show how vesicle
matrix proteins find their way into the secretory vesicles of the
regulated secretory pathway. Hence, the present results not only
provide a more detailed insight into the aggregation and
membrane-interaction property of the vesicle matrix proteins, but also
strengthen the case for the critical roles of chromogranins A and B in
vesicle biogenesis (Huttner et al., 1988, 1991a; Yoo, 1993b,
1995a).