©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
pH- and Ca-dependent Aggregation Property of Secretory Vesicle Matrix Proteins and the Potential Role of Chromogranins A and B in Secretory Vesicle Biogenesis (*)

(Received for publication, June 9, 1995; and in revised form, October 16, 1995)

Seung Hyun Yoo (§)

From the Laboratory of Neurochemistry, NIDCD, National Institutes of Health, Bethesda, Maryland 20892-3320

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Cyanogen bromide (CNBr)-activated Sepharose 4B was obtained from Pharmacia Biotech Inc. Immunoglobulin G was from Calbiochem. Chelex 100, acrylamide, and bisacrylamide were from Bio-Rad.

Preparation of the Secretory Vesicle Matrix Proteins and Chromogranins A and B

To obtain the secretory vesicle matrix proteins, the secretory vesicles were prepared as described previously (Yoo and Albanesi, 1990), and the vesicles were resuspended in 40 volumes of 15 mM Tris-HCl, pH 7.5, and frozen and thawed to lyse the vesicles. The lysed vesicles were centrifuged at 48,000 times g for 30 min to separate the matrix proteins from the membrane. The supernatant was collected for the soluble intravesicular matrix proteins, and the lysates were used for purification of CGA (Yoo and Albanesi, 1990) and CGB (Yoo, 1995b).

Aggregation Experiment

The secretory vesicle matrix proteins, chromogranin A, and chromogranin B in either 15 mM sodium acetate, pH 5.5, or 15 mM MOPS, pH 7.5, were separately treated with Chelex 100 to remove residual Ca. Each protein solution then was titrated with concentrated CaCl(2), and the Ca-induced aggregation was monitored by measuring the turbidity change at 320 nm with a Beckman DU 70 spectrophotometer. All measurements were done at 22 °C.

Coupling of Chromogranin A to CNBr-Sepharose 4B

Coupling of the purified bovine chromogranin A to CNBr-activated Sepharose 4B (Pharmacia) was done as described previously (Yoo, 1994). Following this procedure, it was estimated that 1.35 mg of CGA were coupled per ml of Sepharose 4B (wet volume).

Two-dimensional Polyacrylamide Gel Electrophoresis (PAGE)

One-dimensional isoelectric focusing was carried out in the pH range of 4-8, and two-dimensional PAGE was carried out on a 10% SDS-gel according to Laemmli(1970).

Protein Isolation and Sequencing

The proteins separated on a two-dimensional gel were cut out and electroeluted. The eluted proteins were subjected either to amino acid sequencing or to trypsin digestion, followed by the separation of the tryptic fragments by high performance liquid chromatography. The amino acid sequences were determined by the Edman degradation method using an Applied Biosystems 470A protein sequencer and a 120A parathyroid hormone analyzer, and 5-8 amino acids from the N terminus of each protein were determined.


RESULTS

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 (circle), and 0.4 mg/ml (Delta). 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 times 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 (circle) and 7.5 (Delta) 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 times 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 (bullet), vesicle lysate (up triangle), and CGA (circle) 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 (circle-circle) the elution was carried out with a pH 7.5 buffer (buffer B) (bullet-bullet). B, after the loading and washing step in the absence of Ca (circle-circle) the elution was carried out with the same pH 7.5 buffer (buffer B) containing 2 mM EGTA (up triangle-up triangle). 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.




DISCUSSION

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).


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Neurochemistry, 5 Research Court, 2A37, NIDCD/NIH, Bethesda, MD 20892-3320. Tel.: 301-402-4223; Fax: 301-402-5475.

(^1)
The abbreviations used are: TGN, trans-Golgi network; CGA, chromogranin A; CGB, chromogranin B; PAGE, polyacrylamide gel electrophoresis; MOPS, 3-(N-morpholino)-2-hydroxypropanesulfonic acid; ANS, 1-anilino-8-naphthalenesulfonate.


REFERENCES

  1. Anderson, R. G. W., and Pathak, R. K. (1985) Cell 40, 635-643 [Medline] [Order article via Infotrieve]
  2. Bauer, J. W., and Fischer-Colbrie, R. (1991) Biochim. Biophys. Acta 1089, 124-126 [Medline] [Order article via Infotrieve]
  3. Benedum, U. M., Baeuerle, P. A., Konecki, D. S., Frank, R., Powell, J., Mallet, J., and Huttner, W. B. (1986) EMBO J. 5, 1495-1502 [Abstract]
  4. Bulenda, D., and Gratzl, M. (1985) Biochemistry 24, 7760-7765 [Medline] [Order article via Infotrieve]
  5. Casey, R. P., Njus, D., Radda, G. K., and Sehr, P. A. (1977) Biochemistry 16, 972-977 [Medline] [Order article via Infotrieve]
  6. Chanat, E., and Huttner, W. B. (1991) J. Cell Biol. 115, 1505-1519 [Abstract]
  7. Fischer-Colbrie, R., Gutierrez, J., Hsu, C. M., Iacangelo, A., and Eiden, L. E. (1990) J. Biol. Chem. 265, 9208-9213 [Abstract/Free Full Text]
  8. Gerdes, H.-H., Rosa, P., Phillips, E., Baeuerle, P. A., Frank, R., Argos, P., and Huttner, W. B. (1989) J. Biol. Chem. 264, 12009-12015 [Abstract/Free Full Text]
  9. Gorr, S.-U., Shioi, J., and Cohn, D. V. (1989) Am. J. Physiol. 257, E247-E254
  10. Huttner, W. B., Friederich, E., Gerdes, H.-H., Niehrs, E., and Rosa, P. (1988) in Progress in Endocrinology (Imura, H., Shizume, K., and Yoshida, S., eds) pp. 325-328, Excerpta Medica, Elsevier Science Publishers, Amsterdam
  11. Huttner, W. B., Gerdes, H.-H., and Rosa, P. (1991a) Trends Biochem. Sci. 16, 27-30 [CrossRef][Medline] [Order article via Infotrieve]
  12. Huttner, W. B., Gerdes, H.-H., and Rosa, P. (1991b) in Markers for Neural and Endocrine Cells, Molecular and Cell Biology, Diagnostic Application (Gratzl, M., and Langley, K., eds) pp. 93-131, VCH, Weinheim, Germany
  13. Hutton, J. C. (1989) Diabetologia 32, 271-281 [Medline] [Order article via Infotrieve]
  14. Iacangelo, A., Affolter, H.-U., Eiden, L. E., Herbert, E., and Grimes, M. (1986) Nature 323, 82-86 [Medline] [Order article via Infotrieve]
  15. Johnson, R. G., and Scarpa, A. (1976) J. Biol. Chem. 251, 2189-2191 [Abstract]
  16. Laemmli, U. K. (1970) Nature 227, 680-685 [Medline] [Order article via Infotrieve]
  17. Mata, M., and Fink, D. J. (1989) J. Histochem. Cytochem. 37, 971-980 [Abstract]
  18. O'Connor, D. T., and Frigon, R. P. (1984) J. Biol. Chem. 259, 3237-3247 [Abstract/Free Full Text]
  19. Orci, L., Ravazolla, M., Amherdt, M., Madsen, O., Perrelet, A., Vasalli, J.-D., and Anderson, R. G. W. (1986) J. Cell Biol. 103, 2273-2281 [Abstract]
  20. Pimplikar, S. W., and Huttner, W. B. (1992) J. Biol. Chem. 267, 4110-4118 [Abstract/Free Full Text]
  21. Reiffen, F. U., and Gratzl, M. (1986) FEBS Lett. 195, 327-330 [CrossRef][Medline] [Order article via Infotrieve]
  22. Roos, N. (1988) Scanning Microsc. 2, 323-329 [Medline] [Order article via Infotrieve]
  23. Simon, J.-P., and Aunis, D. (1989) Biochem. J. 262, 1-13 [Medline] [Order article via Infotrieve]
  24. Song, L., and Fricker, L. D. (1995) J. Biol. Chem. 270, 7963-7967 [Abstract/Free Full Text]
  25. Winkler, H., and Fischer-Colbrie, R. (1992) Neuroscience 49, 497-528 [CrossRef][Medline] [Order article via Infotrieve]
  26. Winkler, H., and Westhead, E. (1980) Neuroscience 5, 1803-1823 [CrossRef][Medline] [Order article via Infotrieve]
  27. Yoo, S. H. (1993a) Biochemistry 32, 8213-8219 [Medline] [Order article via Infotrieve]
  28. Yoo, S. H. (1993b) Biochim. Biophys. Acta 1179, 239-246 [Medline] [Order article via Infotrieve]
  29. Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006 [Abstract/Free Full Text]
  30. Yoo, S. H. (1995a) J. Biol. Chem. 270, 12578-12583 [Abstract/Free Full Text]
  31. Yoo, S. H. (1995b) Biochemistry 34, 8680-8686 [Medline] [Order article via Infotrieve]
  32. Yoo, S. H., and Albanesi, J. P. (1990) J. Biol. Chem. 265, 14414-14421 [Abstract/Free Full Text]
  33. Yoo, S. H., and Albanesi, J. P. (1991) J. Biol. Chem. 266, 7740-7745 [Abstract/Free Full Text]
  34. Yoo, S. H., and Ferretti, J. A. (1993) FEBS Lett. 334, 373-377 [CrossRef][Medline] [Order article via Infotrieve]
  35. Yoo, S. H., and Lewis, M. S. (1992) J. Biol. Chem. 267, 11236-11241 [Abstract/Free Full Text]
  36. Yoo, S. H., and Lewis, M. S. (1995) Biochemistry 34, 632-638 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.