©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium- and pH-dependent Aggregation of Carboxypeptidase E (*)

(Received for publication, September 26, 1994; and in revised form, February 6, 1995)

Lixin Song Lloyd D. Fricker (§)

From the Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Carboxypeptidase E (CPE) is involved with the biosynthesis of numerous peptide hormones and neurotransmitters. Several forms of CPE have been previously detected in neuroendocrine cells, including a form which is soluble at pH 5.5 (S-CPE), a form which can be extracted from membranes with 1 M NaCl at pH 5.5 (M1-CPE), and a form which requires both 1% Triton X-100 and 1 M NaCl for extraction from membranes at pH 5.5 (M2-CPE). Like other peptide processing enzymes, CPE is known to be sorted into peptide-containing secretory vesicles of the regulated pathway. One mechanism that has been proposed to be important for sorting of regulated pathway proteins is Ca and pH-induced aggregation. CPE purified from bovine pituitary membranes aggregates at pH 5.5 when the concentration of CPE is 0.3 µg/µl or higher, but not when the concentration is 0.01 µg/µl. Aggregation of CPE is pH-dependent, with very little aggregation occurring at pH 6 or above. At pH 5.0-5.5, the M2 form of CPE shows a greater tendency to aggregate than the other two forms. At pH 6, Ca concentrations from 1-30 mM increase the aggregation of M1- and M2-CPE, but not S-CPE. The aggregation of M2-CPE does not explain the apparent membrane binding of this protein since the aggregate is solubilized by 1% Triton X-100 at pH 5.5 or by pH 6.0, whereas M2-CPE is not extracted from membranes under these conditions. Taken together, these results are consistent with a model in which the decreasing pH and increasing Ca levels in the trans Golgi network induce the aggregation of CPE, which contributes to the sorting of this protein into regulated pathway secretory vesicles.


INTRODUCTION

Many peptide hormones and neurotransmitters are produced by the selective proteolysis of prohormone precursors (Steiner, 1991). Initially, endopeptidases cleave the precursor at basic amino acid consensus sites (Lindberg and Hutton, 1991; Devi, 1991), and then a carboxypeptidase removes these basic residues from the peptide intermediates (Fricker, 1988a, 1991). Several mammalian peptide-processing endopeptidase have been reported, including prohormone convertase 1 (PC1, also known as PC3), PC2, PACE 4, PC4, PC5 (also known as PC6), and furin (Smeekens and Steiner, 1990; Seidah et al., 1990, 1991; Smeekens et al., 1991; Kiefer et al., 1991; Nakayama et al., 1992, 1993). In contrast to the multitude of endopeptidases, only a single carboxypeptidase is thought to be involved with intracellular peptide processing (Fricker, 1988a, 1991). Carboxypeptidase E (CPE) (^1)is present in all neuroendocrine tissues, and in several tissues this enzyme has been localized to the peptide-containing secretory vesicles (Fricker, 1988a, 1991).

Peptide processing is thought to begin in the trans Golgi network and continue in the secretory vesicle (Orci et al., 1987; Schnabel et al., 1989). Thus, it is essential that the peptide processing enzymes and the substrates are co-segregated into secretory vesicles. Two distinct mechanisms of sorting have been proposed; one mechanism requires a specific protein which binds to and sorts the proteins destined for the secretory vesicles (Kelly, 1985). Although a prohormone-specific sorting protein was reported (Chung et al., 1989), this protein has been identified as chymotrypsinogen (Gorr et al., 1992), and it is unlikely that this protein participates in the sorting of most secretory proteins. The second mechanism proposed for the sorting of secretory proteins is the selective aggregation hypothesis (Chanat and Huttner, 1991; Reaves and Dannies, 1991). In this model, proteins sort themselves by forming aggregates in the environment of the trans Golgi network (Bauerfeind and Huttner, 1993). This compartment is slightly acidic (Anderson and Pathak, 1985), and has been estimated to contain mM concentrations of Ca (Chanat and Huttner, 1991), based on the Ca concentrations in other organelles of the secretory pathway (Bulenda and Gratzl, 1985; Roos, 1988; Sambrook, 1990). Evidence for this aggregation model of protein sorting is provided by the finding that several secretory granule proteins undergo aggregation at slightly acidic pH and in the presence of mM concentrations of Ca (Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991; Shennan et al., 1994). For example, chromogranin A and B and secretogranin II form aggregates in the presence of 10-20 mM Ca at pH 5-6, but not at neutral pH (Chanat and Huttner, 1991). Recently PC2 has been found to undergo pH- and Ca-induced aggregation (Shennan et al., 1994).

The sorting of CPE into secretory vesicles was hypothesized to proceed by the binding of a C-terminal amphipathic helix to membranes at acidic, but not neutral pH values (Fricker et al., 1990). Evidence that this C-terminal region may contribute to the sorting of CPE was provided by studies using fusion proteins; albumin with the C-terminal region of CPE is sorted into the regulated pathway, although with a low efficiency compared to the sorting of native CPE (Mitra et al., 1994). This result raised the possibility that additional regions of CPE are involved in the efficient sorting of this protein. CPE has homology to the Ca-binding region of the bacterial metalloenzyme carboxypeptidase T, and CPE has recently been found to bind Ca (Nalamachu et al., 1994). Since bacteria and mammals diverged approximately 1.8 billion years ago, this homology between the Ca-binding region of carboxypeptidase T and CPE implies that the binding of Ca to CPE is important. However, Ca does not substantially alter CPE activity, and has only a small effect on CPE stability (Nalamachu et al., 1994). The possibility that Ca influences the aggregation of CPE was previously investigated using low concentrations (0.01-0.1 µg/µl) of protein at pH 5.5 (Nalamachu et al., 1994). Although no evidence for aggregation was found, these concentrations are much lower than the protein concentrations typically used to study aggregation (Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991). In the present study, we find that CPE does aggregate at pH 5.5 when the concentration is 0.3 µg/µl or higher. This aggregation is dependent on pH and is augmented by 1-30 mM Ca, although not all forms of CPE show the same dependence on pH or Ca. Since these concentrations of CPE, Ca, and H are thought to be physiological, these findings raise the possibility that the aggregation of CPE may participate in the sorting of this protein into the regulated pathway.


MATERIALS AND METHODS

Purification of Soluble and Membrane-bound Forms of CPE from Bovine Pituitary

Bovine pituitary glands were extracted by a modification of a previously described procedure (Fricker et al., 1990). Frozen glands (Pel-Freez) were homogenized (Polytron, Brinkman) for 1 min in 5 volumes of 20 mM sodium acetate (NaAc) buffer, pH 5.5, containing 1 mM phenylmethylsulfonyl fluoride, and then centrifuged at 50,000 times g for 30 min at 4 °C. The pellet was resuspended in 5 volumes of 20 mM NaAc buffer, homogenized as above, and centrifuged again at 50,000 times g for 30 min. This second supernatant was combined with the first and the two supernatants (soluble extracts, or ``S-CPE'') were loaded onto a p-aminobenzoyl-Arg Sepharose 6B substrate affinity column, as described (Fricker et al., 1990). The pellet was resuspended in 5 volumes of 20 mM NaAc buffer containing 1 M NaCl, homogenized, and centrifuged at 50,000 times g for 30 min. The pellet was re-extracted with 1 M NaCl in NaAc buffer, and these two supernatants (``membrane-1'' extracts, or ``M1-CPE'') combined and applied to the affinity columns. The pellet was resuspended in 5 volumes of NaAc buffer containing 1 M NaCl and 1% Triton X-100, homogenized, and centrifuged as above. This step was repeated, and the combined supernatants (``membrane-2'' extract, or ``M2-CPE'') were applied to the affinity columns. CPE was eluted from the affinity columns as described (Fricker et al. 1990), and concentrated using a Centricon membrane filtration apparatus (Amicon). The concentrated CPE was washed with 5 mM Tris-Cl, pH 8.0, containing 0.01% Triton X-100. The amount of protein was determined using the Bradford assay. Typically, 20-30% of the total amount of CPE is recovered as S-CPE, 20-30% is recovered as M1-CPE, and 40-60% is recovered as M2-CPE.

Aggregation of CPE

In a typical assay, purified S-, M1-, or M2-CPE was combined with buffer (25 mM final concentration) in a volume of 10 µl. Unless indicated, the final concentration of CPE was 1.5 µg/µl, and pH 5.5 NaAc buffer was used, without added CaCl(2). To prevent nonspecific binding of CPE to the 7 times 20-mm polycarbonate ultracentrifuge tube, 0.01% Triton X-100 was included in the mixture. The tubes were incubated for 2 h at 37 °C and then centrifuged at 100,000 times g for 30 min at 4 °C in a Beckman L7 ultracentrifuge. Pellets could be visually observed for samples containing 3 µg or more total CPE under conditions where aggregation occurred.

For experiments where the amount of protein was analyzed only by gel electrophoresis, the supernatant was removed using microcapillary pipette tips (Marsh) and diluted with 10 volumes of SDS gel loading buffer. A similar volume of SDS gel loading buffer was added to the pellet fraction, the tubes were mixed, and aliquots were taken for gel electrophoresis. Samples were heated at 95 °C for 5 min and analyzed on a denaturing 10% polyacrylamide gel. Following electrophoresis, the protein was detected using the silver staining procedure (Morrissey, 1981). Quantitation of the silver-stained gels was performed using an image analysis system consisting of a Cohu video camera, a PC visions frame grabber, and Jandel Scientific's JAVA software.

For experiments where the amount of CPE was quantitated by enzyme and protein assays in addition to the gel electrophoresis, the supernatant from the 100,000 times g centrifugation step was diluted 1:10 into 5 mM Tris-Cl, pH 8.0, containing 0.01% Triton X-100. An aliquot was further diluted 1:200 in 100 mM NaAc buffer at pH 5.5 containing 0.01% Triton X-100, and then 20 µl were used to measure CPE activity using the substrate dansyl-Phe-Ala-Arg as described by Fricker(1994). The amount of protein in the supernatant was determined using the Bradford assay with 20 µl of the 1:10 diluted supernatant. An aliquot of the 1:10 diluted supernatant was also analyzed on denaturing polyacrylamide gels, along with an aliquot of the pellet fraction after extraction with SDS gel loading buffer, as described above. All CPE activity and protein measurements were performed in triplicate; variation was typically less than 5%. Each experiment was repeated a minimum of two times with comparable results.

Extraction of CPE from Bovine Pituitary Membranes and Western Blot Analysis-Bovine pituitary glands (Pel-Freez) were homogenized (Polytron, Brinkman) and centrifuged as described above for the purification of CPE. Pellets after the extraction of S-CPE and M1-CPE were homogenized in various buffers, as indicated in Fig. 5. The homogenate/buffer mixture was mixed and then centrifuged for 30 min at 50,000 times g at 4 °C. The supernatant was removed, and a 100-µl aliquot analyzed on 10% denaturing polyacrylamide gels. Following electrophoresis, the protein was transferred to nitrocellulose and Western blots were performed as described previously (Fricker et al., 1990). CPE was detected using a 1:1000 dilution of an antiserum raised against a peptide corresponding to the N-terminal region of CPE (Fricker et al., 1990), using I-protein A to detect the primary antisera. An aliquot of the supernatant was diluted with 100 mM NaAc, pH 5.5, buffer and used for CPE activity determinations, as described above.


Figure 5: Effect of different buffers on the extraction of M2-CPE from bovine pituitary membranes. Bovine pituitary glands were homogenized in 5 volumes of 20 mM NaAc buffer containing 1 M NaCl and centrifuged at 50,000 times g for 30 min. This step was repeated one time, and then the pellets were resuspended in 5 volumes of 20 mM NaAc buffer, homogenized, and centrifuged again. The pellets were combined with various buffers: 100 mM NaAc at pH 5.5 or 6.0; 100 mM Tris-Cl at pH 7.0, 8.0, or 9.0; and in the presence (+) or absence(-) of 1 M NaCl or 1% Triton X-100. The mixture was homogenized, and then centrifuged at 50,000 times g for 30 min. An aliquot of the supernatant was analyzed on a denaturing 10% polyacrylamide gel followed by Western blot analysis as described under ``Materials and Methods.'' The positions and molecular masses of prestained molecular weight standards (Bio-Rad) are indicated.




RESULTS

When purified M2-CPE was incubated at a concentration of 0.01 µg/µl for 2 h at 37 °C at pH 5.5 and then centrifuged at 100,000 times g for 30 min, nearly all of the protein was recovered in the supernatant (Fig. 1). This finding is consistent with a previous study which found no evidence for the aggregation of CPE under these conditions (Nalamachu et al. 1994). However, when the concentration of the protein was increase to 0.3 µg/µl, a large amount of the M2-CPE was recovered in the pellet (Fig. 1). For this analysis, the samples were diluted after centrifugation so that equal amounts of protein could be loaded onto the gel. Concentrations of M2-CPE from 1 to 3 µg/µl showed slightly more protein in the pellet, compared with the 0.3 µg/µl sample (Fig. 1). These results indicate that CPE aggregates at high protein concentrations at pH 5.5 in the absence of added Ca.


Figure 1: Effect of CPE concentration on the aggregation of M2-CPE. Purified M2-CPE at the indicated concentration was incubated for 2 h at 37 °C in 25 mM NaAc buffer, pH 5.5, and then centrifuged at 100,000 times g for 30 min, as described under ``Materials and Methods.'' The entire supernatant (S) and the pellet (P) fractions from the 0.01 µg/µl group were used without dilution. The sets containing higher amounts of CPE were diluted according to the initial amount of protein so that each set would contain equal amounts of protein for the gel. The positions and molecular masses of protein markers (Bio-Rad) are shown (M).



The aggregation of CPE was not found to be substantially different when samples were incubated at 37 or at 4 °C (data not shown), and so the physiologically relevant 37 °C was used in subsequent experiments. Samples which were not incubated before centrifugation showed no aggregate, with the maximal amount of aggregate observed after 1-2 h of incubation (data not shown). To test the effect of pH on the aggregation of CPE, 1.5 µg/µl purified M2-CPE was incubated for 2 h, at pH values between 5 and 8, and then centrifuged and analyzed on a denaturing polyacrylamide gel (Fig. 2, top). At pH 5.0, nearly all of the M2-CPE was recovered in the pellet fraction (Fig. 2, top). Approximately 50% of the M2-CPE was particulate at pH 5.5, and very little was particulate at pH values of 6.0 or higher (Fig. 2, top). The amounts of CPE in the supernatant and pellet fractions were quantitated by three different methods; the silver stained polyacrylamide gels were quantitated using an image analysis system, the amount of protein in the supernatant was determined using the Bradford protein assay, and the amount of CPE activity in the supernatant was determined using an enzyme assay (after diluting the sample into pH 5.5 buffer). All three of these methods gave comparable results, and the enzyme assay was found to be the most convenient and sensitive. Using the enzyme assay to quantitate the amount of CPE in the supernatant, the different forms of CPE were compared for the pH-dependent aggregation (Fig. 2, bottom). All forms of CPE showed pH-dependent aggregation, although S-CPE and M1-CPE did not produce as much aggregate as M2-CPE (Fig. 2, bottom). At pH 5.0, approximately 30-40% of the S- and M1-CPE was recovered in the soluble faction. These observations were verified by polyacrylamide gel electrophoresis of the soluble and pellet fractions of each form of CPE (data not shown).


Figure 2: Effect of pH on the aggregation of CPE. Top, polyacrylamide gel analysis of the supernatant and pellet fractions after incubation of 1.5 µg/µl M2-CPE at the indicated pH for 2 h at 37 °C and then centrifugation at 100,000 times g for 30 min, as described under ``Materials and Methods.'' NaAc buffers were used for the samples at pH 5-6.5, and Tris-Cl buffers were used for the pH 7 and 8 groups. The positions and molecular masses of protein markers (Bio-Rad) are shown. Bottom, quantitation of the amount of CPE activity in the supernatant after incubation at the indicated pH and centrifugation. Samples were diluted 1:200 into 100 mM NaAc, pH 5.5, prior to CPE activity analysis. CPE activities are shown in relative units, which were adjusted to 100 for the pH 7.0 data since the polyacrylamide gels revealed that virtually all of the CPE was soluble at this pH. Error bars show range of data from two separate determinations. Points without error bars had range values smaller than the symbol size.



To test the Ca dependence of the aggregation of CPE, 1.5 µg/µl CPE was incubated with 0.3 to 30 mM CaCl(2) at pH 6.0, and then centrifuged and analyzed by gel electrophoresis (Fig. 3, top) or by enzyme assay after dilution with pH 5.5 buffer (Fig. 3, bottom). As found in Fig. 2, very little M2-CPE was detected in the pellet when tested at pH 6.0 in the absence of Ca (Fig. 3, top). However, when CaCl(2) is included in the incubation mixture, a moderate amount of M2-CPE was detected in the pellet fraction (Fig. 3, top). Quantitation of the amount of CPE in the supernatant fraction showed that 10-30 mM CaCl(2) reduced this amount by approximately 30%, while 1 mM CaCl(2) reduced this amount by approximately 20% (Fig. 3, bottom). Similar results were obtained using M1-CPE, whereas the amount of S-CPE in the supernatant did not change by more than 10% for any concentration of CaCl(2) (Fig. 3, bottom).


Figure 3: Effect of CaCl(2) on the aggregation of CPE. Top, polyacrylamide gel electrophoresis of the supernatant and pellet fractions after incubation of M2-CPE with the indicated concentration of CaCl(2) and centrifugation, as described under ``Materials and Methods.'' The positions and molecular masses of protein markers (Bio-Rad) are shown. Bottom, quantitation of the amount of CPE activity in the supernatant after incubation of M2-CPE with the indicated amount of CaCl(2) and centrifugation. Samples were diluted 1:200 into 100 mM NaAc, pH 5.5, prior to the enzyme assay. CPE activities are shown in relative units, which were adjusted to 100 for the samples incubated in the absence of CaCl(2) since the polyacrylamide gel analysis revealed that nearly all of the CPE was soluble under this condition. Error bars show standard error: n = 3 for S-CPE and M1-CPE, and n = 5 for M2-CPE. Points without error bars had standard error smaller than the symbol size.



The finding that aggregation of S-, M1-, and M2-CPE differ in their pH and/or Ca dependence ( Fig. 2and Fig. 3, bottom) raises the possibility that aggregation, and not membrane binding, may account for the presence of CPE in the particulate fraction of cell extracts. For example, the M2-form of CPE might not represent a membrane-bound form, as hypothesized, but instead might be due to an insoluble aggregate. To test this, we compared the ability of various buffers to solubilize aggregates of M2-CPE. The aggregates were produced by incubating 1.5 µg/µl M2-CPE at pH 5.0 for 2 h at 37 °C, and then the aggregates were collected by centrifugation. When the aggregates were mixed with sodium acetate buffer at pH 5.5 and then re-centrifuged, the majority of CPE was present in the pellet (Fig. 4). Addition of 1 M NaCl to the buffer did not substantially alter the amount of CPE which could be extracted from the aggregate, whereas 1% Triton X-100 was very effective in solubilizing the aggregate (Fig. 4). The combination of 1 M NaCl and 1% Triton X-100 was slightly more effective than Triton X-100 alone (Fig. 4). As found for the pH-dependence of the formation of the aggregate, the dissolution of the aggregate was also pH-dependent, with nearly all of the aggregate extracted at pH 6.0 (Fig. 4). Higher pH buffers (7.0 and 8.0) were similar to pH 6 buffers in extracting CPE from the aggregate (data not shown).


Figure 4: Effect of different buffers on the extraction of CPE from aggregates of M2-CPE. The aggregates of M2-CPE were obtained by incubating 1.5 µg/µl M2-CPE for 2 h at 37 °C in 25 mM NaAc buffer, pH 5.0, containing 10 mM CaCl(2). The aggregates were collected by centrifugation at 100,000 times g for 30 min. The pellets were combined with 100 mM NaAc buffer at the indicated pH, either containing (+) or not containing(-) 1 M NaCl and/or 1% Triton X-100, as indicated. The mixture was sonicated 20 s and then centrifuged again at 100,000 times g for 30 min at 4 °C. Both supernatant and pellet were diluted and analyzed on a denaturing polyacrylamide gel, as described under ``Materials and Methods.'' The positions and molecular masses of protein markers (Bio-Rad) are shown.



The conditions which extract M2-CPE from the aggregate are distinct from the conditions previously reported to extract CPE activity from the membranes (Supattapone et al., 1984; Fricker, 1988b; Fricker et al., 1990). Specifically, very little CPE activity is extracted by pH 6.0 or by 1% Triton X-100 alone at pH 5.5; efficient extraction of membrane-bound CPE requires either a combination of NaCl and Triton X-100 at pH 5.5 or pH values in the neutral to basic range (Fricker, 1988b; Fricker et al., 1990). However, these previous studies measured enzyme activity and not the extraction of CPE protein. To test whether the extraction of CPE protein from membranes is similar to that previously reported for the enzyme activity (and distinct from the conditions that solubilize the M2-CPE aggregate), bovine pituitary membranes were extracted with various buffers and the CPE protein identified using both enzyme assays and Western blot analysis. In this procedure, the bovine pituitary membranes were first washed with 1 M NaCl to remove the S-CPE and M1-CPE, so that the analysis would detect only the M2-CPE form. When membranes were extracted with pH 5.5 buffer alone, virtually no CPE could be detected in the extract (Fig. 5). Either 1 M NaCl or 1% Triton X-100 alone at pH 5.5 only extracted a small amount of M2-CPE, whereas the combination of salt and detergent extracted a large amount of M2-CPE (Fig. 5). Importantly, very little CPE could be extracted from the membranes at pH 6.0, although large amounts were extracted at pH 7.0 or higher (Fig. 5). The amounts of CPE protein in the various extracts, as detected by Western blotting, were comparable to the amount of CPE activity detected in the extracts (data not shown). These results indicate that the conditions for the extraction of M2-CPE from membranes is distinct from the conditions which extract M2-CPE from aggregates.


DISCUSSION

The major finding of this study is that CPE can aggregate under conditions that resemble those found in the regulated secretory pathway. It is difficult to assign exact values for the pH and Ca concentration of the trans Golgi apparatus, and it has been estimated that the pH is slightly acidic (6.2-6.5) and Ca concentrations are around 10 mM (Chanat and Huttner, 1991). Mature secretory vesicles are even more acidic, with an internal pH of 5.0-5.5 and Ca concentrations of 20 mM or higher (Bulenda and Gratzl, 1985; Roos, 1988). The concentration of CPE in the secretory pathway is difficult to calculate; if secretory vesicles and the trans Golgi network constitute 0.5-5% of the total volume of the pituitary, then the concentration of CPE would be approximately 0.5-5 µg/µl (based on the typical recovery of 50 µg of CPE per pituitary used for the purification of CPE). Thus, it is likely that the conditions used for the present study resemble those inside the secretory pathway of a cell, and that CPE aggregates in vivo. Support for this hypothesis was provided by immunocytochemical studies of rat islet cells; both immunoreactive CPE and insulin were found to be co-localized in discreet patches on the outside of cells (Aguilar-Diosdado et al., 1994). The presence of cell surface insulin, and presumably CPE, is thought to reflect the secretion and incomplete dissolution of a dense-core aggregate (AguilarDiosdado et al., 1994).

If aggregation of CPE occurs in the trans Golgi network, this could be a mechanism which contributes to the sorting of CPE into the regulated pathway. The combination of low pH and high Ca is likely to be important for CPE to aggregate in vivo in the trans Golgi network since little aggregation of CPE occurs in the absence of Ca at pH 6. The combination of low pH and high Ca is thought to be involved with aggregation of other proteins found in the regulated secretory pathway. Several studies have found that chromogranin A and B, and secretogranin II aggregate at acidic pH values in the presence of Ca (Gorr et al., 1989; Yoo and Albanesi, 1990; Chanat and Huttner, 1991). For example, Chanat and Huttner(1991) reported that aggregation of secretogranin II was stimulated by 1-10 mM Ca at pH 6.4, whereas little aggregation occurred even in the presence of Ca at pH 6.9 or 7.4. Similar to our findings with CPE, the aggregation of these other secretory granule proteins is not complete at pH 6-7, even with elevated levels of Ca (Gorr et al., 1989; Chanat and Huttner, 1991). It will be important to determine whether CPE and the other secretory granule proteins can form co-aggregates. One study has reported that CPE is present in a complex with glycoprotein III in bovine adrenal chromaffin granules (Palmer and Christie, 1992). However, this complex is not likely to be an insoluble aggregate since it was soluble upon extraction of the membranes at pH 7.0 with 2% octylglucoside and centrifugation at 100,000 times g (Palmer and Christie, 1992).

According to the selective aggregation hypothesis, once the protein aggregate forms in the trans Golgi network it must somehow bind to membranes (Bauerfeind and Huttner, 1993). In the case of chromogranin A and B, both soluble and membrane bound forms have been detected in neuroendocrine cells (Settleman et al., 1985; Pimplikar and Huttner, 1992). CPE is also found in soluble and membrane-bound forms (Fricker and Snyder, 1982; Supattapone et al., 1984). The membrane binding domain of CPE is thought to reside in the C-terminal region, possibly due to an amphiphilic helix (Fricker et al., 1990). This membrane binding of CPE is pH-dependent, with substantial binding observed at pH 5-6, and much less binding observed at pH values of 7 or higher (Fricker, 1988b; Fricker et al., 1990). It is likely that the combination of pH-dependent aggregation and membrane binding both contribute to the efficient sorting of CPE into the regulated pathway. This model would explain why a fusion protein containing only the C-terminal region of CPE was sorted into the regulated pathway with low efficiency (Mitra et al., 1994).

The different aggregation properties of the various forms of CPE were unexpected. All forms of CPE bind Ca (Nalamachu et al., 1994), although the affinity and number of Ca binding sites has not been determined for the various forms. It is possible that the highly acidic sequence near the C terminus of CPE (ERKEEEKEE) binds Ca; since this region is thought to be missing from S-CPE (Fricker et al., 1990), this could be responsible for the different effect of Ca on S-CPE, compared with M1- and M2-CPE. The different pH sensitivity of M2-CPE, compared to S- and M1-CPE is not due to differences in the isoelectric points of these proteins because isoelectric focusing of S-, M1-, and M2-CPE showed a similar pattern; all three samples contained a broad distribution of proteins with pI values between 4.8 and 5.2 (data not shown). This is consistent with previous reports that soluble and membrane forms of CPE are each composed of multiple proteins with pI values around 5 (Laslop et al., 1986; Christie and Palmer, 1990; Palmer and Christie, 1992). The molecular basis for the different forms of CPE upon isoelectric focusing is not known. Further studies examining the various forms of CPE, and the interaction of these forms with Ca are in progress.


FOOTNOTES

*
This work was supported by National Institute on Drug Abuse grants R01 DA-04494 and K02 DA-00194 and by an Irma T. Hirschl Career Scientist Award (to L. D. F.). 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: Dept. of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-4225; Fax: 718-829-8705.

(^1)
The abbreviations used are: CPE, carboxypeptidase E; S-CPE, soluble CPE; M1-CPE, membrane 1 CPE; M2, membrane 2 CPE; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.


REFERENCES

  1. Aguilar-Diosdado, M., Parkinson, D., Corbett, J. A., Kwon, G., Marshall, C. A., Gingerich, R. L., Santiago, J. V., and McDaniel, M. L. (1994) Diabetes 43, 418-425 [Abstract]
  2. Anderson, R. G. W., and Pathak, R. K. (1985) Cell 40, 635-643 [Medline] [Order article via Infotrieve]
  3. Bauerfeind, R., and Huttner, W. B. (1993) Curr. Opin. Cell Biol. 5, 628-635 [Medline] [Order article via Infotrieve]
  4. Bulenda, D., and Gratzl, M. (1985) Biochemistry 24, 7760-7765 [Medline] [Order article via Infotrieve]
  5. Chanat, E., and Huttner, W. B. (1991) J. Cell Biol. 115, 1505-1519 [Abstract]
  6. Christie, D. L., and Palmer, D. J. (1990) Biochem. J. 270, 57-61 [Medline] [Order article via Infotrieve]
  7. Chung, K., Walter, P., Aponte, G. W., and Moore, H. H. (1989) Science 243, 192-197 [Medline] [Order article via Infotrieve]
  8. Devi, L. (1991) in Peptide Biosynthesis and Processing (Fricker, L. D., ed) pp. 175-198, CRC Press, Boca Raton, FL
  9. Fricker, L. D. (1988a) Annu. Rev. Physiol. 50, 309-321 [CrossRef][Medline] [Order article via Infotrieve]
  10. Fricker, L. D. (1988b) J. Cell. Biochem. 38, 279-289 [Medline] [Order article via Infotrieve]
  11. Fricker, L. D. (1991) in Peptide Biosynthesis and Processing (Fricker, L. D., ed) pp. 199-230, CRC Press, Boca Raton, FL
  12. Fricker, L. D. (1994) Neuroprotocols 5, in press
  13. Fricker, L. D., and Snyder, S. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3886-3890 [Abstract]
  14. Fricker, L. D., Das, B., and Angeletti, R. H. (1990) J. Biol. Chem. 265, 2476-2482 [Abstract/Free Full Text]
  15. Gorr, S., Shioi, J., and Cohn, D. V. (1989) Am. J. Physiol. 257, E247-E252
  16. Gorr, S., Hamilton, J. W., and Cohn, D. V. (1992) J. Biol. Chem. 267, 21595-21600 [Abstract/Free Full Text]
  17. Kelly, R. B. (1985) Science 230, 25-32 [Medline] [Order article via Infotrieve]
  18. Kiefer, M. C., Tucker, J. E., Joh, R., Landsberg, K. E., Saltman, D., and Barr, P. J. (1991) DNA Cell Biol. 10, 757-769 [Medline] [Order article via Infotrieve]
  19. Laslop, A., Fischer-Colbrie, R., Hook, V., Obendorf, D., and Winkler, H. (1986) Neurosci. Lett. 72, 300-304 [Medline] [Order article via Infotrieve]
  20. Lindberg, I. and Hutton, J. C. (1991) in Peptide Biosynthesis and Processing (Fricker, L. D., ed) pp. 141-174, CRC Press, Boca Raton, FL
  21. Mitra, A., Song, L., and Fricker, L. D. (1994) J. Biol. Chem. 269, 19876-19881 [Abstract/Free Full Text]
  22. Morrissey, J. H. (1981) Anal. Biochem. 117, 307-310 [Medline] [Order article via Infotrieve]
  23. Nakagawa, T., Hosaka, M., Torii, S., Watanabe, T., Murakami, K., and Nakayama, K. (1993) J. Biochem. 113, 132-135 [Abstract]
  24. Nakayama, K., Kim, W. S., Torii, S., Hosaka, M., Nakagawa, T., Ikemizu, J., Baba, T., and Murakami, K. (1992) J. Biol. Chem. 267, 5897-5900 [Abstract/Free Full Text]
  25. Nalamachu, S. R., Song, L., and Fricker, L. D. (1994) J. Biol. Chem. 269, 11192-11195 [Abstract/Free Full Text]
  26. Orci, L., Ravazzola, M., Storch, M. J., Anderson, R. G. W., Vassalli, J. D., and Perrelet, A. (1987) Cell 49, 865-868 [Medline] [Order article via Infotrieve]
  27. Palmer, D. J., and Christie, D. L. (1992) J. Biol. Chem. 267, 19806-19812 [Abstract/Free Full Text]
  28. Pimplikar, S. W., and Huttner, W. B. (1992) J. Biol. Chem. 267, 4110-4118 [Abstract/Free Full Text]
  29. Reaves, B. J., and Dannies, P. S. (1991) Mol. Cell. Endocrinol. 79, C141-C145
  30. Roos, N. (1988) Scanning Microsc. 2, 323-329 [Medline] [Order article via Infotrieve]
  31. Sambrook, J. F. (1990) Cell 61, 197-199 [Medline] [Order article via Infotrieve]
  32. Schnabel, E., Mains, R. E., and Farquhar, M. G. (1989) Mol. Endocrinol. 3, 1223-1235 [Abstract]
  33. Seidah, N. G., Gaspar, L., Mion, P., Marcinkiewicz, M., Mbikay, M., and Chretien, M. (1990) DNA Cell Biol. 9, 415-424 [Medline] [Order article via Infotrieve]
  34. Seidah, N. G., Marcinkiewicz, M., Benjannet, S., Gaspar, L., Beaubien, G., Mattei, M. G., Lazure, C., Mbikay, M., and Chretien, M. (1991) Mol. Endocrinol. 5, 111-122 [Abstract]
  35. Settleman, J., Nolan, J., and Angeletti, R. H. (1985) J. Biol. Chem. 260, 1641-1644 [Abstract]
  36. Shennan, K. I. J., Taylor, N. A., and Docherty, K. (1994) J. Biol. Chem. 269, 18646-18650 [Abstract/Free Full Text]
  37. Smeekens, S. P., and Steiner, D. F. (1990) J. Biol. Chem. 265, 2997-3000 [Abstract/Free Full Text]
  38. Smeekens, S. P., Avruch, A. S., LaMendola, J., Chan, S. J., and Steiner, D. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 340-344 [Abstract]
  39. Steiner, D. F. (1991) in Peptide Biosynthesis and Processing (Fricker, L. D., ed) pp. 1-16, CRC Press, Boca Raton, FL
  40. Supattapone, S., Fricker, L. D., and Snyder, S. H. (1984) J. Neurochem. 42, 1017-1023 [Medline] [Order article via Infotrieve]
  41. Yoo, S. H., and Albanesi, J. P. (1990) J. Biol. Chem. 265, 14414-14421 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.