A hallmark of secretory granules is the presence within them of condensed cores containing aggregated content proteins. Morphological studies have indicated that the formation of these cores begins in dilated extensions of the trans-Golgi network (TGN) ()(1, 2) and continues in the immature secretory granules/condensing vacuoles (ISG) that are intermediates in the granule formation process(3) . Progressive condensation of the content proteins occurs during maturation of the ISGs into mature secretory granules.

During the process of secretory granule formation, the granule content and membrane proteins are segregated from molecules constitutively transported to the plasma membrane. This segregation is thought to occur both in the TGN and in the ISGs(4, 5, 6) . A major unresolved issue is what role spontaneous protein aggregation has in this sequestration of the granule-specific proteins, particularly for the content proteins. Most of the available information concerning the aggregative properties of granule content proteins has derived from the observed in vitro behavior of the chromo/secretogranins (Cg), which can aggregate at mildly acidic pH in the presence of calcium(4, 7, 8) , conditions thought to resemble those existing in the TGN(9) .

If aggregation mediated sorting is to be a general mechanism for the segregation of regulated from constitutively secretory proteins then several criteria should be met. First, granule content proteins should aggregate under the ionic and pH conditions thought to be present in the TGN even in cells where Cgs are minor components or not present. Second, constitutively secreted proteins should not be able to co-aggregate with the granule content proteins. In previous studies where the aggregation of Cgs was examined either in vitro or in detergent-treated cell extracts, the behavior of the other granule content proteins or well characterized constitutively secreted proteins was not examined systematically(4, 7, 8) .

To test whether aggregation of content proteins can potentially mediate the targeting of proteins to secretory granules, we have developed assays to measure protein aggregation in vitro. In these assays, granule content proteins undergo pH-dependent aggregation in a process that excludes constitutively secreted proteins. Some of the content proteins aggregate at low pH when assayed individually in the absence of the other proteins, while others require an aggregating partner to precipitate. Soluble forms of membrane-associated proteins also undergo co-aggregation with the content proteins under conditions where they do not self-associate. The results are consistent with a potential role for protein aggregation in the segregation of both secretory and membrane proteins to storage granules.

MATERIALS AND METHODS

Antibodies and Purified Granule Content Proteins

Preparation of Granule Content Proteins

In Vitro Aggregation Assay

Immunoblotting and Iodination

RESULTS

Secretory Granule Content Proteins Precipitate in a pH-dependent Manner

Figure 1: Pituitary granule content proteins aggregate when the pH is reduced. Secretory granule content (2 mg/ml) was prepared from bovine pituitary gland and equilibrated with 5 mM HEPES, 10 mM MES, pH 7.5. The pH was reduced as indicated and the pellet and supernatant fractions recovered as described under ``Materials and Methods.'' The protein in both fractions was measured and is plotted as the mean and standard error of the protein in the pellet as a percentage of the total (pellet + supernatant). 150 mM KCl and 10 mM CaCl were added where indicated. In both cases, aggregation began by pH 6.5 and increased as the pH was reduced further.

When analyzed by SDS-PAGE, all of the major proteins in the granule content were observed to precipitate as the pH was reduced (Fig. 2). This effect was also reversed when the pH was retitrated back to 7.5 (Fig. 2, right panel). The two most prominent proteins were identified as Prl and GH based on the migration positions of the purified proteins and immunoblotting using specific antibodies (data not shown). Thus, these granules, as is typical for purified pituitary granules(14) , consist primarily of those from somatotrophs and mammotrophs, the most abundant cells in the pituitary. However, other known pituitary proteins could be identified by immunoblot analysis using specific antibodies. A pH-dependent aggregation was observed for LH, FSH, and chromogranin A (CgA) (Fig. 3). In addition, a 37-kDa protein reacting with anti-ACTH antibodies underwent pH-dependent aggregation as well (Fig. 3). This protein co-migrated on polyacrylamide gels with authentic porcine POMC. In general, all of these pituitary proteins aggregated as well as did GH and Prl in the same experiments.

Figure 2: The major pituitary secretory granule content proteins aggregate at mildly acidic pH. Granule content from bovine pituitary gland was utilized in the standard assay (see Fig. 1). Left panel, the pH was reduced to 6.2 or 5.5. The entire pellet (P) (lanes c, f, and i), 20% of the initial sample (I) (lanes a, d, and g), and 20% of the remaining supernatant (S) after centrifugation (lanes b, e, and h) were then subjected to SDS-PAGE and the gel was stained with Coomassie Blue. The migration positions of the indicated proteins, prolactin (Prl) and growth hormone (GH), were determined from those of the corresponding purified proteins and confirmed by immunoblotting using appropriate antibodies. Note the greatly increased appearance of the major proteins in the pellets after pH reduction (lanes f and i). For Prl, the percent aggregation at pH 7.5, 6.2, and 5.5 was 4, 14, and 21%, respectively, and the corresponding values for GH were 7, 35 and 44%. The migration positions of prestained marker proteins are indicated at the right margin. Presented is one of three similar experiments. Right panel, the reversibility of the pH-dependent aggregation was measured after reduction of the pH to 5.5, incubation for 30 min, and retitration back to pH 7.5. Control samples were maintained for 30 min at pH 7.5 and 5.5, respectively. The amounts of Prl and GH in the pellets and supernatants were measured by densitometric scanning of digitalized images of the Coomassie-stained gels and are presented as the overall mean and standard error of three separate experiments.

Figure 3: The major pituitary hormones precipitate at mildly acidic pH. Granule content (3 mg/ml) prepared from bovine pituitary glands was subjected to the aggregation assay at either pH 7.5 (lanes a and b) or 5.6-5.8 (lanes c and d). Pellet (P; lanes a and c) and 30% of the supernatant (S; lanes b and d) fractions were subjected to SDS-PAGE and transfer to nitrocellulose. The indicated proteins were identified by immunoblotting using specific antibodies followed by the ECL procedure (in the case of POMC) or by I-protein A and autoradiography. All of the content proteins underwent pH-dependent aggregation, including FSH (4% at pH 7.5 versus 50% at pH 5.6), LH (3% at pH 7.5 versus 24% at pH 5.6), and chromogranin A (CgA; 2% at pH 7.5 versus 48% at pH 5.6). As expected, GH, included as a control, was found predominantly in the pellet fraction at low pH (6% at pH 7.5 versus 42% at 5.6). 37-kDa POMC, from a separate experiment, also underwent pH-dependent aggregation (6% at pH 7.5 versus 39% at pH 5.6 after normalization for differences in Prl/GH aggregation). In each case, the results represent one of two similar experiments.

To extend these results to other types of secretory granule content, the same assay was conducted with purified adrenal chromaffin granule content proteins, consisting almost entirely (>90%) of the Cgs. Unlike what was observed using the pituitary granule content, the chromaffin granule proteins when used at 2 mg/ml did not undergo pH-dependent aggregation (Fig. 4). Aggregation did occur, however, when calcium was added (Fig. 4) and this effect was greater as the pH was reduced. The aggregation was also reversible. In three separate experiments where the pH was lowered to 5.6 in the presence of 20 mM CaCl and, after a 30-min incubation, titrated back to pH 7.5, the recovery of CgA in the pellets was reduced to the level of the pH 7.5 control (data not shown). The concentration of calcium required for maximal aggregation was 20 mM (not shown), higher than the 10 mM believed to be present in the TGN (4) but lower than the 40 mM present in mature chromaffin granules(15) . The high calcium requirement is likely to be due to the relatively low concentrations of protein employed, far lower than that of mature granules (>100 mg/ml). It has been already established that aggregation of CgA in vitro requires less calcium as the protein concentration is increased(8) . Thus, the overall aggregation of adrenal content is consistent with that expected for the chromogranins themselves.

Figure 4: Adrenal chromaffin granule content proteins aggregate in the presence of calcium. Granule content (2 mg/ml) was obtained from bovine adrenal medulla as described under ``Materials and Methods.'' This content was subjected to the standard aggregation assay in the presence or absence of 40 mM CaCl. Total protein in the pellets was measured and is plotted as the mean ± S.E. Little precipitation of the chromaffin granule content was observed in the absence of CaCl, or in the presence of 150 mM KCl and 10 mM CaCl (not shown). However, when high concentrations of CaCl were used, aggregation did occur, with increased aggregation at lower pH.

Constitutively Secreted Proteins Are Excluded from the Precipitate Formed by Granule Content Proteins

A similar set of experiments was conducted using chromaffin granule content proteins induced to aggregate in the presence of calcium. As shown in Fig. 5, none of the three constitutive markers, IgG, angiotensinogen, or BSA, co-aggregated effectively (<5%) with the adrenal content proteins, whereas CgA, the major content protein, did precipitate well (30-39% at pH 5.6). Taken together, these data show that protein-protein interactions occurring during protein aggregation are specific and that constitutively secreted proteins aggregate less efficiently than granule content proteins, at least in vitro.

Figure 5: Constitutively secreted proteins do not co-aggregate with adrenal chromaffin granule content proteins. Granule content (2 mg/ml) from adrenal medulla was subjected to the aggregation assay at the indicated pH values in the presence of 25 mM CaCl after addition of I-labeled angiotensinogen (Ang) and unlabeled rabbit IgG (panel A) or I-labeled BSA (panel B). Pellets (P; lanes a and c) and 20% of the supernatants (S; lanes b and d) were analyzed after SDS-PAGE (no reducing agent was added) by autoradiography on a PhosphorImager (panels A and B, top) or by Coomassie Blue staining of the gel (panels A and B, bottom). These markers of the constitutive pathway did not co-precipitate significantly with granule content proteins in the presence of CaCl. At pH 5.6, 5% of angiotensinogen, 4% of IgG, and 0% of BSA were detected in the pellet fractions as compared to 30% (panel A) and 39% (panel B) for CgA. For each constitutive protein, the results represent one of two similar experiments.

Insulin-like Growth Factor I (IGF-1) Also Co-aggregates with Granule Content Proteins

Figure 6: IGF-1 also co-aggregates with granule content proteins. I-Labeled IGF-1 (10,000 cpm) was added to the aggregation assays together with content proteins from pituitary (panel A) or adrenal medulla (heat-treated; panel B). The assays were conducted as described in the legends to Fig. 1and Fig. 4for pituitary and adrenal, respectively, at the pH values indicated with 40 mM calcium in the adrenal samples at pH 6.4 and 5.9. Total protein in the pellet and supernatant fractions was measured using a BCA assay while I was determined by counting on a -counter. The mean and standard error of triplicate samples are presented after correcting for free I (10%). IGF-1 co-aggregated with both the pituitary and adrenal content proteins at low pH.

A Role for Self-aggregating Proteins in Content Protein Precipitation

Figure 7: Prolactin, but not other pituitary granule content proteins, homotypically self-aggregates at reduced pH. Bovine Prl, human GH, and ovine LH were subjected to the standard aggregation assay (see Fig. 1). 150 mM KCl and 10 mM CaCl was included in one set of the samples as indicated. Total protein in the supernatants and pellets was measured in triplicate samples and is plotted as the mean ± S.E., which in most cases was too small to be represented on the graph. Prolactin underwent a strong self-aggregation as the pH was reduced <6.5. This aggregation was not affected by the addition of salt. GH did not aggregate well, although some pH-dependent aggregation was detected in the absence of salt (up to 12% in some experiments). By contrast, LH and the constitutively secreted proteins, IgG and BSA, did not self-aggregate detectably under these conditions.

CgA, GH, and LH, which did not self-associate under these conditions, did undergo substantial aggregation in the pituitary content itself (Fig. 2), implying that their aggregation, at least in vitro, is dependent on their interaction with other proteins in the mixture, perhaps self-aggregating proteins like prolactin. To investigate these types of protein-protein interactions, Cgs, in the form of adrenal content, and purified LH were added to Prl and the pH reduced so as to initiate Prl aggregation. Prl itself was capable of driving the aggregation of the major chromaffin granule proteins, including CgA (data not shown), suggesting that these proteins could potentially interact during granule formation. LH, on the other hand, remained mostly if not entirely soluble when mixed with Prl alone in the assay (Fig. 8A), illustrating the specificity of the protein-protein interactions that occur at low pH. The aggregation of LH could be induced, however, by including LH together with adrenal content and Prl. In other words, the aggregation of LH was dependent upon its interaction with proteins in the adrenal content, most likely chromogranins, which in turn can associate with the aggregating Prl. Addition of the same amount of LH to assays conducted with pituitary extracts led to a similar level of aggregation of LH (not shown). To analyze directly the potential interaction of LH with adrenal content proteins, this hormone was added to adrenal content and Cg aggregation was induced in the presence of calcium. As depicted in Fig. 8B, LH was also induced to co-precipitate together with the Cgs in the presence of calcium, whereas neither LH nor the Cgs aggregated in its absence.

Figure 8: Co-aggregation of luteinizing hormone with adrenal components occurs in the presence of prolactin or calcium. Panel A, aggregation assays were performed as described in the legend to Fig. 2at pH 5.8 using 2 mg/ml LH alone (lanes a and b), Prl alone (lanes c and d), Prl together with LH (lanes e and f), Prl, LH together with adrenal content (lanes g and h), and LH together with pituitary content (lanes i and j). Pellet (P; lanes a, c, e, g, and i) and 20% of the supernatant (S; lanes b, d, f, h, and j) fractions were subjected to SDS-PAGE in the absence of reducing agent (lanes g and h are from a separate gel). Prl by itself failed to induce the aggregation of LH (lanes e and f). However, LH did sediment when added together to adrenal content in the presence of Prl (lanes g and h). CgA also sedimented when Prl was mixed with adrenal content under these conditions whether LH was present (lanes g and h) or not (not shown). Panel B, the aggregation assays were performed as in Fig. 5. LH (lanes e and f), adrenal content (lanes c and d, g and h), and a mixture of both (lanes c and d, i and j) were incubated in the absence (lanes a-d) or presence (lanes e-j) of 25 mM CaCl. In the absence of CaCl both LH and CgA failed to aggregate. In the presence of calcium, however, LH co-precipitated with the aggregating adrenal proteins, including CgA. The percent aggregation (% Aggreg.) obtained for each of the indicated proteins is listed at the bottom. The results indicate that LH can interact directly with chromaffin content proteins at low pH. It will sediment when the aggregation of Cgs is induced either by calcium or by Prl. Presented is one of two similar experiments.

These results are in agreement with observations made in vivo concerning the packaging of these proteins in secretory granules. Cgs and Prl are packaged together in the granules of pituitary-derived cell lines in culture and in rat mammotrophs, while LH and chromogranins appear in the same granules in pituitary gonadotrophs(20, 21, 22) .

Co-aggregation of the Luminal Domains of Granule Membrane Proteins with Content Proteins

As can be seen in Fig. 9, the endogenous soluble form of DBH in adrenal extracts aggregated only modestly when the chromaffin granule content was titrated to pH 6.0 in the absence of calcium. When adrenal content was added to pituitary granule extracts prior to the assay, the major content proteins, including CgA, underwent aggregation at low pH in the absence of calcium (Fig. 9A, lanes k and l). In this case, DBH was also prominent in the pellet fractions. This result indicates that under conditions where DBH does not self-aggregate well, it can interact with other granule content proteins. When adrenal content aggregation was induced by addition of calcium to the assay, DBH sedimented (Fig. 9B). Although homotypic self-aggregation of DBH in the presence of calcium cannot be ruled out, the data suggest that DBH is in fact co-aggregating with the Cgs in the chromaffin extracts.

Figure 9: The soluble form of the chromaffin granule membrane protein dopamine--hydroxylase aggregates together with pituitary and adrenal content proteins. Panel A, aggregation assays were performed exactly as described in the legend to Fig. 1at the indicated pH values using either 2 mg/ml adrenal content alone (lanes a-d), 2 mg/ml pituitary content (lanes e-h), or 0.5 mg/ml adrenal mixed with 2 mg/ml pituitary content protein (lanes i-l). Pellets (P; lanes a, c, e, g, i, and k) and 30% of the supernatant (S; lanes b, d, f, h, j, and l) fractions were subjected to SDS-PAGE in the absence of reducing agent. The samples were transfered to nitrocellulose and stained with Ponceau S (bottom). The nitrocellulose was probed with rabbit antibodies to bovine DBH and I-protein A and the radioactive bands detected on a PhosphorImager (top). The adrenal content proteins (including CgA) do not aggregate at low pH in the absence of calcium (lanes c and d) but do aggregate when mixed with the pituitary extract (lanes k and l). Top, the 150-kDa adrenal DBH dimer precipitated in the pituitary extract in a pH-dependent fashion but only modestly in the adrenal content itself (compare top, lanes c and d, k, and l). The migration positions of molecular mass standards are noted at the right. Panel B, adrenal chromaffin granule content proteins were subjected to the aggregation assay at pH 7.5 (lanes a and b) or 5.8 (lanes c and d) in the presence of 25 mM CaCl as described in the legend to Fig. 6. Samples were analyzed as in panel A but using the ECL immunoblotting procedure. DBH aggregated at low pH in the presence of calcium as did CgA. Thus, DBH can co-aggregate with the pituitary content at low pH under conditions where it self-aggregates poorly. It also aggregates in the adrenal extracts in the presence of calcium, conditions that promote self-aggregation of the chromogranins. The percentage of the indicated proteins in the pellet fractions (% Aggreg.) is listed at the bottom of each panel. Presented is one of two similar experiments.

PAM, in contrast to DBH, could undergo a vigorous pH-dependent aggregation in the absence of other granule components. A significant fraction of purified PAM3 (a soluble isoform consisting of virtually the entire luminal domain) was observed to sediment after reduction of the pH to 5.8 regardless of whether the assay was conducted in the presence of pituitary or adrenal granule content, or nonaggregating proteins such as hemoglobin (Fig. 10) or BSA (not shown). However, PAM self-aggregation was inhibited by CaCl (Fig. 10, lanes e and f). This inhibition was specific for calcium and not observed in the presence of the same molar equivalents of potassium ion (data not shown). When added to the pituitary or adrenal assays in the presence of calcium, PAM did sediment effectively (Fig. 10, lanes k and l), indicating that it can interact with other granule content proteins.

Figure 10: A soluble form of peptidyl glycine -amidating monooxygenase aggregates at low pH both with itself and with other granule content proteins. Panel A, purified PAM3 (1-2 µg/ml), a soluble form of the protein (see text), was added to either 1.5 mg/ml hemoglobin (globin; lanes a-f) or pituitary content (lanes g-l) and the standard aggregation assays conducted at pH 7.5 (lanes a and b, g and h) or 5.8 (lanes c-f and i-l) as described under ``Materials and Methods.'' 25 mM CaCl was included in one set of samples in each case (lanes e and f, k and l). Pellet (P; lanes a, c, e, g, i, and k) and 20% of the supernatant (S; lanes b, d, f, h, j, and l) samples were analyzed by SDS-PAGE on a 12% gel followed by transfer to nitrocellulose and staining with Ponceau S (bottom) and immunoblotting using the ECL procedure (top). At low pH, PAM underwent a strong homophilic aggregation (35% at pH 5.8) in the presence of globin, which did not itself precipitate (3%, lanes c and d). In comparison to the samples containing globin, PAM aggregated only slightly better when incubated with pituitary content proteins at pH 5.8 (41%, lanes i and j). However, in the presence of calcium, the self-aggregation of PAM was reduced (13%, lanes e and f) and under these conditions PAM still precipitated strongly in the pituitary samples (57%, lanes k and l). By comparison, the aggregation of Prl and GH together at pH 5.8 was 19% in the absence and 17% in the presence of calcium. Panel B, purified PAM3 was added to 1.5 mg/ml adrenal content proteins or globin and the assay was performed as described in the legend to Fig. 9. Samples were analyzed as described in panel A using a 10% polyacrylamide gel. In the absence of calcium at pH 5.8, 23% of PAM aggregated in the globin sample and 32% was found in the pellets of the adrenal samples. In the presence of calcium, PAM aggregated much better when mixed with adrenal content (55%, lanes k and l) than with globin (3%, lanes e and f). CgA recovered in the pellet was only 2% in the absence of calcium but 33% in its presence. Endogenous PAM did not contribute to the results as it was not readily detectable in immunoblots of pituitary and adrenal content under these conditions (not shown). Thus, PAM is capable of associating with granule content proteins at low pH. The results represent one of two similar experiments.

DISCUSSION

Proteins traversing the TGN and ISG, the sites of sorting of constitutive from regulated proteins, are exposed to mildly acidic pH and high concentrations of calcium ions(9, 28, 29, 30, 31) . The maintenance of an acidic pH in the TGN and ISG is known to be important for granule biogenesis. Treatment of secretory cells with lysosomotropic amines to dissipate pH gradients blocks maturation of secretory granules, as has been shown in both pancreatic acinar cells (32, 33) and islet cells (34) . Moreover, incubation of AtT20 cells with high concentrations of chloroquine promotes the diversion of POMC from the regulated to the constitutive secretory pathway(35) . The results presented here showing spontaneous aggregation of granule proteins in vitro support the idea that the ionic milieu of the TGN and ISG are prime contributors to the selective targeting of proteins to granules and to the condensation of secretory material that is observed to occur by electron microscopy(1, 2) .

Aggregation of Granule Content Proteins

The conditions promoting the precipitation are somewhat different in each type of granule content. In pituitary extracts, calcium ion is not required. All of the major pituitary content proteins tested (CgA, prolactin, growth hormone, POMC, FSH, and LH) precipitate. An important finding was that the constitutively secreted proteins IgG, angiotensinogen, and BSA do not co-precipitate with the pituitary content proteins. Moreover, as demonstrated previously(12) , a secretory form of the pancreatic membrane protein GP2, which is not packaged in granules in endocrine cells, does not co-aggregate with pituitary content proteins. Thus, the packaging of content proteins in storage granules correlates well with the behavior of the regulated and constitutive secretory proteins in the aggregation assays in vitro.

Adrenal content proteins, consisting primarily of the Cgs(15) , do not precipitate under the same ionic conditions as those of the anterior pituitary gland. Their precipitation requires calcium, as does the major constituents of these granules, the chromogranins. Further evidence that it is Cg aggregation being measured in our assay comes from experiments conducted using a preparation that consists almost entirely of soluble Cgs, obtained after chromaffin granule extracts were heated to 100 °C and centrifuged(37) . The same level of Ca-dependent aggregation of CgA was observed (e.g.Fig. 6). However, a recent study has provided evidence that Ca is not required for the incorporation of proteins into ISGs(38) . The transport of secretogranin II (CgC) from the TGN to ISGs in permeabilized PC12 cells was found to be insensitive to chelation of cytosolic Ca and to the addition of the Ca-H ionophore A23187. This suggests that either some protein sorting in the TGN can occur without aggregation of the Cgs or that sorting in this system largely occurs later, after formation of ISGs themselves, where Ca could still play a role. In favor of the latter explanation is the appearance in the ISGs, with a sorting index comparable to that of secretogranin II, of the constitutively secreted protein, heparan sulfate proteoglycan in this study(38) . It is also possible, however, that interaction of the granule content proteins with membrane proteins, a process that our data using DBH and PAM3 suggest may occur at mildly acidic pH even in the absence of calcium, is sufficient to insure some preferential sorting of these proteins in the TGN. In favor of this view, CgA and CgB have been reported to bind to chromaffin granule membranes at low pH in the absence of Ca(39, 40) .

Further support for the notion that interactions between granule content proteins themselves are involved in selective packaging of proteins in granules has been inferred from the properties of the Cgs. Cgs have been shown to self-aggregate when calcium is present(7, 8) . Calcium and low pH can also stabilize the Cg-containing aggregates formed in PC12 and GH4 cells(4) . At neutral pH in the absence of Ca, the S O-labeled Cgs, while still in the TGN, were extracted from detergent-permeabilized cells but at pH 6.4 in the presence of 10 mM Ca, they remained sedimentable. The behavior of content proteins other than Cgs was not reported(4) . More importantly, the use of BiP/GRP78, protein disulfide isomerase, and free glycosaminoglycans as markers of the constitutive secretory pathway that did not efficiently co-aggregate with Cgs is problematic. BiP and protein disulfide isomerase are normally resident endoplasmic reticulum proteins and have not been shown to exit endocrine secretory cells via the constitutive pathway. Glycosaminoglycans, in contrast to bona fide constitutively secreted proteins, are exported from the cell by both constitutive and regulated pathways(41) . Similarly, Gorr et al.(7) reported that ovalbumin did not co-aggregate with CgA. However, ovalbumin has not been expressed in endocrine or neuroendocrine secretory cells; hence it is not known whether it is a regulated or constitutive secretory protein. Thus it is difficult to directly relate aggregation experiments using ovalbumin, BiP, protein disulfide isomerase, or glycosaminoglycan chains to the general problem of protein sorting to secretory granules. It should be noted that the aggregation of pancreatic zymogen granule content proteins in vitro at low pH has been previously reported(42) . IgG added to the assay was also shown not to co-aggregate with amylase and other pancreatic zymogens.

The Role of Self-aggregating Content Proteins

The Luminal Domains of Membrane Proteins Co-aggregate with Content Proteins

Protein Aggregation and Selective Packaging of Proteins in Secretory Granules

FOOTNOTES

*

This work was supported in part by National Institutes of Health Grant DK44238. 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.

§

Recipient of an Ella Fitzgerald Award from the American Heart Association, NYC affiliate.

¶

Crawford-Maynard Established Investigator of the American Heart Association, NYC affiliate. To whom correspondence should be addressed: Dept. Cell Biology, NYU Medical Center, 550 First Ave., New York, NY 10016. Tel: 212-263-5812; Fax: 212-263-8139.

()

The abbreviations used are: TGN, trans-Golgi network; ISG, immature secretory granule; IgG, immunoglobulin G; BSA, bovine serum albumin; GH, growth hormone; Prl, prolactin; Cg, chromo/secretogranin; DBH, dopamine -hydroxylase; PAM, peptidyl glycine -amidating monooxygenase; FSH, follicle stimulating hormone; POMC, pro-opiomelanocortin; IGF-1, insulin-like growth factor 1; ACTH, adrenocorticotropic hormone; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; LH, luteinizing hormone.

ACKNOWLEDGEMENTS

REFERENCES

1. Schnabel, E., Mains, R. E., and Farquhar, M. G. (1989) Mol. Endocrinol. 3, 1223-1235 [Abstract/Free Full Text]
2. Rambourg, A., Clermont, Y., Chretien, M., and Olivier, L. (1992) Anat. Rec. 232, 169-179 [CrossRef][Medline] [Order article via Infotrieve]
3. Jamieson, J. D., and Palade, G. E. (1971) J. Cell Biol. 48, 503-522 [Abstract/Free Full Text]
4. Chanat, E., and Huttner, W. B. (1991) J. Cell Biol. 115, 1505-1519 [Abstract/Free Full Text]
5. Grimes, M., and Kelly, R. B. (1992) J. Cell Biol. 117, 539-549 [Abstract/Free Full Text]
6. Kuliawat, R., and Arvan, P. (1992) J. Cell Biol. 118, 521-529 [Abstract/Free Full Text]
7. Gorr, S. U., Shioi, J., and Cohn, D. V. (1989) Am. J. Physiol. 257, E247-254
8. Yoo, S. H., and Albanesi, J. P. (1991) J. Biol. Chem. 266, 7740-7745 [Abstract/Free Full Text]
9. Orci, L., Ravazzola, M., Storch, M. J., Anderson, R. G., Vassalli, J. D., and Perrelet, A. (1987) Cell 49, 865-868 [CrossRef][Medline] [Order article via Infotrieve]
10. Nolan, J., Fonseca, R., and Angeletti, R. H. (1985) Arch. Biochem. Biophys. 240, 257-264 [CrossRef][Medline] [Order article via Infotrieve]
11. Smith, A. D., and Winkler, H. (1967) Biochem. J. 103, 480-482 [Medline] [Order article via Infotrieve]
12. Colomer, V., Lal, K., Hoops, T. C., and Rindler, M. J. (1994) EMBO J. 13, 3711-3719 [Medline] [Order article via Infotrieve]
13. Kadner, S. S., Katz, J., Berliner, M., Levitz, M., and Finlay, T. H. (1984) Endocrinology 155, 2406-2417
14. Giannattasio, G., Zanini, A., and Meldolesi, J. (1975) J. Cell Biol. 64, 246-251 [Abstract/Free Full Text]
15. Winkler, H., Apps, D. K., and Fischer, C. R. (1986) Neuroscience 18, 261-290 [CrossRef][Medline] [Order article via Infotrieve]
16. Rosa, P., Weiss, U., Pepperkok, R., Ansorge, W., Niehrs, C., Stelzer, E. H., and Huttner, W. B. (1989) J. Cell Biol. 109, 17-34 [Abstract/Free Full Text]
17. Deschepper, C. F., and Reudelhuber, T. L. (1990) Hypertension 16, 147-153 [Abstract/Free Full Text]
18. Mitra, A., Song, L., and Fricker, L. D. (1994) J. Biol. Chem. 269, 19876-19881 [Abstract/Free Full Text]
19. Schmidt, W. K., and Moore, H.-P. H. (1994) J. Biol. Chem. 269, 27115-27124 [Abstract/Free Full Text]
20. Bassetti, M., Huttner, W. B., Zanini, A., and Rosa, P. (1990) J. Histochem. Cytochem. 38, 1353-1363 [Abstract]
21. Scammell, J. G., Rosa, P., Hille, A., and Huttner, W. B. (1990) J. Histochem. Cytochem. 38, 949-956 [Abstract]
22. Ozawa, H., Picart, R., Barret, A., and Tougard, C. (1994) J. Histochem. Cytochem. 42, 1097-1107 [Abstract]
23. Dhawan, S., Duong, L. T., Ornberg, R. L., and Fleming, P. J. (1987) J. Biol. Chem. 262, 1869-1875 [Abstract/Free Full Text]
24. Eipper, B. A., May, V., and Braas, K. M. (1988) J. Biol. Chem. 263, 8371-8399 [Abstract/Free Full Text]
25. May, V., Cullen, E. I., Braas, K. M., and Eipper, B. A. (1988) J. Biol. Chem. 263, 7550-7554 [Abstract/Free Full Text]
26. Braas, A. R., Stoffers, D. A., Eipper, B. A., and May, V. (1989) Mol. Endocrinol. 3, 1387-1398 [Abstract/Free Full Text]
27. Milgram, S. L., Johnson, R. C., and Mains, R. E. (1992) J. Cell Biol. 117, 717-728 [Abstract/Free Full Text]
28. Orci, L., Ravazzola, M., and Anderson, R. G. (1987) Nature 326, 77-79 [CrossRef][Medline] [Order article via Infotrieve]
29. Mata, M., Staple, J., and Fink, D. J. (1987) Histochem 87, 339-349 [CrossRef]
30. Roos, N. (1988) Scanning Microscopy 2, 323-329 [Medline] [Order article via Infotrieve]
31. Sambrook, J. F. (1990) Cell 61, 197-199 [CrossRef][Medline] [Order article via Infotrieve]
32. von Zastrow, M., Castle, A. M., and Castle, J. D. (1989) J. Biol. Chem. 264, 6566-6571 [Abstract/Free Full Text]
33. Sachs, E., and Jamieson, J. D. (1992) Am. J. Physiol. 262, G257-266
34. Orci, L., Halban, P., Perrelet, A., Amherdt, M., Ravazzola, M., and Anderson, R. G. W. (1994) J. Cell Biol. 126, 1149-1156 [Abstract/Free Full Text]
35. Moore, H. P., Gumbiner, B., and Kelly, R. B. (1983) Nature 302, 434-436 [CrossRef][Medline] [Order article via Infotrieve]
36. Orci, L., Ravazzola, M., Amherdt, M., Madsen, O., Perrelet, A., Vassalli, J. D., and Anderson, R. G. (1986) J. Cell Biol. 103, 2273-2281 [Abstract/Free Full Text]
37. 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]
38. Carnell, L., and Moore, H.-P. H. (1994) J. Cell Biol. 127, 693-706 [Abstract/Free Full Text]
39. Yoo, S. H. (1993) Biochemistry 32, 8213-8319 [CrossRef][Medline] [Order article via Infotrieve]
40. Yoo, S. H. (1993) Biochim. Biophys. Acta 1179, 239-246 [Medline] [Order article via Infotrieve]
41. Miller, S. G., and Moore, H. P. (1991) J. Cell Biol. 112, 39-54 [Abstract/Free Full Text]
42. Leblond, F. A., Viau, G., Laine, J., and Lebel, D. (1993) Biochem. J. 291, 289-296
43. Kaarsholm, N. C., Havelund, S., and Hougaard, P. (1990) Arch. Biochem. Biophys. 283, 496-502 [CrossRef][Medline] [Order article via Infotrieve]
44. Mayadas, T. N., and Wagner, D. D. (1989) J. Biol. Chem. 264, 13497-13503 [Abstract/Free Full Text]
45. Gorr, S. U., Dean, W. L., Radley, T. L., and Cohn, D. V. (1988) Bone Miner. 4, 17-25
46. Yoo, S. H. (1994) J. Biol. Chem. 269, 12001-12006 [Abstract/Free Full Text]
47. Chanat, E., Weiss, U., and Huttner, W. B. (1994) FEBS Lett. 351, 225-230 [CrossRef][Medline] [Order article via Infotrieve]

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