Secretogranin III is a sulfated protein undergoing proteolytic processing in the regulated secretory pathway.

Secretogranin III (SgIII) is an acidic protein of unknown function that is present in the storage vesicles of many neuroendocrine cells. It is coexpressed with the prohormone proopiomelanocortin in the intermediate pituitary of Xenopus laevis. We developed an antiserum to investigate the biosynthesis of SgIII in pulse-chase incubated Xenopus neurointermediate lobes. SgIII was synthesized as a 61- or 63-kDa (N-glycosylated) protein and processed to a 48-kDa form which, in turn, was partially cleaved to fragments of 28 and 20 kDa. The 48-, 28-, and 20-kDa cleavage products, but not their precursors, were secreted. This secretion is regulated and can be blocked in parallel with that of proopiomelanocortin-derived peptides by the hypothalamic factors dopamine, γ-aminobutyric acid, and neuropeptide Y. Coexpression of Xenopus SgIII with prohormone convertase (PC)1 or PC2 in transfected fibroblasts was sufficient to reconstitute the processing events observed in the neurointermediate lobes. Site-directed mutagenesis revealed that Xenopus SgIII is cleaved at two dibasic sites, namely Lys68-Arg69 and Arg237-Arg238. Pulse-chase incubations of lobes with Na2[35S]SO4 showed that SgIII is sulfated in the trans-Golgi network before it is processed. Finally, SgIII processing was found in several neuroendocrine cell types from various species. We conclude that SgIII is a precursor protein and that the intact molecule can only have an intracellular function, whereas an extracellular role can only be attributed to its cleavage products.

A hallmark of neuroendocrine cells is their ability to synthesize, store, and release biologically active peptides in a regulated fashion. Most neuropeptides and peptide hormones are generated from inactive precursor proteins that are proteolytically processed at pairs of basic amino acids and often further modified to yield a functional product (1). The bulk of these modifications occurs subsequent to the sorting of peptide precursors, along with their processing enzymes, into secretory granules. These specialized storage vesicles deliver their contents to the cell surface only in response to an external signal. Besides peptides and processing enzymes, secretory granules contain a group of acidic secretory proteins, collectively known as the granin (chromogranin/secretogranin) family (2). Within this family, only chromogranin A (CgA) 1 and CgB (secretogra-nin I, SgI) show a structural relationship. Granins are characterized by acidic isoelectric points and by the presence of numerous pairs of basic amino acids, some of which are used by endoproteolytic enzymes. Unlike peptide hormones and processing enzymes, which have well defined functions in the neuroendocrine system, the physiological role of the granins is unclear. One hypothesis is that granins themselves are precursors for biologically active peptides. This notion is supported by the observation that peptides derived from proteolytic processing of CgA and SgII are capable of modulating secretion in an autocrine or paracrine manner (3)(4)(5). However, recent experimental evidence suggests that at least some granins function intracellularly as helper proteins in the sorting and proteolytic processing of prohormones. For instance, overexpression of CgB in anterior pituitary-derived AtT20 cells was found to promote the aggregation-dependent sorting of proopiomelanocortin (POMC)-derived cleavage products into secretory granules (6). Furthermore, the neuroendocrine protein 7B2 (SgV) physically interacts with the proform of prohormone convertase PC2 and seems to regulate both transport and activation of this processing enzyme in the secretory pathway (7)(8)(9)(10).
One of the granins whose function has remained elusive is SgIII. Its transcript was originally identified from rat brain during a search for mRNAs that are exclusively expressed in the central nervous system (11). The protein was detected in many brain areas, especially in neurons participating in auditory, olfactory, and extrapyramidal motor functions, as well as in those related to the hypothalamic-pituitary axis. Moreover, SgIII was found in cells of the intermediate and anterior pituitary, whereas ultrastructural studies demonstrated its presence in intracellular vesicles (11). Genetic ablation of the gene in mice revealed that animals lacking SgIII survive without any obvious impairment in viability, fertility, or locomotor behavior (12). Consequently, many of the cell types that normally express SgIII can function in the absence of this protein, perhaps because its physiological role can be replaced by that of another gene product. Recently, we cloned the first nonmammalian homolog of SgIII from the amphibian Xenopus laevis. This was achieved by a differential screening strategy designed to identify genes coexpressed with POMC in the melanotrope cells of the intermediate pituitary (13). In these cells, the production levels of POMC-derived melanophore-stimulating peptides can be manipulated in vivo by changing the background color of the toad. When the animal is placed on a black background, the mRNA levels of SgIII in the intermediate pituitary increase dramatically (up to 35-fold) and in parallel with that of POMC (30-fold increase) (14), suggesting that SgIII has a role in the production and release of peptide hormones.
In the present study, we investigate the biosynthesis of SgIII in Xenopus melanotropes, taking advantage of the high metabolic activity of these cells in black-adapted animals. We find that SgIII is a sulfated precursor protein and demonstrate that proteolytic processing occurs at two dibasic sites that are recognized by the prohormone convertases PC1 and PC2.

MATERIALS AND METHODS
Animals-South-African clawed toads, Xenopus laevis, were adapted to a black background by keeping them in black buckets under constant illumination for at least 3 weeks at 22°C.
Production of Recombinant SgIII Protein and Generation of Antiserum-A polyclonal antiserum was raised against recombinant Xenopus SgIII produced in Escherichia coli using the Qiagen expression system (Qiagen Inc., Chatsworth, CA). For this purpose, a 1.5-kb BamHI fragment of Xenopus SgIII cDNA clone X8596 (14) was ligated into the BamHI site of the prokaryotic expression vector pQE-30. This allowed the production of recombinant protein composed of Xenopus SgIII residues Ϫ8 to 437 with a hexahistidine tail at its amino terminus. Following purification of the protein by Ni 2ϩ -NTA agarose affinity chromatography, a 500-g initial dose emulsified with Freund's complete adjuvant was administered to rabbits at 20 subcutaneous sites. After 3 and 6 weeks, rabbits were boosted with 250 g of protein in Freund's incomplete adjuvant. The production of specific antibodies was monitored by enzyme-linked immunosorbent assay.
Metabolic Labeling of Xenopus NILs and Immunoprecipitation Analysis-NILs from black-adapted Xenopus were dissected and preincubated in incubation medium (IM, 112 mM NaCl; 2 mM KCl; 2 mM CaCl 2 ; 15 mM Hepes, pH 7.4, 0.3 mg/ml bovine serum albumin, 2 mg/ml glucose, pH 7.4) at 22°C for 20 min. Pulse labeling of newly synthesized proteins was performed by incubating lobes in IM containing 1.7 mCi/ml Tran[ 35 S]-label (ICN Radiochemicals) for the indicated periods at 22°C. Subsequent chase incubations were in IM containing 5 mM L-methionine. Pulse labeling of sulfated proteins was achieved by incubating lobes in 1.3 mCi/ml Na 2 [ 35 S]SO 4 (ICN Radiochemicals). Subsequent chase incubations were in IM containing 1 mM Na 2 SO 4 . Drugs and secretagogues described in the text were added in the 20-min preincubation period and remained present during subsequent pulse and chase incubations. Lobes were homogenized on ice in lysis buffer (50 mM Hepes, pH 7.2, 140 mM NaCl, 10 mM EDTA, pH 8.0, 1% Tween 20, 0.1% Triton X-100, 0.1% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mg/ml soybean trypsin inhibitor). Homogenates were cleared by centrifugation (10,000 ϫ g, 7 min at 4°C), supplemented with 0.1 volume of 10% SDS and diluted 10-fold in lysis buffer before addition of anti-SgIII antiserum (1:5000 dilution). Immune complexes were precipitated with protein A-Sepharose (LKB-Pharmacia) and resolved by SDS-PAGE. Radiolabeled proteins were visualized by fluorography.
Eukaryotic Expression Plasmids-A 1.7-kb BamHI fragment of Xenopus SgIII cDNA clone X8596 -1 encoding the entire protein (14) was subcloned downstream of the cytomegalovirus promotor into the BamHI site of the eukaryotic expression vector pcDNA3 (Invitrogen, San Diego, CA). A 2.7-kb HindIII-EcoRV fragment of plasmid pIP83 covering the entire open reading frame of human PC1 (a generous gift of Dr. A. Roebroek, University of Leuven) (15) was subcloned in the HindIII/EcoRV sites of pcDNA3. The pcDNA3 vector containing a fulllength 2.2-kb human PC2 cDNA was obtained from Dr. J. Creemers (University of Cambridge, United Kingdom). DNA for transfection studies was isolated using the Qiagen plasmid kit (Qiagen Inc.).
Site-directed Mutagenesis of SgIII-A 1.7-kb BamHI fragment of Xenopus SgIII cDNA clone X8596 -1 was subcloned into the BamHI site of the pALTER-1 vector, and oligonucleotide-directed mutagenesis was performed on single-stranded DNA using the pALTER system (Promega, Madison, WI). The desired mutations were checked by restriction enzyme digestion and double-stranded DNA sequencing. Correctly mutagenized SgIII DNA inserts were subcloned into pcDNA3. Oligonucleotides used to create amino acid substitutions in dibasic cleavage sites of SgIII were 5Ј-GGTGATGTGACTAGTAGTGGAAGC-3Ј (KR 69 3 TS 69 ) and 5Ј-CTGTGGAGAGCTCGAACGAATTA-3Ј (RR 238 3 SS 238 ).
Cell Culture -All cell culture media were obtained from Life Technologies, Inc. and supplemented with 10% (v/v) fetal calf serum. Green monkey CV-1 kidney fibroblasts were cultured in Iscove's modified Eagle's medium, mouse anterior pituitary-derived AtT20 cells, and mouse glucagon-producing ␣TC4 cells in high glucose Dulbecco's modified Eagle's medium, and mouse insuloma ␤TC3 cells in low glucose Dulbecco's modified Eagle's medium.
Cell Transfection, Metabolic Labeling, and Immunoprecipitation Analysis-Transfection of CV-1 and AtT20 cells was accomplished by the calcium-phosphate precipitation method (16). AtT20 cells were transfected with the Xenopus SgIII-pcDNA3 construct and after 48 h selected for stable expression of SgIII in medium containing 750 g/ml neomycin (Boehringer Mannheim). For transient expression studies, CV-1 cells were plated in 20-mm culture dishes, grown until 30% confluency, and transfected with 2.5 g of DNA per construct per dish. CV-1 cells (48 h after transfection) and other cell types were starved for 60 min in methionine-and cysteine-free Dulbecco's modified Eagle's medium (ICN Biochemicals) supplemented with 10% (v/v) dialyzed fetal calf serum (Life Technologies, Inc.) and subsequently pulsed for 180 min in the same medium with 0.2 mCi/ml Tran[ 35 S]-label. Cells were lysed on ice in lysis buffer, and the cell lysates and incubation media were processed for immunoprecipitation analysis as described above.

Biosynthesis and Proteolytic Processing of SgIII in Xenopus
Melanotropes-To investigate the biosynthesis of SgIII in the neurointermediate lobe (NIL) of Xenopus, we raised a polyclonal antiserum against a recombinant protein comprising Xenopus SgIII residues Ϫ8 to 437 (Fig. 1). In immunofluorescence studies on primary cultures of NILs dissected from blackadapted animals, the antiserum gave a bright staining of the melanotrope cells, whereas no immunostaining above background was detected in other (minor) cell types (e.g. stellate cells, endothelial cells). 2 These findings suggest that the melanotropes constitute the primary site of SgIII production in the NIL. When NILs from black animals were pulsed for 15 min with Tran[ 35 S]-label and subjected to immunoprecipitation analysis with the antiserum, two major radiolabeled proteins of 63 and 61 kDa were detected (Fig. 2, lane 2). These proteins were not immunoprecipitated with preimmune serum (Fig. 2, lane 1), indicating that they represent newly synthesized SgIII. Pulse-chase incubations revealed that these proteins are proteolytically processed, first yielding a 48-kDa product which is then partially cleaved into fragments of 28 and 20 kDa (Fig. 2,  lanes 3-5). Analysis of the chase media showed that only the 48-, 28-, and 20-kDa cleavage products, but not the 63-and 61-kDa precursors, are released into the medium (Fig. 2, lane  9). Collectively, these data demonstrate that SgIII is a secretory precursor protein and suggest that at least two of the seven potential dibasic cleavage sites present within its sequence ( Fig. 1) are used by endoproteolytic enzymes.
When NILs were preincubated and pulsed in the presence of tunicamycin (a blocker of N-linked glycosylation), the mobility of the 63-kDa precursor protein increased by 2 kDa, whereas the migration of the 61-kDa precursor and the three cleavage products remained unchanged. 2 This result indicates that SgIII is partially glycosylated at Asn 47 , the single putative acceptor site for N-linked glycosylation found within its primary sequence (Fig. 1). Moreover, it can be concluded that . Black boxes correspond to a conserved internally repeated sequence element, DSTK. Arginine, lysine, and methionine residues are indicated by the singleletter codes R, K, and M, respectively. This schematic representation is based on the amino acid sequence deduced from a Xenopus SgIII cDNA which is available from the EMBL data base under accession number X92872.
when intact SgIII is converted to the 48-kDa cleavage product, the glycosylated amino-terminal portion of the protein is cleaved.
The mobility of nonglycosylated intact SgIII produced by Xenopus melanotropes in SDS-PAGE gels (61 kDa) is considerably slower than one would predict from its amino acid sequence (calculated molecular mass, 49,744 Da). Similar discrepancies were found for other members of the granin family (17,18). At present, it is unclear to what extent post-translational modifications contribute to this phenomenon (i.e. phosphorylation, O-linked glycosylation, and sulfation) or whether the highly acidic nature of these proteins causes anomalous behavior on SDS-PAGE in the Laemmli system.
Melanotropes Release SgIII-and POMC-derived Cleavage Products in a Coordinated Fashion-The release of the POMCderived peptide ␣-MSH from melanotrope cells is negatively controlled by various factors of hypothalamic origin. These include dopamine, GABA, and neuropeptide Y (NPY) (19 -21). To investigate whether secretion of the SgIII-derived cleavage products is a regulated event, NILs were pulsed for 60 min and chased for 210 min in the absence or presence of these secretagogues. Secretion of the SgIII cleavage products was completely blocked by the D 2 dopamine receptor agonist apomorphine, as well as by GABA and NPY (Fig. 3). In all three cases, the inhibition led to an accumulation of SgIII cleavage products in the lobes while the precursor proteins did not accumulate, indicating that these secretagogues had not interfered with SgIII processing. Analysis of the incubation media and lobe extracts showed that the transport of POMC-derived cleavage products was affected in a similar way. These results demonstrate that the secretion of SgIII-and POMC-derived peptides by Xenopus melanotropes is regulated in a coordinated manner.
Cellular Compartments Involved in SgIII Processing-To determine the secretory compartment in which processing of SgIII occurs, we conducted pulse-chase experiments on NILs in the presence or absence of the fungal metabolite brefeldin A (BFA) or the sodium ionophore monensin. BFA causes an accumulation of newly synthesized secretory proteins in the endoplasmic reticulum (22), whereas monensin is known to interfere with protein transport between Golgi compartments (23).
Treatment of the lobes with these drugs strongly inhibited the generation of SgIII cleavage products and completely blocked their release into the medium, whereas an intracellular accumulation of the SgIII precursor forms was observed (Fig. 4). These findings suggest that the first proteolytic conversion of SgIII does not occur before the protein has reached the distal part of the Golgi apparatus. The minor amount of 48-kDa cleavage product formed in the drug-treated lobes indicates that some SgIII had escaped from the blocks. This was also found for some newly synthesized POMC, 2 a prohormone whose processing is known to occur distal to the site of action of these drugs (24).
Tyrosine sulfation is a post-translational modification mediated by a protein tyrosine-sulfotransferase found in the TGN (25). This modification may affect the biological activity or intracellular transport of a protein (26). We noticed that the Xenopus SgIII sequence contains a putative sulfation site on Tyr 110 (Fig. 1), and we decided to analyze sulfated forms of SgIII in order to further define the compartment where its processing occurs. After a 10-min pulse of NILs with Na 2 [ 35 S]SO 4 , the majority of immunoprecipitable radioactivity was found associated with the 61-and 63-kDa precursor forms of SgIII (Fig. 5). Following a 20-min pulse, only a small amount of the 48-kDa cleavage product was observed. After an additional chase of 40 min, most of the radioactivity was associated with the 48-kDa cleavage product. A chase of 120 min allowed detection of both the 48-and 20-kDa cleavage products but not the 28-kDa fragment, whereas the radioactivity associated with the 61-and 63-kDa precursors further decreased. Only the 48-and 20-kDa forms could be detected in the chase medium. Together, these data demonstrate that sulfation of SgIII precedes its proteolytic processing and that the protein reaches the TGN in an intact form.
Tooze et al. (27) showed that in rat pheochromocytoma PC12 cells the sulfation and subsequent sorting of SgII from the TGN to immature secretory granules occurs within a time interval of 20 min. Our pulse-chase analysis revealed a lag period of about 20 min between SgIII sulfation and the appearance of the first cleavage product (Fig. 5). This finding suggests that SgIII processing in Xenopus melanotropes starts in the immature secretory granules. SgIII Is a Substrate for Prohormone Convertases PC1 and PC2-To determine if the proteolytic system responsible for SgIII processing is restricted to cells of neuroendocrine origin, CV-1 kidney fibroblasts were transfected with a Xenopus SgIII cDNA construct and pulsed with Tran[ 35 S]-label for 180 min. Immunoprecipitation analysis of the cell lysate and incubation medium revealed two newly synthesized proteins of 61 and 63 kDa (Fig. 6, lanes 1 and 2) whose migrations on SDS-PAGE are identical to those of the intact SgIII precursors produced in Xenopus NILs (Fig. 6, lane 3). Both lysates and incubation media of transfected cells were devoid of SgIII-derived cleavage products, indicating that CV-1 fibroblasts are not equipped with the proteolytic system by which SgIII is processed in Xenopus melanotropes.
The prohormone convertases PC1 (also termed PC3) and PC2 represent two neuroendocrine-specific members of the subtilisin family of endoproteases and are responsible for the proteolytic conversion of a wide range of prohormones and other peptide precursors at pairs of basic amino acids (28,29). To investigate their possible involvement in SgIII processing, CV-1 cells were cotransfected with SgIII and PC1 or PC2 cDNA constructs, pulse-labeled, and analyzed for the biosynthesis of SgIII. The incubation medium of cells cotransfected with SgIII and PC2 contained, in addition to the SgIII precursor forms, three smaller immunoreactive proteins whose sizes were indistinguishable from the SgIII-derived cleavage products generated in Xenopus NILs (Fig. 6, compare lanes 3 and 6). The same set of radiolabeled proteins could be immunoprecipitated from the incubation medium of cells cotransfected with SgIII and PC1 (Fig. 6, lane 8). The 20-kDa cleavage product generated in CV-1 cells often appeared as a smear in the gel (e.g. see Fig. 6, lane 7), hampering its detection in some of our experiments. This smearing, which was also evident for the 63-kDa precursor form, may relate to additional post-translational modifications of SgIII when produced in fibroblasts. Nevertheless, our results demonstrate that both PC1 and PC2 recognize SgIII as a suitable substrate. Unlike PC1, PC2 is a highly abundant protein in Xenopus melanotropes (13,30) and therefore represents the most likely enzyme to be responsible for the processing of SgIII in these cells.
Identification of the Cleavage Sites Involved in SgIII Processing-Xenopus SgIII contains seven potential dibasic cleavage sites (Fig. 1). As mentioned above, the first cleavage yielding the 48-kDa product is in the amino-terminal region of the protein, yet carboxyl-terminal of the N-linked glycosylation site (Asn 47 ). We noticed that upon cleavage of the 48-kDa product, 2-to 3-times more radioactivity remains associated with the 28-kDa fragment than with the 20-kDa fragment (see Fig. 2 or 3). Combined with the distribution of methionines in the SgIII polypeptide sequence (Fig. 1), these observations led us to predict that the first cleavage of SgIII occurs at the Lys 68 -Arg 69 site and the second cleavage at the Arg 237 -Arg 238 site. To test this hypothesis, amino acid substitutions were introduced at these sites, and the mutated proteins were coexpressed with PC2 in CV-1 fibroblasts. The mutant in which the Lys 68 -Arg 69 site was substituted by Thr 68 -Ser 69 gave rise to the SgIII precursor forms and a single cleavage product of 28 kDa (Fig. 7,  lane 3). Other potential SgIII fragments were undetectable,  (Fig. 7, lane 4). The intact precursors but no cleavage products were observed after expressing the mutant that carried substitutions at both sites (Fig. 7, lane 5). Together, these results confirm our prediction that the endoproteolytic conversion of SgIII in Xenopus melanotropes results from cleavages at Lys 68 -Arg 69 and Arg 237 -Arg 238 . A schematic representation of SgIII processing in Xenopus melanotropes is given in Fig. 8.
SgIII Is Processed in Several Neuronal and Endocrine Cell Types from Various Species-Western blot analysis revealed that Xenopus brain and anterior pituitary lobes contain the same set of SgIII-derived cleavage products as those generated in Xenopus NILs, 2 indicating that SgIII is processed in a variety of neuroendocrine cell types. Following immunoprecipitation analysis of pulse-labeled NILs and anterior pituitary lobes from rat, a single SgIII precursor of 62 kDa and two cleavage products of 48 and 31 kDa were detected. 2 Radiolabeled proteins of the same sizes were also immunoprecipitated from cell lysates and incubation media of pulsed mouse insuloma ␤TC3 cells, glucagon-producing ␣TC4 cells, and anterior pituitaryderived AtT20 cells (Fig. 9, lanes 1-6). When AtT20 cells were stably transfected with Xenopus SgIII, additional products of 63-, 28-, and 20 kDa were found (Fig. 9, lanes 7 and 8). Thus, the dissimilarities in SgIII cleavage patterns observed between Xenopus and rodent cells probably result from species-related differences in the primary structures of the proteins. For example, rodent SgIII lacks the functional Lys 68 -Arg 69 cleavage site found in the Xenopus protein (14). The identification of a bovine chromaffin granule-associated peptide corresponding to the region amino-terminal of the dibasic Arg 95 -Lys 96 in rodent SgIII (31), a pair not present in the Xenopus protein, further substantiates the notion that SgIII processing in the various species involves dibasic sites that are not conserved during vertebrate evolution.
Ottiger et al. (11) previously reported on a single SgIII protein of ϳ57 kDa which was detected by Western blot analysis in various regions of the rat brain. We did not observe a newly synthesized SgIII-related protein of this size in rat pituitary or mouse endocrine cells. In view of the present data, the possi-bility should be considered that the protein detected by Ottiger and co-workers (11) does not represent intact SgIII but is a SgIII-derived cleavage product.
In this study, we have shown that SgIII is a sulfated precursor protein whose endoproteolytic processing is a wide-spread phenomenon in the neuroendocrine system of vertebrates. At present, the significance of SgIII processing is unclear. The possibility that it serves to liberate functionally important peptides, conforming to what has been proposed for CgA and SgII (3)(4)(5), is unlikely. First, the majority of potential dibasic cleavage sites in SgIII is not conserved during vertebrate evolution The amino-terminal (partially N-glycosylated) fragment formed after cleavage at the KR 69 site was not detected in our pulse-chase experiments, probably because it is not recognized by the antiserum. ER, endoplasmic reticulum; TGN, trans-Golgi network; SG, secretory granules. Other designations are as in Fig. 1. See text for details.
FIG. 9. SgIII processing in mouse endocrine cell lines. Mouse insuloma ␤TC3 cells, glucagon-producing ␣TC4 cells, anterior pituitary-derived AtT20 cells, and AtT20 cells stably transfected with Xenopus SgIII (AtT20/XSgIII) were pulsed for 180 min with Tran[ 35 S]label. Cell lysates (C) and incubation media (M) were subjected to immunoprecipitation analysis using anti-SgIII antiserum. Migration positions of mouse SgIII precursor and cleavage products are indicated on the left and those of Xenopus SgIII forms on the right. The arrowhead indicates a nonspecifically reacting protein also recognized by antisera raised against other His-tagged recombinant proteins. 2 (14). Moreover, our present data demonstrate that Xenopus SgIII is fully processed at the nonconserved Lys 68 -Arg 69 site, whereas only partial cleavage occurs at the conserved Arg 237 -Arg 238 site (as diagrammed in Fig. 8). Therefore, if SgIII belongs to the group of prohormones and neuropeptide precursors, it would represent a notable exception since the members of this class are generally cleaved at conserved dibasic sites (32). Second, a comparative analysis of SgIII protein sequences from Xenopus and rodents showed that regions with the highest degree of sequence identity (over 90%) are not flanked by dibasic sites (14), in contrast to what one would expect for a genuine peptide precursor. In fact, the two functional cleavage sites in Xenopus SgIII each reside within a poorly conserved region where the degree of sequence identity has dropped below 30%.
If not to liberate bioactive peptides, what then is the purpose of SgIII processing? It may terminate a function exerted by the intact protein in the early secretory compartments. An interesting example in this respect concerns the neuroendocrine protein 7B2 (SgV). When travelling through the endoplasmic reticulum and Golgi compartments, the uncleaved form of 7B2 is associated with and appears to prevent premature activation of pro-PC2 (7,8,10). Upon arrival in the TGN, 7B2 is cleaved and dissociates from pro-PC2, allowing the proenzyme to mature. Given the existence of a private chaperone for PC2, it is conceivable that additional helper proteins interact with other enzymes in the secretory pathway. If SgIII represents such a helper protein, then its processing could trigger complex dissociation. Another possibility is that SgIII promotes the selective aggregation of luminal proteins and their subsequent packaging into secretory granules, whereby its processing serves to dissolve and/or facilitate maturation of the granular content. Alternatively, the proteolytic conversion of SgIII may simply reflect the fate of the protein in the regulated secretory pathway rather than being essential for its mechanism of action.
The function of SgIII remains to be established. Neither the genetic ablation of its gene in mice (12) nor its overexpression in cultured neuroendocrine cells 2 has provided any clue with respect to the role of this protein in the neuroendocrine system. The results of our present study show that SgIII itself is a precursor molecule and can only have an intracellular function, whereas an extracellular role can only be attributed to SgIIIderived peptides.