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Golgi-to-plasma-membrane trafficking of synaptic-like microvesicle (SLMV) proteins, vesicular acetylcholine transporter (VAChT) and synaptophysin (SYN), and a large dense-core vesicle (LDCV) protein, chromogranin A (CgA), was investigated in undifferentiated neuroendocrine PC12 cells. Live cell imaging and 20°C block-release experiments showed that VAChT-GFP, SYN-GFP and CgA-RFP specifically and transiently cohabitated in a distinct sorting compartment during cold block and then separated into synaptic protein transport vesicles (SPTVs) and LDCVs, after release from temperature block. We found that in this trans-Golgi subcompartment there was colocalization of SPTV and LDCV proteins, most significantly with VAMP4 and Golgin97, and to some degree with TGN46, but not at all with TGN38. Moreover, some SNAP25 and VAMP2, two subunits of the exocytic machinery, were also recruited onto this compartment. Thus, in neuroendocrine cells, synaptic vesicle and LDCV proteins converge briefly in a distinct trans-Golgi network subcompartment before sorting into SPTVs and LDCVs, ultimately for delivery to the plasma membrane. This specialized sorting compartment from which SPTVs and LDCVs bud might facilitate the acquisition of common exocytic machinery needed on the membranes of these vesicles.

Synaptotagmin IV (Syt IV) is a fourth member of the Syt family and has been shown to regulate some forms of memory and learning by analysis of Syt IV null mutant mice (Ferguson, G. D., Anagnostaras, S. G., Silva, A. J., and Herschman, H. R. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5598-5603). However, the involvement of Syt IV protein in vesicular trafficking and even its localization in secretory vesicles are still matters of controversy. Here we present several lines of evidence showing that the Syt IV protein in PC12 cells is normally localized in the Golgi or immature vesicles at the cell periphery and is sorted to fusion-competent mature dense-core vesicles in response to short nerve growth factor (NGF) stimulation. (i) In undifferentiated PC12 cells, Syt IV protein is mainly localized in the Golgi and small amounts are also present at the cell periphery, but according to the results of an immunocytochemical analysis, they do not colocalize with conventional secretory vesicle markers (Syt I, Syt IX, Rab3A, Rab27A, vesicle-associated membrane protein 2, and synaptophysin) at all. By contrast, limited colocalization of Syt IV protein with dense-core vesicle markers is found in the distal parts of the neurites of NGF-differentiated PC12 cells. (ii) Immunoelectron microscopy with highly specific anti-Syt IV antibody revealed that the Syt IV protein in undifferentiated PC12 cells is mainly present on the Golgi membranes and immature secretory vesicles, whereas after NGF stimulation Syt IV protein is also present on the mature dense-core vesicles. (iii) An N-terminal antibody-uptake experiment indicated that Syt IV-containing vesicles in the neurites of NGF-differentiated PC12 cells undergo Ca(2+)-dependent exocytosis, whereas no uptake of the anti-Syt IV-N antibody was observed in undifferentiated PC12 cells. Our results suggest that Syt IV is a stimulus (e.g. NGF)-dependent regulator for exocytosis of dense-core vesicles.

The binding of pro-opiomelanocortin,(POMC), pro-insulin, pro-enkephalin and chromogranin A (CGA) to the regulated secretory pathway sorting receptor, carboxypeptidase E (CPE), in bovine pituitary secretory granule (SG) membranes was investigated. N-POMC1-26, which contains the POMC sorting signal, bound to CPE in the SG membranes with low affinity and the binding was ion independent. Pro-insulin bound CPE with similar kinetics. Pro-enkephalin, but not CGA bound to CPE with similar IC50 as pro-insulin and N-POMC1-26. Crosslinking studies showed that pro-insulin and pro-enkephalin bound specifically to SG membrane CPE, similar to N-POMC1-26 reported previously. CPE was extracted from the SG membranes with NaHCO3 or KSCN, but not Triton X-100/1 M NaCl. The results show that CPE is tightly associated with SG membranes and binds several prohormones, but not CGA, with similar kinetics, providing further evidence that membrane CPE has the characteristics to function as a common sorting receptor for targeting prohormones to the regulated secretory pathway.

A proposed mechanism for sorting secretory proteins into granules for release via the regulated secretory pathway in endocrine-neuroendocrine cells involves binding the proteins to a sorting receptor at the trans-Golgi network, followed by budding and granule formation. We have identified such a sorting receptor as membrane-associated carboxypeptidase E (CPE) in pituitary Golgi-enriched and secretory granule membranes. CPE specifically bound regulated secretory pathway proteins, including prohormones, but not constitutively secreted proteins. We show that in the Cpe(fat) mutant mouse lacking CPE, the pituitary prohormone, pro-opiomelanocortin, was missorted to the constitutive pathway and secreted in an unregulated manner. Thus, obliteration of CPE, the sorting receptor, leads to multiple endocrine disorders in these genetically defective mice, including hyperproinsulinemia and infertility.

The subcellular localization and functionality of transfected yeast aspartic protease 3 (YAP3p) in a mammalian cell line were investigated. The complementary DNAs encoding the prohormone-processing enzyme (YAP3p) and a prohormone, bovine POMC, were cotransfected into PC12 (rat pheochromocytoma) cells. Immunocytochemical analysis of the cells using a YAP3p antibody showed a perinuclear punctate distribution of YAP3p in the cell body as well as immunostaining in the tips of the neurites. This pattern of immunostaining indicates localization of YAP3p in secretory granules. Analysis of the processing of POMC showed that in cells transfected with the POMC complementary DNA alone, only POMC was found, indicating a lack of processing of the prohormone. However, in cells coexpressing YAP3p, the POMC was completely processed to yield ACTH-(1-39) and ACTH-(1-14), consistent with the specificity of YAP3p found in vitro. Pulse-chase studies showed that POMC was processed after 20 min of chase, suggesting that processing occurred in the late Golgi network and continued in the secretory granules. Western blot analysis determined that YAP3p was secreted from the cells in a regulated manner. This study provides the first demonstration that a yeast prohormone-processing enzyme (YAP3p) of the aspartic protease class can be sorted correctly to secretory granules and activated to process a prohormone (POMC) in a highly efficient manner in mammalian cells.

Mouse pituitary neurointermediate lobes were pulse-incubated in [3H] arginine or [3H] lysine for 10 min and then chase-incubated for periods 0 to 4h. The labeled peptides from the lobes were analysed by immunoprecipitation with specific antisera, and thereafter, by acid-urea polyacrylamide gel electrophoresis. Using this paradigm, the synthesis of a prohormone common to adrenocorticotropin (ACTH) and endorphin was detected in 10 min pulse labeled lobes. Following a chase period, processing of the prohormone to several forms of ACTH (mol. wt. 25000, 23000, and 13000), beta-lipotropin and beta-endorphin was observed. To determine the intracellular site of processing of the prohormone, subcellular fractionation studies of labeled lobes were carried out. Analysis of the fractions from the pulse-labeled lobes revealed that the newly synthesized labeled prohormone was primarily localized in a granule-enriched fraction. In lobes that were pulsed and then chase-incubated for 1 h, there was a decrease in the amount of prohormone and an appearance of processed products in the granule-enriched fraction. In another paradigm, where the secretory granule-fraction was isolated from pulse-labeled lobes and then incubated in vitro for 6 h at pH 5.5, processing of the endogenous labeled prohormone within the isolated granule fraction was observed. These data suggest, that in the mouse neurointermediate lobe, the ACTH/endorphin prohormone (pro-opiocortin) is rapidly packaged into secretory granules after synthesis and processed intragranularly.

Lysates from purified secretory granules of rat anterior pituitary glands were incubated with [3H]phenylalanine or [3H]arginine-labeled toad proopiocortin. The processed products formed were identified by immunoprecipitation with ACTH and beta-endorphin antisera, and by comigration with known markers on acid-urea polyacrylamide gels. Proopiocortin was cleaved by the secretory granule lysate primarily to 21,000 mol wt ACTH, 13,000 mol wt ACTH, 16,000 mol wt NH2-terminal glycopeptide, beta-lipotropin, a beta-endorphin-like peptide, and beta-endorphin. Characterization of the anterior pituitary proopicortin-converting activity shows that it: (1) cleaves specifically at the peptide bond on the carboxy side of the lysine-arginine residues of proopiocortin, (2) has a pH optimum in the acidic range, (3) is present in membrane and soluble fractions of the granule lysate, and (4) is inhibited by leupeptin, pepstatin A, and 2,2' dithiodipyridine, but not by p-chloromercuribenzoate, diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, L-1-tosylamide-2-phenylethyl-chloromethyl ketone, or EDTA.

The prohormone, pro-opiomelanocortin (POMC) is synthesized on ribosomes, subsequently routed to the Golgi apparatus and finally packaged into secretory granules where it is processed to various biologically active hormones (alpha-melanotropin, adrenocorticotropin, beta-endorphin and beta-lipotropin). We report here that in frog and mouse pars intermedia cells, newly synthesized [3H]Arg-labeled POMC is associated with the secretory granule membrane prior to processing. This association with the secretory granule membrane may be related to the intracellular transport and packaging of POMC and/or the facilitation of processing of the prohormone within the organelle.

The neuropeptides arginine vasopressin and oxytocin are generated from their prohormones in the hypothalamoneurohypophysial system by enzymatic cleavages at paired basic residues (i.e. Lys-Arg). This study describes the purification of an enzyme from bovine neural lobe secretory vesicles, the putative site of this processing, which is capable of cleaving several prohormones at paired basic residues. The enzyme is a glycoprotein of Mr approximately 70,000 and has an acidic pH maximum. It processes the heterologous precursors pro-opiomelanocortin and insulin at paired basic residues in a manner similar to a pro-opiomelanocortin-converting enzyme derived from bovine intermediate lobe secretory vesicles which has been described previously. In addition, the neural lobe-derived converting enzyme cleaves the human vasopressin prohormone in vitro to yield arginine vasopressin-Gly10-Lys11-Arg12 as the major vasopressin cleavage product. This indicates that the enzymatic cleavage in the vasopressin precursor occurred primarily on the carboxyl side of the arginine in the pair of Lys-Arg basic residues separating the vasopressin peptide from the neurophysin moiety in the precursor. The properties of the neural and intermediate lobe-derived enzymes are virtually identical, raising the possibility that a family of similar enzymes may be responsible for cleaving a number of prohormones at paired basic residues in different tissues.

Peptide hormones are synthesized from larger precursors by cleavages at paired basic residues. We have isolated a pro-hormone converting enzyme from bovine neural and intermediate lobe secretory vesicles that cleaves pro-vasopressin and pro-opiomelanocortin at Lys-Arg residues to yield vasopressin, and adrenocorticotropin/endorphin-related peptides, respectively. The enzyme from both lobes is an aspartyl protease of approximately 70,000 Da, is a glycoprotein and has an optimum pH range of 4.0-5.0. Present within the same secretory vesicles is an aminopeptidase B-like enzyme which is a metalloprotease that is inhibited by Co2+ and Zn2+. This enzyme may play a role in trimming off the N-terminal extended basic residues from peptides liberated by the pro-hormone converting enzyme.

Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.

Neurosecretory granules (NSGs) from neural lobes of bovine pituitary glands were isolated in a highly purified form by metrizamide-sucrose gradient centrifugation. The purified NSGs were lysed and centrifuged, and the supernatants were further fractionated by gel filtration on Sephadex G-75. Proopiocortin-converting enzyme activity was assayed by incubation of [3H]arginine- or [3H]phenylalanine-labeled toad proopiocortin with NSG supernatant fractions. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea gel electrophoresis. The optimum pH for the enzyme-mediated conversion was around pH 5.0. Conversion of toad proopiocortin by NSG converting enzyme activity was inhibited by leupeptin, antipain, p-chloromercuribenzoate, and pepstatin A, but not by diisopropyl fluorophosphate, EDTA, or N-alpha-p-tosyl-L-lysine-chloromethyl ketone HCl. The results suggest that the proopiocortin-converting enzyme activity in bovine neurosecretory granules is due to an acid-thiol protease which may contain secondary hydrophobic binding sites that are involved in substrate recognition.

The ability of bovine intermediate lobe secretory vesicle membrane-associated enzyme(s) and purified, soluble paired basic residue-specific, pro-opiomelanocortin converting enzyme (Loh, Y.P., Parish, D. C., and Tuteja, R. (1985) J. Biol. Chem. 260, 7194-7205) to cleave bovine NH2-terminal pro-opiomelanocortin1-77 (N-POMC 1-77) was investigated. Purified pro-opiomelanocortin converting enzyme and an enzyme activity associated with the secretory vesicle membrane were shown to cleave bovine N-POMC1-77 to two major products: N-POMC1-49 and Lys-gamma 3-melanotropin (MSH), and one minor product, gamma 3-MSH. These products were identified by their retention times on high performance liquid chromatography, immunological characteristics, and for Lys-gamma 3-MSH, amino acid composition. The products generated indicate cleavage preferentially between Arg 49-Lys 50 of bN-POMC1-77 (where b indicates bovine), which is identical to the processing pattern found in the bovine intermediate lobe in situ. The membrane converting activity was shown to be stimulated by 5 mM Ca2+ and has a pH optimum of 4-5 and an inhibitor profile characteristic of an aspartic protease. This suggests that the membrane-associated enzyme involved is very similar or identical to the purified, soluble pro-opiomelanocortin converting enzyme, which has previously been reported to be an acidic, aspartic protease responsible for the initial steps of POMC processing. The results of this study lead to the proposal that the lack of processing of the Arg49-Lys50 site in POMC in the anterior lobe versus the intermediate lobe of the pituitary in vivo may be due to other regulatory mechanisms rather than invoking the existence in the intermediate lobe of another enzyme specific for this site, different from pro-opiomelanocortin converting enzyme.

Pro-opiomelanocortin (ACTH/endorphin prohormone) is processed within the secretory vesicles of pituitary intermediate lobe cells to alpha-melanotropin and beta-endorphin. In order to learn more about the microenvironment in which processing occurs, a method was developed to purify large quantities of bovine intermediate lobe secretory vesicles (ILSV) using isoosmolar metrizamide-sucrose gradients. Analysis of alpha-melanotropin and marker enzymes revealed that the gradients provided a 94-fold purification of secretory vesicles with respect to lysosomes and a 15-fold purification with respect to mitochondria. Pro-opiomelanocortin-converting enzyme activity in the ILSVs was assayed and found to be maximally active around pH 5. From measuring the delta pH across the intact ILSV membrane using 9-aminoacridine fluorescence quenching, the internal pH of ILSVs was determined to be less than 5.6, consistent with the operating pH range of the converting enzyme activity. The pH gradient of the ILSVs collapsed in the presence of ammonium sulfate or by using a combination of nigericin and K+, when the external medium pH was 7. Using the voltage-sensitive dye, oxanol VI, Mg2+ ATP was shown to cause a marked change in the ILSV membrane potential with the inside being positive which was reversed by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Thus, the ILSVs appear to have a Mg2+ ATP-dependent electrogenic proton-translocating system.

Lysates of secretory granules from rat pituitary neurointermediate lobes were incubated with [3H]arginine- or [3H]phenylalanine-labeled toad pro-opiocortin. The processed products formed were identified by immunoprecipitation with adrenocorticotropin (ACTH) and endorphin antisera and by migration behavior on acid/urea/polyacrylamide gels. Pro-opiocortin was cleaved by the proteolytic activity in the secretory granule fraction to approximately 21,000 Mr ACTH, approximately 13,000 Mr ACTH, alpha-melanotropin, 16,000 Mr NH2-terminal glycopeptide, beta-lipotropin, and an endorphin-related peptide. Characterization of this pro-opiocortin-converting activity shows that it (i) is present in membrane and soluble fractions of the granule lysates, (ii) has a pH optimum of 5.0, (iii) appears to cleave at pairs of basic amino acid residues in the precursor, and (iv) is inhibited by leupeptin, pepstatin A, and p-chloromercuribenzoate but not diisopropyl fluorophosphate, N alpha-p-tosyl-L-lysine chloromethyl ketone hydrochloride, chloroquine, or EDTA. These inhibitor studies suggest that the converting-enzyme activity is due to an acid thiol, arginyl protease, distinct from any known cathepsin B-like activity.

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.