Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment.

We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway.     

Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.     

Sorting ourselves out: seeking consensus on trafficking in the beta-cell

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans -Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.     

Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells

In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.     

Proinsulin modified by analogues of arginine and lysine is degraded rapidly in pancreatic B-cells.

Modified cytosolic proteins are known to be degraded more rapidly than their native counterparts. In order to determine whether the same applies to a modified protein within the potentially protective environment of secretory granules, rat islets were labelled [( 3H]leucine) in the presence or absence (controls) of 3 mM-canavanine and 3 mM-thialysine (analogues of arginine and lysine respectively), followed by a 24h 'chase' period without analogues. The results showed the following. (1) Incorporation of the analogues into newly synthesized labelled proinsulin inhibited its conversion into insulin during the chase period. (2) Despite this block in conversion, the modified proinsulin was released from islets at the same rate as native proinsulin and insulin from control islets. (3) Morphometric analysis of high-resolution autoradiographs showed that products labelled in the presence of analogues were sequestered into secretory granules at the same rate as native products in control B-cells. (4) Only 7% of prelabelled proinsulin had been degraded within islet cells during the chase period in control islets, compared with 36% for proinsulin prelabelled in the presence of analogues. (5) Control experiments showed that the analogues had no effect on the release or intracellular degradation of unmodified stored insulin (present in islets before exposure to the analogues). (6) Despite sequestration into secretory granules, modified proinsulin, if not released from B-cells, is thus degraded more rapidly than native products.     

Ammonium chloride alters secretory protein sorting within the maturing exocrine storage compartment

Addition of 20 mM ammonium chloride during in vitro chase incubation of [35S]methionine pulse-labeled parotid tissue does not perturb the magnitude or radiochemical composition of secretion stimulated by isoproterenol. An apparent inhibition of stimulated output of radiolabeled secretory proteins that was observed when ammonium chloride was added immediately postpulse (but not at later time points prior to stimulation) could be accounted for by slowdown in Golgi transit of exocrine secretory protein at a stage prior to completion of terminal glycosylation. Thus, ammonium chloride does not block entry of newly synthesized secretory proteins into the secretagogue-releasable storage granule compartment. By contrast, ammonium chloride increases the output and substantially alters the relative composition of newly synthesized protein in unstimulated secretion. The latter effects could be assigned to stages of intracellular transport that normally occur at chase times greater than 60 min postpulse and thus are focused within the maturing acinar storage granule. Notably, the compositional alterations cannot reflect the preferential exocytosis of immature granules. Taken together, these results suggest that the sorting of exocrine secretory proteins into the secretagogue-regulated pathway may not involve positive selection by a pH-based process initiated in a pregranule compartment. Rather, unstimulated secretion may arise by a negative sorting (or exclusion) process that occurs during compaction of proteins for storage within maturing granules and that is perturbed by weak base addition. Sorted (or excluded) proteins would appear to follow the vesicular (nongranular) secretory pathway that originates in maturing granules (von Zastrow, M., and Castle, J.D. (1987) J. Cell Biol. 105, 2675-2684).     

Reduction of the disulfide bond of chromogranin B (secretogranin I) in the trans-Golgi network causes its missorting to the constitutive secretory pathways.

The role of the single, highly conserved disulfide bond in chromogranin B (secretogranin I) on the sorting of this regulated secretory protein to secretory granules was investigated in the neuroendocrine cell line PC12. Treatment of PC12 cells with dithiothreitol (DTT), a membrane permeable thiol reducing agent known to prevent disulfide bond formation in intact cells, resulted in the secretion of newly synthesized chromogranin B, but only slightly decreased the intracellular storage of newly synthesized secretogranin II, a regulated secretory protein devoid of cysteines. The secretion of newly synthesized chromogranin B in the presence of DTT occurred with similar kinetics to those of a heparan sulfate proteoglycan, a known marker of the constitutive secretory pathway in PC12 cells. Analysis of the various secretory vesicles derived from the trans-Golgi network (TGN) indicated that DTT treatment diverted newly synthesized chromogranin B to constitutive secretory vesicles, whereas the packaging of secretogranin II into immature secretory granules was unaffected by the reducing agent. The chromogranin B molecules diverted to constitutive secretory vesicles, in contrast to those stored in secretory granules, were found to contain free sulfhydryl residues. The effect of DTT on chromogranin B occurred in the TGN rather than in the endoplasmic reticulum. We conclude that the sorting of CgB in the TGN to secretory granules is dependent upon the integrity of its single disulfide bond.     

Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ~90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.     

Disruption of a receptor-mediated mechanism for intracellular sorting of proinsulin in familial hyperproinsulinemia

In familial hyperproinsulinemia, specific mutations in the proinsulin gene are linked with a profound increase in circulating plasma proinsulin levels. However, the molecular and cellular basis for this disease remains uncharacterized. Here we investigated how these mutations may disrupt the sorting signal required to target proinsulin to the secretory granules of the regulated secretory pathway, resulting in the unregulated release of proinsulin. Using a combination of molecular modeling and site-directed mutagenesis, we have identified structural molecular motifs in proinsulin that are necessary for correct sorting into secretory granules of endocrine cells. We show that membrane carboxypeptidase E (CPE), previously identified as a prohormone-sorting receptor, is essential for proinsulin sorting. This was demonstrated through short interfering RNA-mediated depletion of CPE and transfection with a dominant negative mutant of CPE in a beta-cell line. Mutant proinsulins found in familial hyperproinsulinemia failed to bind to CPE and were not sorted efficiently. These findings provide evidence that the elevation of plasma proinsulin levels found in patients with familial hyperproinsulinemia is caused by the disruption of CPE-mediated sorting of mutant proinsulins to the regulated secretory pathway.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Secretory lysosomes - a special mechanism of regulated secretion in haemopoietic cells

The secretory granules o f cells derived from the haemopoietic lineage are 'secretory lysosomes' containing both lysosomal hydrolases and secretory proteins. Studies on cytotoxic T lymphocytes (CTLs) have elucidated several of the mechanisms that regulate protein sorting to, and secretion from, this unusual secretory organelle. In particular, recent findings from a CTL mutant have led to the hypothesis that CTLs, and other cells of the haemopoietic lineage, use specialized sorting and secretory mechanisms in which the lysosome functions as a regulated secretory granule.     

Substrate-favored lysosomal and proteasomal pathways participate in the normal balance control of insulin precursor maturation and disposal in β-cells

Our recent studies have uncovered that aggregation-prone proinsulin preserves a low relative folding rate and maintains a homeostatic balance of natively and non-natively folded states (i.e., proinsulin homeostasis, PIHO) in β-cells as a result of the integration of maturation and disposal processes. Control of precursor maturation and disposal is thus an early regulative mechanism in the insulin production of β-cells. Herein, we show pathways involved in the disposal of endogenous proinsulin at the early secretory pathway. We conducted metabolic-labeling, immunoblotting, and immunohistochemistry studies to examine the effects of selective proteasome and lysosome or autophagy inhibitors on the kinetics of proinsulin and control proteins in various post-translational courses. Our metabolic-labeling studies found that the main lysosomal and ancillary proteasomal pathways participate in the heavy clearance of insulin precursor in mouse islets/β-cells cultured at the mimic physiological glucose concentrations. Further immunoblotting and immunohistochemistry studies in cloned β-cells validated that among secretory proteins, insulin precursor is heavily and preferentially removed. The rapid disposal of a large amount of insulin precursor after translation is achieved mainly through lysosomal autophagy and the subsequent basal disposals are carried out by both lysosomal and proteasomal pathways within a 30 to 60-minute post-translational process. The findings provide the first clear demonstration that lysosomal and proteasomal pathways both play roles in the normal maintenance of PIHO for insulin production, and defined the physiological participation of lysosomal autophagy in the protein quality control at the early secretory pathway of pancreatic β-cells.     

Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C- peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.     

Imaging secretory vesicles by fluorescent protein insertion in propeptide rather than mature secreted peptide

We combined confocal and live-cell imaging with a novel molecular strategy aimed at revealing mechanisms underlying glucose-regulated insulin vesicle secretion. The 'Ins-C-GFP' reporter monitors secretory peptide targeting, trafficking, and exocytosis without directly tagging the mature secreted peptide. We trapped a green fluorescent protein (GFP) reporter in equimolar quantity within the secretory vesicle by fusing it within the C peptide of proinsulin which only after nascent vesicle sealing and acidification is cleaved from the mature secreted A and B chains of insulin. Ins-C-GFP expression in mouse islets without fail exhibited punctate distribution of green fluorescence by confocal microscopy. Ins-C-GFP colocalized GFP with insulin at vesicle dense cores by immuno-electron microscopy. Glucose stimulation decreased vesicle fluorescence coordinately with enhanced secretion from islets of C-GFP detected by anti-GFP Western blots, and of insulin detected by anti-insulin radioimmunoassay. An insulin secretagogue with a red fluorescent label, glibenclamide BODIPY^®TR, was applied to islets expressing Ins-C-GFP. The stimulus response was imaged as a rise in red secretagogue leading to marked loss in green granules. Since neuropeptides as well as peptide hormones are processed from propeptides after sealing of secretory granules, vesicle trapping likely is widely applicable for studies on targeting, trafficking, and regulated release of secretory peptides.     

Processing of proinsulin by transfected hepatoma (FAO) cells.

Rat hepatoma (FAO) cells were stably transfected with the gene encoding either rat proinsulin II (using the DOL retroviral vector) or human proinsulin (using the RSV retroviral vector). Using the DOL vector, production of insulin immunoreactive material was stimulated up to 30-fold by dexamethasone (5 x 10(-7) M). For both proinsulins, fractional release of immunoreactive material relative to cellular content was high, in keeping with the absence of any storage compartment for secretory proteins in these cells. Pulse-chase experiments showed kinetics of release of newly synthesized products in keeping with release via the constitutive pathway. High performance liquid chromatography analysis showed immunoreactivity in the medium distributed between three peaks. For rat proinsulin II, the first coeluted with intact proinsulin; the second coeluted with des-64,65 split proinsulin (the product of endoproteolytic attack between the insulin A-chain and C-peptide followed by trimming of C-terminal basic residues by carboxypeptidase); the third (and minor peak) coeluted with native (fully processed) insulin. For human proinsulin, by contrast, the second peak coeluted with des-31,32 split proinsulin (split and trimmed at the B-chain/C-peptide junction). Analysis of cellular extracts showed intact proinsulin as the major product. The generation of the putative conversion intermediates and insulin was not due to proteolysis of proinsulin after its release but rather to an intracellular event. The data suggest that proinsulin, normally processed in secretory granules and released via the regulated pathway, may also be processed, albeit less efficiently, by the constitutive pathway conversion machinery. The comparison of the sites preferentially cleaved in rat II or human proinsulin suggests cleavage by endoprotease(s) with a preference for R/KXR/KR as substrate.     

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.     

Proinsulin endoproteolysis confers enhanced targeting of processed insulin to the regulated secretory pathway

Recently, two different prohormone-processing enzymes, prohormone convertase 1 (PC1) and carboxypeptidase E, have been implicated in enhancing the storage of peptide hormones in endocrine secretory granules. It is important to know the extent to which such molecules may act as "sorting receptors" to allow the selective trafficking of cargo proteins from the trans-Golgi network into forming granules, versus acting as enzymes that may indirectly facilitate intraluminal storage of processed hormones within maturing granules. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, proinsulin readily enters the immature granules of these cells. Interestingly, GH4C1 clones that stably express modest levels of PC1 store more proinsulin-derived protein in granules. Even in the presence of PC1, a sizable portion of the proinsulin that enters granules goes unprocessed, and this portion largely escapes granule storage. Indeed, all of the increased granule storage can be accounted for by the modest portion converted to insulin. These results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is expressed endogenously at higher levels). An in vitro assay of protein solubility indicates a difference in the biophysical behavior of proinsulin and insulin in the PC1 transfectants. We conclude that processing to insulin, facilitated by the catalytic activities of granule proteolytic enzymes, assists in the targeting (storage) of the hormone.     

Mutant proinsulin that cannot be converted is secreted efficiently from primary rat beta-cells via the regulated pathway

Prohormones are directed from the trans-Golgi network to secretory granules of the regulated secretory pathway. It has further been proposed that prohormone conversion by endoproteolysis may be necessary for subsequent retention of peptides in granules and to prevent their release by the so-called "constitutive-like" pathway. To address this directly, mutant human proinsulin (Arg/Gly(32):Lys/Thr(64)), which cannot be cleaved by conversion endoproteases, was expressed in primary rat islet cells by recombinant adenovirus. The handling of the mutant proinsulin was compared with that of wild-type human proinsulin. Infected islet cells were pulse labeled and both basal and stimulated secretion of radiolabeled products followed during a chase. Labeled products were quantified by high-performance liquid chromatography. As expected, the mutant proinsulin was not converted at any time. Basal (constitutive and constitutive-like) secretion was higher for the mutant proinsulin than for wild-type proinsulin/insulin, but amounted to <1% even during a prolonged (6-h) period of basal chase. There was no difference in stimulated (regulated) secretion of mutant and wild-type proinsulin/insulin at any time. Thus, in primary islet cells, unprocessed (mutant) proinsulin is sorted to the regulated pathway and then retained in secretory granules as efficiently as fully processed insulin.     

Ultrastructural localization of insulin and C-peptide antigenic sites in rat pancreatic B cell obtained by applying the quantitative high-resolution protein A-gold approach

Insulin and C-peptide antigenic sites have been revealed in rat pancreatic B cells by applying immunohistochemical and cytochemical techniques. Fluorescein and rhodamine stains at the light-microscope level have detected both antigens in the same B cells. With the protein A-gold technique, labeling for both antigens was found in the cisternae of the rough endoplasmic reticulum, in those of the transitional elements, in all the cisternae of the Golgi apparatus except in the trans-most one, in the smooth but not in the coated vesicles, in the immature and mature secretory granules, and in some lysosomal (multigranular) structures. The fixation procedure used yielded excellent ultrastructural preservation which allowed for high resolution. The various control experiments demonstrated the high specificity of the results. Quantitative evaluations confirmed the qualitative observations in that they documented the specificity of the label and revealed the presence of an increasing gradient for both antigenic sites along the endoplasmic reticulum-Golgi-granule secretory pathway. The quantification also demonstrated various sites in which an increased labeling occurs: the rough endoplasmic reticulum, the smooth vesicles, the trans-cisternae of the Golgi apparatus, and the immature and the mature secretory granules. The Golgi apparatus was composed of three different subcompartments distinguished by their concentration of label. These include the cisternae on the cis-side, those on the trans-side, and the trans-most rigid cisternae. Since insulin and C-peptide form the proinsulin chain, their antigenic sites were found in the same locations along the secretory pathway; differences in location appeared only in the secretory granules, where insulin was concentrated in the core, while C-peptide was found in both the core and the halo of the granules. Furthermore, in the mature secretory granules displaying a crystalline core, insulin was restricted to the core, while C-peptide was confined to the halo. These results are in accord with the biochemical data, which indicate that simultaneous localization of both antigenic sites in compartments upstream to the immature secretory granules reflects their presence in the form of proinsulin. However, upon dissociation of proinsulin into insulin and C-peptide, both antigenic sites are segregated in different locations. The peptides appear to share parallel pathways and a fate which includes secretion through exocytosis or degradation by the lysosomal system.     

Prohormone-convertase 1 processing enhances post-Golgi sorting of prothyrotropin-releasing hormone-derived peptides

Rat prothyrotropin-releasing hormone (pro-TRH) is endoproteolyzed within the regulated secretory pathway of neuroendocrine cells yielding five TRH peptides and seven to nine other unique peptides. Endoproteolysis is performed by two prohormone convertases, PC1 and PC2. Proteolysis of pro-TRH begins in the trans-Golgi network and forms two intermediates that are then differentially processed as they exit the Golgi and are packaged into immature secretory granules. We hypothesized that this initial endoproteolysis may be necessary for downstream sorting of pro-TRH-derived peptides as it occurs before Golgi exit and thus entry into the regulated secretory pathway. We now report that when pro-TRH is transiently expressed in GH4C1 cells, a neuroendocrine cell line lacking PC1, under pulse-chase conditions release is constitutive and composed of more immature processing intermediates. This is also observed by radioimmunoassay under steady-state conditions. When a mutant form of pro-TRH, which has the dibasic sites of initial processing mutated to glycines, is expressed in AtT20 cells, a neuroendocrine cell line endogenously expressing PC1, both steady-state and pulse-chase experiments revealed that peptides derived from this mutant precursor are secreted in a constitutive fashion. A constitutively secreted form of PC1 does not target pro-TRH peptides to the constitutive secretory pathway but results in sorting to the regulated secretory pathway. These results indicated that initial processing action of PC1 on pro-TRH in the trans-Golgi network, and not a cargo-receptor relationship, is important for the downstream sorting events that result in storage of pro-TRH-derived peptides in mature secretory granules.