Mature Secretory Granules Research Articles

Identification of Secretory Granule Phosphatidylinositol 4,5-Bisphosphate-interacting Proteins Using an Affinity Pulldown Strategy

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis is required for calcium-dependent exocytosis in neurosecretory cells. We developed a PtdIns(4,5)P2 bead pulldown strategy combined with subcellular fractionation to identify endogenous chromaffin granule proteins that interact with PtdIns(4,5)P2. We identified two synaptotagmin isoforms, synaptotagmins 1 and 7; spectrin; alpha-adaptin; and synaptotagmin-like protein 4 (granuphilin) by mass spectrometry and Western blotting. The interaction between synaptotagmin 7 and PtdIns(4,5)P2 and its functional relevance was investigated. The 45-kDa isoform of synaptotagmin 7 was found to be highly expressed in adrenal chromaffin cells compared with PC12 cells and to mainly localize to secretory granules by subcellular fractionation, immunoisolation, and immunocytochemistry. We demonstrated that synaptotagmin 7 binds PtdIns(4,5)P2 via the C2B domain in the absence of calcium and via both the C2A and C2B domains in the presence of calcium. We mutated the polylysine stretch in synaptotagmin 7 C2B and demonstrated that this mutant domain lacks the calcium-independent PtdIns(4,5)P2 binding. Synaptotagmin 7 C2B domain inhibited catecholamine release from digitonin-permeabilized chromaffin cells, and this inhibition was abrogated with the C2B polylysine mutant. These data indicate that synaptotagmin 7 C2B-effector interactions, which occur via the polylysine stretch, including calcium-independent PtdIns(4,5)P2 binding, are important for chromaffin granule exocytosis.

Syntaxin6 separates from GM1a-rich membrane microdomain during granule maturation

Since it was reported that components of immature secretory granules (ISGs) are different from those of mature secretory granules (MSGs) in rat parotid acinar cells, we have been considering that components of secretory granules (SGs) change dynamically during granule maturation. As the first step to understand the mechanism of granule maturation, we separated low-density detergent-resistant membrane fractions (DRMs) from purified SGs of rat parotid gland. When SGs were lysed by the detergent Brij-58, syntaxin6 and VAMP4 were found in DRMs that were different from the GM1a-rich DRMs containing VAMP2. Because syntaxin6 and VAMP4 are known to be related to granule formation, we attempted to separate DRMs from ISGs. To enrich for ISGs, glands were removed from rats 5 h after intraperitoneal injection of isoproterenol and used to purify the newly synthesized granules. Compared to mature granules prepared without injection, these newly formed granules were lower in density and contained higher concentrations of syntaxin6, VAMP4, and γ-adaptin. This composition is consistent with the characterizations of ISGs. DRMs isolated from the newly formed granules were GM1a-rich and contained syntaxin6, VAMP4, and VAMP2 together. Thus, our findings suggest that syntaxin6 and VAMP4 associate with a GM1a-rich membrane microdomain during granule formation but enter a separate membrane microdomain before transport from granules during maturation.

Secretory cytotoxic granule maturation and exocytosis require the effector protein hMunc13-4

Cytotoxic T lymphocytes and natural killer cells exert their cytotoxic activity through the polarized secretion of cytotoxic granules at the immunological synapse. Rab27a and hMunc13-4 are critical effectors of the exocytosis of cytotoxic granules. Here we show that the cytotoxic function of lymphocytes requires the cooperation of two types of organelles: the lysosomal cytotoxic granule and the endosomal 'exocytic vesicle'. Independently of Rab27a, hMunc13-4 mediated the assembly of Rab11(+) recycling and Rab27(+) late endosomal vesicles, constituting a pool of vesicles destined for regulated exocytosis. It also primed cytotoxic granule fusion, possibly through interaction with active Rab27a. Cytotoxic T lymphocyte-target cell recognition induced rapid polarization of both types of organelles, which coalesced near the cell-cell contact area. Our data provide insight into the regulation of the generation and release of cytotoxic granules by effector cytotoxic T lymphocytes and natural killer cells.

Serglycin Is the Major Secreted Proteoglycan in Macrophages and Has a Role in the Regulation of Macrophage Tumor Necrosis Factor-α Secretion in Response to Lipopolysaccharide

It has recently been shown that serglycin is essential for maturation of mast cell secretory granules. However, serglycin is expressed also by other cell types, and in this study we addressed the role of serglycin in macrophages. Adherent cells were prepared from murine peritoneal cell populations and from spleens, and analyzed for proteoglycan synthesis by biosynthetic labeling with [35S]sulfate. Conditioned media from serglycin-/- peritoneal macrophages and adherent spleen cells displayed a 65-80% reduction of 35S-labeled proteoglycans, compared with corresponding material from serglycin+/+ cells, indicating that serglycin is the dominant secretory proteoglycan in macrophages of these origins. In contrast, the levels of intracellular proteoglycans were similar in serglycin+/+ and serglycin-/- cells, suggesting that serglycin is not stored intracellularly to a major extent in macrophages. This is in contrast to mast cells, in which serglycin is predominantly stored intracellularly. Transmission electron microscopy revealed that the absence of serglycin did not cause any major morphological effects on peritoneal macrophages, in contrast to dramatic defects in intracellular storage vesicles in peritoneal mast cells. Several secretory products were not found to be affected by the lack of serglycin. However, the secretion of tumor necrosis factor-alpha in response to lipopolysaccharide stimulation was markedly higher in serglycin-/- cultures than in those of serglycin+/+. The present report thus demonstrates that serglycin is the major proteoglycan secreted by peritoneal macrophages and suggests that the macrophage serglycin may have a role in regulating secretion of tumor necrosis factor-alpha.

Synaptotagmin IV is necessary for the maturation of secretory granules in PC12 cells

In neuroendocrine PC12 cells, immature secretory granules (ISGs) mature through homotypic fusion and membrane remodeling. We present evidence that the ISG-localized synaptotagmin IV (Syt IV) is involved in ISG maturation. Using an in vitro homotypic fusion assay, we show that the cytoplasmic domain (CD) of Syt IV, but not of Syt I, VII, or IX, inhibits ISG homotypic fusion. Moreover, Syt IV CD binds specifically to ISGs and not to mature secretory granules (MSGs), and Syt IV binds to syntaxin 6, a SNARE protein that is involved in ISG maturation. ISG homotypic fusion was inhibited in vivo by small interfering RNA–mediated depletion of Syt IV. Furthermore, the Syt IV CD, as well as Syt IV depletion, reduces secretogranin II (SgII) processing by prohormone convertase 2 (PC2). PC2 is found mostly in the proform, suggesting that activation of PC2 is also inhibited. Granule formation, and the sorting of SgII and PC2 from the trans-Golgi network into ISGs and MSGs, however, is not affected. We conclude that Syt IV is an essential component for secretory granule maturation.

GGA function is required for maturation of neuroendocrine secretory granules

Secretory granule (SG) maturation has been proposed to involve formation of clathrin-coated vesicles (CCVs) from immature SGs (ISGs). We tested the effect of inhibiting CCV budding by using the clathrin adaptor GGA (Golgi-associated, gamma-ear-containing, ADP-ribosylation factor-binding protein) on SG maturation in neuroendocrine cells. Overexpression of a truncated, GFP-tagged GGA, VHS (Vps27, Hrs, Stam)-GAT (GGA and target of myb (TOM))-GFP led to retention of MPR, VAMP4, and syntaxin 6 in mature SGs (MSGs), suggesting that CCV budding from ISGs is inhibited by the SG-localizing VHS-GAT-GFP. Furthermore, VHS-GAT-GFP-overexpression disrupts prohormone convertase 2 (PC2) autocatalytic cleavage, processing of secretogranin II to its product p18, and the correlation between PC2 and p18 levels. All these effects were not observed if full-length GGA1-GFP was overexpressed. Neither GGA1-GFP nor VHS-GAT-GFP perturbed SG protein budding from the TGN, or homotypic fusion of ISGs. Reducing GGA3 levels by using short interfering (si)RNA also led to VAMP4 retention in SGs, and inhibition of PC2 activity. Our results suggest that inhibition of CCV budding from ISGs downregulates the sorting from the ISGs and perturbs the intragranular activity of PC2.

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.

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.

Serglycin-deficient Cytotoxic T Lymphocytes Display Defective Secretory Granule Maturation and Granzyme B Storage

Cytotoxic T lymphocytes eliminate infected and tumor cells mainly by perforin/granzyme-induced apoptosis. Earlier studies suggested that serglycin-proteoglycans form macromolecular complexes with granzymes and perforin in the cytotoxic granule. Serglycin-proteoglycans may also be involved in the delivery of the cytolytic machinery into target cells. We have developed a serglycin-deficient mouse strain, and here we studied the importance of serglycin-proteoglycans for various aspects of cytotoxic T lymphocyte function. 35SO4(2-) radiolabeling of serglycin-deficient cells demonstrated a dramatic reduction of incorporated label as compared with wild type cells, indicating that serglycin is by far the dominating proteoglycan species produced by the cytotoxic T lymphocyte. Moreover, lack of serglycin resulted in impaired ability of cytotoxic T lymphocytes to produce secretory granule of high electron density, although granule of lower electron density were produced both in wild type and serglycin-deficient cells. The serglycin deficiency did not affect the mRNA expression for granzyme A, granzyme B, or perforin. However, the storage of granzyme B, but not granzyme A, Fas ligand, or perforin, was severely defective in serglycin-deficient cells. Serglycin-deficient cells did not display defects in late cytotoxicity toward target cell lines. Taken together, these results point to a key role for serglycin in the storage of granzyme B and for secretory granule maturation but argue against a major role for serglycin in the apoptosis mediated by cytotoxic T lymphocytes.

Phosphatidylinositol 3-kinase C2alpha is essential for ATP-dependent priming of neurosecretory granule exocytosis.

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2alpha (PI3K-C2alpha) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2alpha is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2alpha function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2alpha enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2alpha mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2alpha is required for acquisition of fusion competence in neurosecretion.

Parotid secretory granules: crossroads of secretory pathways and protein storage.

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins-including amylase, proline-rich proteins, and parotid secretory protein (PSP)-to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.

Identification of a novel aquaporin, AQP12, expressed in pancreatic acinar cells

Members of the aquaporin (AQP) water channel family are widely distributed in various tissues and contribute to the water permeability of epithelial and endothelial cells. Currently 11 members of the AQP family (AQP0–10) have been reported in mammals. Here we report the identification of AQP12, which we found by performing a BLAST program search. Northern blot analysis revealed that AQP12 was specifically expressed in the pancreas. Further analysis by in situ hybridization and RT-PCR studies showed that AQP12 was selectively localized in the acinar cells of the pancreas. To analyze the cellular localization and function of AQP12, we expressed AQP12 in Xenopus oocytes and cultured mammalian cells. Immunocytochemistry revealed that AQP12 was not targeted to the plasma membrane. The selective localization of AQP12 in pancreatic acinar cells and possibly in the intracellular organelles suggests a role of AQP12 in digestive enzyme secretion such as maturation and exocytosis of secretory granules.

Core Formation and the Acquisition of Fusion Competence are Linked During Secretory Granule Maturation in Tetrahymena

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.

Miniglucagon (MG)-Generating Endopeptidase, which Processes Glucagon into MG, Is Composed of N-Arginine Dibasic Convertase and Aminopeptidase B

Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.

Secretory protein trafficking: genetic and biochemical analysis.

Acute insulin secretion from stimulated pancreatic beta-cells is derived from the intracellular pool of insulin secretory granules wherein insulin is packaged in a highly concentrated (and in some species, crystalline) state. Here we review experimental work, principally from our laboratory, on the question of biogenesis of mature secretory granules within the broader context of intracellular protein trafficking. Events occurring in the lumen of organelles at various stages of intracellular transport within the secretory pathway and events at the limiting membrane of newly forming secretory granules each contribute to formation of the insulin storage compartment comprising the readily releasable pool.

Serglycin Is Essential for Maturation of Mast Cell Secretory Granule

To address the biological function of the scarcely studied intracellular proteoglycans, we targeted the gene for serglycin (SG), the only known committed intracellular proteoglycan. SG-/- mice developed normally and were fertile, but their mast cells (MCs) were severely affected. In peritoneum there was a complete absence of normal granulated MCs. Furthermore, peritoneal cells and ear tissue from SG-/- animals were devoid of the various MC-specific proteases. However, mRNA for the proteases was present in SG+/+, SG+/-, and SG-/- tissues, indicating that SG is essential for the storage, but not expression, of the MC proteases. Experiments, in which the differentiation of bone marrow stem cells into mature MCs was followed, showed that secretory granule maturation was compromised in SG-/- cells. Moreover, SG+/+ and SG+/- cells, but not SG-/- cells, synthesized proteoglycans of high anionic charge density. Taken together, we demonstrate a key role for SG proteoglycan in MC function.

Activity-dependent dynamics of coexisting brain-derived neurotrophic factor, pro-opiomelanocortin and alpha-melanophore-stimulating hormone in melanotrope cells of Xenopus laevis.

Brain-derived neurotrophic factor (BDNF) is involved as an autocrine factor in the regulation of the secretory activity of the neuroendocrine pituitary melanotrope cells of Xenopus laevis. We studied the subcellular distribution of BDNF in Xenopus melanotropes using a combination of high-pressure freezing, cryosubstitution and immunoelectron microscopy. Presence of BDNF, pro-opiomelanocortin (POMC) and alpha-melanophore-stimulating hormone (alphaMSH) within melanotrope secretory granules was studied by triple-labelling immunoelectron microscopy. In addition, intracellular processing of BDNF was investigated by quantifying the number of immunogold particles in different stages of secretory granule maturation, in animals adapted to black or white background light conditions. The high-pressure freezing technique provides excellent preservation of both cellular ultrastructure and antigenicity. BDNF coexists with POMC and alphaMSH within secretory granules. BDNF-immunoreactivity increases along the secretory granule maturation axis (i.e. from electron-dense, via moderately electron-dense, to electron-lucent secretory granules). Immature, low immunoreactive, electron-dense secretory granules are assumed to contain mainly or even exclusively proBDNF. Strongly immunoreactive electron-lucent secretory granules represent the mature granule stage in which proBDNF has been processed to mature BDNF. Furthermore, in moderately electron-dense secretory granules, immunoreactivity is markedly (+79%) higher in black-adapted than in white-adapted animals, indicating that stimulation of melanotrope cell activity by the black background condition speeds up processing of BDNF from its precursor in this granule stage. It is concluded that, in the Xenopus melanotrope, BDNF biosynthesis and processing occur along the secretory granule maturation axis, together with that of POMC-derived alphaMSH, and that the environmental light condition not only controls the biosynthesis and secretion of BDNF and of POMC end-products, but also regulates the rate of their intragranular processing.

Comparative aspects of intracellular proteolytic processing of peptide hormone precursors: studies of proopiomelanocortin processing.

In this review, the mechanisms underlying the intracellular processing of peptide hormone precursors, with a focus on proopiomelanocortin (POMC), were discussed on the basis of recent information. POMC as well as other prohormones is processed to active peptides through proteolytic cleavage by prohormone convertases PC1 and/or PC2. However, the cleavage-specificity of PC1 and PC2 in mammals is somewhat different from that in amphibians. From the comparative endocrinological point of view, expression and tissue distribution of PC1 and PC2 were discussed here. In mammals, proteolytic processing of POMC occurs coordinately with the maturation of secretory granules. Studies using immunoelectron microscopy with DAMP (3-[2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as a pH probe revealed that the acidic pH in the secretory granules, generated by vacular type-H+-ATPase, provides a favorable environment for activating PC1 in AtT-20 cells, a mouse corticotrope tumor cell line. Recent data indicate that the 7B2 protein serves as a chaperone in the regulation of PC2 activation and to control the timing for activating the convertase. Together, secretory granules in endocrine and neuroendocrine cells provide proper sites for biosynthesizing hormones in addition to serving as storage sites and vehicles for the transport of peptide hormones.

Localization of VIP36 in the post-Golgi secretory pathway also of rat parotid acinar cells.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.