Biogenesis of secretory granules. Implications arising from the immature secretory granule in the regulated pathway of secretion

In endocrine cells the regulated secretion of hormones, peptides, enzymes and neurotransmitters into the external medium occurs when mature secretory granules fuse with the plasma membrane. Secretory granules form at the trans-Golgi network (TGN) by envelopment of the dense-core aggregate of regulated secretory proteins by a specific membrane. The secretory granules initially formed at the TGN, referred to here as immature secretory granules, are morphologically and biochemically distinct from mature secretory granules. The functional similarities and differences between the immature secretory granule and the mature secretory granule, and the events involved in the maturation of the secretory granules are briefly discussed.     

Regulation of secretory granule size by the precise generation and fusion of unit granules

Morphometric evidence derived from studies of mast cells, pancreatic acinar cells and other cell types supports a model in which the post-Golgi processes that generate mature secretory granules can be resolved into three steps: (1) fusion of small, Golgi-derived progranules to produce immature secretory granules which have a highly constrained volume; (2) transformation of such immature granules into mature secretory granules, a process often associated with a reduction in the maturing granule’s volume, as well as changes in the appearance of its content and (3) fusion of secretory granules of the smallest size, termed ‘unit granules’, forming granules whose volumes are multiples of the unit granule’s volume. Mutations which perturb this process can cause significant pathology. For example, Chediak–Higashi syndrome / lysosomal trafficking regulator (CHS)/(Lyst) mutations result in giant secretory granules in a number of cell types in human beings with the Chediak–Higashi syndrome and in ‘beige’ (Lyst^bg/Lyst^bg) mice. Analysis of the secretory granules of mast cells and pancreatic acinar cells in Lyst-deficient beige mice suggests that beige mouse secretory granules retain the ability to fuse randomly with other secretory granules no matter what the size of the fusion partners. By contrast, in normal mice, the pattern of granule–granule fusion occurs exclusively by the addition of unit granules, either to each other or to larger granules. The normal pattern of fusion is termed unit addition and the fusion evident in cells with CHS/Lyst mutations is called random addition. The proposed model of secretory granule formation has several implications. For example, in neurosecretory cells, the secretion of small amounts of cargo in granules constrained to a very narrow size increases the precision of the information conveyed by secretion. By contrast, in pancreatic acinar cells and mast cells, large granules composed of multiple unit granules permit the cells to store large amounts of material without requiring the amount of membrane necessary to package the same amount of cargo into small granules. In addition, the formation of mature secretory granules that are multimers of unit granules provides a mechanism for mixing in large granules the contents of unit granules which differ in their content of cargo.     

Intracellular transport and storage of secretory proteins in relation to cytodifferentiation in neoplastic pancreatic acinar cells

The pancreatic acinar carcinoma established in rat by Reddy and Rao (1977, Science 198:78-80) demonstrates heterogeneity of cytodifferentiation ranging from cells containing abundant well- developed secretory granules to those with virtually none. We examined the synthesis intracellular transport and storage of secretory proteins in secretory granule-enriched (GEF) and secretory granule-deficient (GDF) subpopulations of neoplastic acinar cells separable by Percoll gradient centrifugation, to determine the secretory process in cells with distinctly different cytodifferentiation. The cells pulse-labeled with [3H]leucine for 3 min and chase incubated for up to 4 h were analyzed by quantitative electron microscope autoradiography. In GEF neoplastic cells, the results of grain counts and relative grain density estimates establish that the label moves successively from rough endoplasmic reticulum (RER) leads to the Golgi apparatus leads to post-Golgi vesicles (vacuoles or immature granules) leads to mature secretory granules, in a manner reminiscent of the secretory process in normal pancreatic acinar cells. The presence of approximately 40% of the label in association with secretory granules at 4 h postpulse indicates that GEF neoplastic cells retain (acquire) the essential regulatory controls of the secretory process. In GDF neoplastic acinar cells the drainage of label from RER is slower, but the peak label of approximately 20% in the Golgi apparatus is reached relatively rapidly (10 min postpulse). The movement of label from the Golgi to the post- Golgi vesicles is evident; further delineation of the secretory process in GDF neoplastic cells, however, was not possible due to lack of secretory granule differentiation. The movement of label from RER leads to the Golgi apparatus leads to the post-Golgi vesicles suggests that GDF neoplastic cells also synthesize secretory proteins, but to a lesser extent than the GEF cells. The reason(s) for the inability of GDF cells to concentrate and store exportable proteins remain to be elucidated.     

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.     

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.     

Not all secretory granules are created equal: Partitioning of soluble content proteins

Secretory granules carrying fluorescent cargo proteins are widely used to study granule biogenesis, maturation, and regulated exocytosis. We fused the soluble secretory protein peptidylglycine alpha-hydroxylating monooxygenase (PHM) to green fluorescent protein (GFP) to study granule formation. When expressed in AtT-20 or GH3 cells, the PHM-GFP fusion protein partitioned from endogenous hormone (adrenocorticotropic hormone, growth hormone) into separate secretory granule pools. Both exogenous and endogenous granule proteins were stored and released in response to secretagogue. Importantly, we found that segregation of content proteins is not an artifact of overexpression nor peculiar to GFP-tagged proteins. Neither luminal acidification nor cholesterol-rich membrane microdomains play essential roles in soluble content protein segregation. Our data suggest that intrinsic biophysical properties of cargo proteins govern their differential sorting, with segregation occurring during the process of granule maturation. Proteins that can self-aggregate are likely to partition into separate granules, which can accommodate only a few thousand copies of any content protein; proteins that lack tertiary structure are more likely to distribute homogeneously into secretory granules. Therefore, a simple "self-aggregation default" theory may explain the little acknowledged, but commonly observed, tendency for both naturally occurring and exogenous content proteins to segregate from each other into distinct secretory granules.     

Characterization of the TGN exit routes in AtT20 cells using pancreatic amylase and serum albumin

The AtT20 pituitary cell is the one that was originally used to define the pathways taken by secretory proteins in mammalian cells. It possesses two secretory pathways, the constitutive for immediate secretion and the regulated for accumulation and release under hormonal stimulation. It is in the regulated pathway, most precisely in the immature granule of the regulated pathway, that proteolytic maturation takes place. A pathway that stems from the regulated one, namely the constitutive-like pathway releases proteins present in immature granules that are not destined for accumulation in mature granules. In AtT20 cells proopiomelanocortin the endogenous precursor of the accumulated adrenocorticotropic hormone, is predominantly secreted in a constitutive manner without proteolytic maturation. In order to better understand by which secretory pathway intact proopiomelanocortin is secreted by a cell line possessing a regulated secretory pathway, it was transfected with rat serum albumin (a marker of constitutive secretory proteins), and pancreatic amylase (a marker of regulated proteins). COS cells were also transfected in order to serve as control of release by the constitutive pathway. It was observed that both the basal and stimulated secretions of albumin and proopiomelanocortin from AtT20 cells are identical. In addition, secretagogue stimulation when POMC is in transit in the trans-Golgi network decreases its constitutive secretion by 50%. It was also observed using cell fractionation and 20 degrees C secretion blocks that albumin and proopiomelanocortin are present in the regulated pathway, presumably in the immature granules, and are secreted by the constitutive-like secretory pathway. These observations show that stimulation can increase sorting into the regulated pathway, and confirm the importance of the constitutive-like secretory pathway in the model AtT20 cell line.     

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.     

Protein sorting among two distinct export pathways occurs from the content of maturing exocrine storage granules

We have developed a method for separating purified parotid secretory granules according to their degree of maturation, and we have used this method to examine the relationship between granule formation and stimulus-independent (constitutive) protein secretion. Constitutive export of pulse-labeled secretory proteins occurs almost entirely after their appearance in newly formed granules, and this secretion can be resolved kinetically into two distinct components. Later-phase secretion is the more prominent component and, according to kinetic and compositional criteria, appears to result from basal exocytosis of mature granules. In contrast, early-phase secretion (1.5-15% of constitutive protein output) appears to originate from maturing granules but differs significantly from granule content in composition; that is, the early component exports individual protein species in different relative amounts. Maturing granules, which are labeled most highly before and during the appearance of early-phase secretion, possess numerous coated membrane evaginations suggestive of vesicular traffic. We propose that, in addition to basal exocytosis of relatively mature granules, constitutive exocrine secretion results from limited, selective removal of content proteins from forming and maturing granules. Thus protein sorting and packaging occur together in granule compartments. Exocrine secretory granules constitute an extension of the post-Golgi sorting system and are not merely terminal depots for proximally targeted polypeptides.     

Membrane modification during secretory granule formation in rat somatotrophs

Somatotrophs from male rat anterior pituitary were used to investigate the formation of secretory granules. When enzymatically dispersed cells were incubated with cationized ferritin (CF) for 15 min, CF labeled immature secretory granules, but not mature granules of somatotrophs. Most immature granules labeled by CF transformed to the mature types within 120 min. This indicates that the fusion of endocytic vesicles with the immature granules occurs during the maturation process of secretory granules. The internalized CF was distributed not only in the immature secretory granules, but also in the peripheral region of trans Golgi cisternae or GERL. Enzyme cytochemistry revealed that acid phosphatase-positive cisternae (GERL) were the main site for secretory granule formation, and was devoid of thiamine pyrophosphatase (TPPase) activity. A small number of secretory granules were also present in the peripheral regions of TPPase-positive Golgi cisternae. The granule-forming sites, however, lacked TPPase activity, while the remaining region of the same cisterna showed the positive enzyme activity. This indicates that the granule-forming region at the periphery of Golgi cisterna is different from the remaining part of the same cisterna in terms of cytochemical properties. This probably results from the insertion of endocytic vesicle membrane, since the same granule-forming sites preferentially fused with CF-labeled small vesicles which lacked cytochemical TPPase activity. Taken together. Our results suggest that the membrane of secretory granules is modified during the granule formation, at least partly by the fusion of endocytic small vesicles with Golgi cisternae (or GERL), and with immature secretory granules.     

Biogenesis of secretory granules

The biogenesis of secretory granules in endocrine, neuroendocrine, and exocrine cells is thought to involve a selective aggregation of the regulated secretory proteins into a dense-cored structure. The dense-core is then enveloped by membrane in the trans-Golgi network and buds, forming an immature secretory granule. The immature secretory granule then undergoes a maturation process which gives rise to the mature secretory granule. The recent data on the processes of aggregation, budding and maturation are summarized here. In addition, the current knowledge about the mature secretory granule is reviewed with emphasis on the biogenesis of the membrane of this organelle.     

Secretory granule biogenesis: rafting to the SNARE

Regulated secretion of hormones occurs when a cell receives an external stimulus, triggering the secretory granules to undergo fusion with the plasma membrane and release their content into the extracellular milieu. The formation of a mature secretory granule (MSG) involves a series of discrete and unique events such as protein sorting, formation of immature secretory granules (ISGs), prohormone processing and vesicle fusion. Regulated secretory proteins (RSPs), the proteins stored and secreted from MSGs, contain signals or domains to direct them into the regulated secretory pathway. Recent data on the role of specific domains in RSPs involved in sorting and aggregation suggest that the cell-type-specific composition of RSPs in the trans-Golgi network (TGN) has an important role in determining how the RSPs get into ISGs. The realization that lipid rafts are implicated in sorting RSPs in the TGN and the identification of SNARE molecules represent further major advances in our understanding of how MSGs are formed. At the heart of these findings is the elucidation of molecular mechanisms driving protein--lipid and protein--protein interactions specific for secretory granule biogenesis.     

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.     

Granule lattice protein 1 (Grl1p), an acidic, calcium-binding protein in Tetrahymena thermophila dense-core secretory granules, influences granule size, shape, content organization, and release but not protein sorting or condensation

The electron-dense cores of regulated secretory granules in the ciliate Tetrahymena thermophila are crystal lattices composed of multiple proteins. Granule synthesis involves a series of steps beginning with protein sorting, followed by the condensation and precise geometric assembly of the granule cargo. These steps may to various degrees be determined by the cargo proteins themselves. A prominent group of granule proteins, in ciliates as well as in vertebrate neuronal and endocrine cells, are acidic, heat-stable, and bind calcium. We focused on a protein with these characteristics named granule lattice protein 1 (Grl1p), which represents 16% of total granule contents, and we have now cloned the corresponding gene. Mutants in which the macronuclear copies of GRL1 have been disrupted continue to synthesize dense-core granules but are nonetheless defective in regulated protein secretion. To understand the nature of this defect, we characterized mutant and wild-type granules. In the absence of Grl1p, the sorting of the remaining granule proteins appears normal, and they condense to form a well-defined core. However, the condensed cores do not demonstrate a visible crystalline lattice, and are notably different from wild type in size and shape. The cellular secretion defect arises from failure of the aberrant granule cores to undergo rapid expansion and extrusion after exocytic fusion of the granule and plasma membranes. The results suggest that sorting, condensation, and precise granule assembly are distinct in their requirements for Grl1p.     

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.     

The protein content and morphogenesis of zymogen granules

When zymogen granules, the secretion granules of pancreatic acinar cells, fill, secretory product is accumulated in immature granules, condensing vacuoles. Mature granules are formed when this product (protein) condenses into an osmotically inactive aggregate and, bulk water is expelled. This hypothesis for granule morphogenesis has two elements. The first is that immature granules are precursors to mature granules. The second is that a particular maturational event, condensation, which involves the aggregation of protein, takes place. These hypotheses lead to two straightforward predictions. One, that condensing vacuoles on average, should contain less protein than filled or mature granules. And two, that, due to condensation, mature granules should contain protein at a common concentration. In the current work, both of these predictions were tested using measurements of the protein content of individual granules acquired by X-ray microscopy. Neither prediction was affirmed by the experimental results. First, there was no distinguishable difference in the distribution of protein between immature and mature granules. Second, the protein concentration of mature granules varied widely between preparations, although granules from the same preparation had similar concentrations. From the data we conclude that: 1) mature granules and condensing vacuoles are different, though not necessarily unrelated, types of secretory vesicle, and not two forms of the same object; 2) as such, condensing vacuoles are not precursors to mature granules; 3) all granules do not contain protein at one particular concentration when full, or mature; 4) granule maturation does not involve a condensation step; 5) concentration is not determined by such physical limits as the space available for protein packing or condensation; and 6) the amount of protein contained is physiologically regulated.     

Biogenesis of secretory granules

Secretory granules of neuroendocrine cells store and release peptide hormones and neuropeptides in response to various stimuli. Generation of granules from the Golgi complex involves the aggregation of cargo proteins and their sorting from non-regulated secretory molecules. Recent findings on knockout mice lacking individual granule constituents have challenged the hypothesis that an 'essential' protein for the assembly of these organelles exists, while studies on polypyrimidine tract-binding protein and ICA512/IA-2 have provided insight into the mechanisms for adjusting granule production in relation to stimulation and secretory activity.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.