seminars in
CELL BIOLOGY, Vol 3, 1992 : pp 357-366
Biogenesis of secretory granules Sharon A . Tooze and Jane C . Stinchcombe of the secretory granule remain largely an enigma : the major proteins of the chromaffin granule have been biochemically identified (see below) but very little is known about the sorting of the membrane proteins to the secretory granule . All cells, including endocrine, exocrine, and neuronal cells also contain a second major secretory pathway referred to as the constitutive pathway . The specific proteins, referred to as constitutive secretory proteins, which follow this pathway are not stored or concentrated within the cell but are transported to the cell surface and secreted immediately independently of an extracellular stimulus . The constitutive secretory pathway is the major secretory pathway in most cell types and serves to allow the rapid and continuous secretion of the constitutive secretory proteins into the extracellular space . This review will focus on the biogenesis of secretory granules in neuroendocrine cells . In particular, the recent findings concerning the aggregation and sorting of both soluble and membrane-associated proteins to the regulated pathway, as well as the formation of secretory granules from the TGN will be discussed . The key steps believed to be involved in the formation of a new secretory granule are summarized in Figure 1 . These will be discussed below in the context of either the soluble secretory proteins or the granule membrane proteins . Much less is known about the biogenesis of the granule membrane than about how the soluble proteins reach the secretory granule, but the available data suggest that the mechanisms differ .
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 densecored 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 .
Key words : secretory granules / biogenesis of secretory granules / regulated secretion / aggregation of secretory granule proteins / secretory granule membrane proteins
THE SECRETORY GRANULE is a specialized cyto-
plasmic storage organelle typically found in endocrine, exocrine, and neuronal cells . Secretory granules have a unique composition of membrane proteins and contain a subset of the secretory proteins, hormones and small molecules synthesized by the cell . These secretory proteins are stored at high concentration, which may reach up to 200-fold of that in the ER, and gives the granules their characteristic electron dense core . The primary function of secretory granules is to store this subset of molecules within the cell, thereby allowing the controlled release of these molecules into the extracellular space . The granules exocytose their content only in response to a specific stimulation of the cell which causes the secretory granule to fuse with the plasma membrane . The membrane of the secretory granule has specific functions in the transport of the organelle, the accumulation of small molecules from the cytoplasm, acidification of the granule, and exocytosis . Consequently it has a specific lipid and protein composition that differs from that of the membranes of other organelles within the cell . The membrane proteins
Synthesis of secretory granule membrane proteins and content Soluble and membrane proteins destined for the secretory granule are synthesized by the RER in the same way as constitutive secretory proteins and proteins destined for the lysosomes or the plasma membrane (for review see ref 1) . All of these different classes of proteins are then vectorally transported through the intermediate compartment and the Golgi complex, to the trans-Golgi network (TGN) . Transport of all these different classes of proteins
From the EMBL, Meyerhofstrasse 1, W-6900 Heidelberg, Germany ©1992 Academic Press Ltd 1043-4682/92/050357 + 10$8 .00/0
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I
Figure 1 . Regulated secretory proteins are synthesized at the RER and transported through the Golgi complex to the trans-Golgi network (TGN) (1) . The initial milieu of the TGN induces the proteins to form aggregates (2) which associate with the membrane via a membrane-bound receptor protein (3) . The aggregate becomes progressively enwrapped with membrane (4 and 5) and finally buds from the TGN to form immature secretory granules (ISG) partially coated with clathrin (6) . The clathrin patches are rapidly lost (7) and may possibly be recycled back to the TGN with proteins involved only in ISG formation and not required in the mature granule (8) . The ISG can either exocytose its contents in response to an extracellular signal (9) or fuse with other ISGs to form a dense-cored mature secretory granule (MSG) (10) . The MSG is stored at the periphery of the cell until the cell receives an external signal which triggers it to fuse with the plasma membrane and release its secretory content (11) . Following exocytosis, the membrane is rapidly internalized (12) and returned to the TGN (13) to be used in the next round of granule formation . The aggregate receptor may be returned to the TGN from the plasma membrane with other membrane proteins present in the MSG or alternatively it might be recycled from the ISG . • , regulated secretory protein ; A, aggregate receptor protein ; T, Clathrin coat ; r °s , ISG membrane ; -, MSG membrane .
Biogenesis of secretory granules
is probably by bulk flow until the TGN, where their sorting is thought to occur (for review see ref 2) . Two aspects of the biosynthesis of regulated secretory proteins are of particular physiological importance . First, many regulated secretory proteins are synthesized as large prohormones (for review see ref 3) . This allows the precursor prohormone to be processed to a variety of different bioactive peptides in a cell-specific manner . The transport of these prohormone molecules through the secretory pathway to their site of processing is indistinguishable from the transport of other regulated secretory molecules . Secondly, in some cell types, the biosynthetic rate of certain regulated secretory proteins may be altered by the physiological state of the cell or organism . For example, an alteration of the glucose concentration can change the biosynthetic rate of proinsulin synthesis by 20-fold in less than an hour . 4 It is not clear if pertubations in the synthetic rate of proinsulin effects the sorting and the efficiency of its packaging into secretory granules . In the rat exocrine pancreas, after a 5-fold increase in synthesis induced by hormonal stimulation, it was found that the zymogens are correctly sorted and packaged into secretory granule .'
Segregation of regulated secretory proteins from other soluble proteins in the TGN
In cells with a regulated secretory pathway, a tripartite sorting event occurs in the TGN . 6 The constitutive secretory proteins, lysosomal enzymes and regulated secretory proteins are segregated from each other and directed to their respective destinations . The constitutive secretory proteins are passively segregated by bulk flow into COP-coated vesicles, while the lysosomal enzymes and regulated secretory proteins are actively sorted to pre-lysosomes and immature secretory granules (ISG), respectively . The mechanism for the sorting of lysosomal enzymes involves the mannose-6-phosphate recognition signal and the mannose-6-phosphate receptor (for review see ref 7) . As discussed below, the sorting of the regulated secretory proteins in the TGN appears to be a consequence of the unique milieu of the TGN, and may also depend on the presence of membrane proteins in the TGN .
359 Sorting of regulated and constitutive secretory proteins in the TGN
In cells with a regulated secretory pathway the first indication that a specialized sorting event had occurred in the TGN, was the morphological identification of dense-cored aggregates in the TGN of exocrine pancreatic cells . These dense-cored aggregates were shown to contain regulated secretory proteins, the zymogens (for review see ref 8) . Likewise in endocrine cells, aggregates have also been found and were shown to contain prohormones, such as proinsulin 9 or POMC . 10 It has been suggested that this precipitation of dense-core aggregates in the TGN could segregate the regulated secretory proteins from the other soluble proteins present in the TGN, and that the precipitation results from two factors working coordinately : the milieu of the TGN and the inherent solubility properties of the regulated secretory proteins .
Aggregation of regulated secretory proteins
The aggregation of regulated secretory proteins in the TGN has been studied using the granin family of proteins . These proteins are found in virtually all secretory granules in neuronal or endocrine cells (for review see ref 11) . The granins are acidic proteins which can precipitate in vitro at an acidic pH in the presence of Cat + , conditions similar to those believed to exist in the lumen of the TGN . 12,13 Recent studies have directly demonstrated that in the lumen of the TGN the granins are in an aggregated state, and this aggregation segregates them from constitutively secreted proteins . 14 In line with earlier reports, where it was demonstrated that in the exocrine pancreas, intracisternal granules found in the RER contain the complete set of zymogens, and exclude soluble RER resident proteins such as BIP, 15 it was also shown that the granins, 14 but not soluble resident ER proteins, can be induced to aggregate in the RER by altering the milieu of this compartment to one similar to the TGN . These results show that the aggregation of the regulated secretory proteins is itself a sorting step and could indeed function to segregate regulated secretory proteins from constitutive secretory proteins .
3 60 The sorting of regulated secretory proteins to the secretory granule has also been investigated in experiments which involve transfection of tissue culture cells with cDNAs encoding foreign prohormones, or hybrid prohormone proteins, followed by assays to determine if the protein is targeted to the regulated secretory pathway . It has been shown that heterologous mammalian prohormones from both endocrine and exocrine sources can be sorted into neuroendocrine secretory granules . 16 This was also found to be the case for a cytoplasmic protien, a-globin, when it was fused to a prohormone domain . 17 Similarly, a constitutive secretory protein, an antibody molecule, can be diverted to secretory granules when it is specific for a granin . 18 One could postulate that these foreign molecules are sorted into secretory granules exclusively by co-aggregation with the endogenous molecules, or as in one case as an antibody-antigen complex . Therefore their correct sorting would require no defined sorting signal in the polypeptide, but rather would be a result of an inherent property of these molecules . This hypothesis is confirmed by experiments which involved transfection of the cDNA encoding a prohormone precursor, ELH, from a marine mollusc into AtT20 cells . The mature, processed ELH was stored in dense-core granules in the AtT20 cells, 19 suggesting that the aggregation of both the mammalian and molluscan regulated secretory proteins requires a similar milieu in the TGN, and utilizes the same mechanism, therefore allowing co-aggregation and correct targeting of the ELH hormone in AtT20 cells . On the other hand, it has also been shown that prohormones from other species, such as anglerfish preprosomatostatin 20 or prodermorphin from frogs, 21 when introduced into mammalian cells, are not sorted into the mammalian secretory granule unless they are fused to a mammalian prohormone . These results suggest that the mechanism for sorting regulated secretory proteins to the secretory granule is more complex than can be explained simply by co-aggregation, and may require either as yet unidentified sorting sequences or additional components, such as molecules in the membrane of the TGN . Association of regulated secretory proteins with the TGN membrane
One unanswered question is whether the densecored aggregate interacts with the membrane of the
S .A . Tooze and J. C. Stinchcombe
TGN, and initiates the budding of a secretory granule . Previous models (see refs 22, 23) have envisaged that the aggregate interacts with a protein, or proteins, in the TGN membrane . It has long been known that a small population of the normally soluble regulated secretory proteins exists as membrane bound molecules (see further discussion below) . These membrane-bound forms of the regulated secretory proteins may link the dense-core aggregate to the membrane by interacting in a homophilic manner . 24 Alternatively, there may be heterophilic interactions between unidentified membrane proteins and the aggregate, similar to a receptor-ligand interaction . This latter hypothesis remains attractive, and is supported by some experimental evidence (reviewed in ref 2) ; to date however, no receptor molecule has been found . The major difficulty in identifying such a receptor molecule may be its low abundance : one would expect that a single receptor molecule would bind to an aggregate containing many secretory proteins . In addition, after budding the receptor molecule may recycle from the ISG to the TGN, and be reused . It may not therefore be present in the dense mature secretory granules which can be isolated by cell fractionation .
Sorting of regulated secretory proteins and lysosomal enzymes in the TGN
In the lumen of the TGN the newly-synthesized lysosomal enzymes bearing the mannose-6-phosphate recognition signal interact with the mannose-6phosphate receptor . These receptor-ligand complexes are then sorted to the pre-lysosome by a transport step which involves clathrin coated vesicles . 7 However, in exocrine pancreatic cells which are highly specialized for the secretion of molecules by the regulated pathway, it appears that the sorting of lysosomal enzymes to the lysosome is perturbed : cathepsin B, a lysosomal enzyme, is found in the dense-core of zymogen granules, and in the pancreatic juice secreted from the cells after an appropriate stimulus . 25 This raises the possibility that in other regulated secretory cells a small percentage of the newly-synthesized lysosomal enzymes may be missorted into the secretory granule . To avoid proteolytic degradation of the regulated secretory proteins by the lysosomal enzymes in the secretory granule, the lysosomal enzymes must
Biogenesis of secretory granules be removed or inactivated . One could postulate that these missorted molecules are removed from the secretory granule by the same mechanism that is utilized in the TGN, that is, clathrin coated vesicles . This could also explain the function of the clathrincoated patches seen not only on regions of the TGN containing dense-cored aggregates but also on the newly formed ISG (see below) . In short, sorting may continue after the ISG has detached from the TGN .
Formation of secretory granules from the TGN After the aggregation and sorting of the regulated secretory proteins from the constitutive secretory proteins, the dense-core is enveloped by membrane and buds from the TGN to form an ISG . Likewise, constitutive secretory vesicles, containing constitutive secretory proteins, also bud from the TGN . This is aided by a cytoplasmic coat, which fully encompasses the budding vesicle while it is still attached to the TGN . The formation of ISGs does not appear to involve a continuous cytoplasmic coat, although patches of clathrin can be detected on the membrane of the TGN, often in regions juxtaposed to the densecored aggregates in the lumen . 26,27 To investigate the requirements for vesicle formation from the TGN, cell-free systems have been developed which reconstitute the budding of vesicles from the TGN . 28,29 One such system monitored the budding of both constitutive and regulated secretory vesicles . 28 Here, . the ISG formed from the TGN contained only regulated secretory proteins, while the constitutive secretory vesicles formed from the TGN contained only constitutive secretory proteins, showing that the regulated secretory proteins are sorted from the constitutive secretory proteins before their exit from the TGN . This cell-free system was further exploited to demonstrate first that the formation of both regulated and constitutive secretory vesicles from the TGN is inhibited by the addition of non-hydrolyzable analogues of GTP, i .e . GTP-yS, 30 and second that budding of both the regulated and constitutive secretory vesicles involves a member of the family of heterotrimeric G-proteins . 31 Properties of immature secretory granules and their maturation The first vesicle of the regulated secretory pathway formed from the TGN has been alternatively referred
361 to as a condensing vacuole, 32 a coated granule, 26 and an ISG (for review see ref 33) . In the neuroendocrine cells, it has been shown that the ISG is an intermediate in the formation of mature secretory granule (MSG) . The process by which an ISG matures to an MSG involves an increase in size of the ISG, perhaps as a result of fusion of multiple ISGs to form one MSG . 34,35 It has also been shown recently that the ISG can be translocated to the plasma membrane, in a microtubule-dependent fashion, and can undergo regulated exocytosis after an appropriate stimulus . 35 The morphological evidence that the ISG has clathrin-coated regions, 26,27 and the biochemical evidence suggesting that ISGs undergo a fusion process during their maturation, imply that the ISG is a dynamic intermediate . Indeed, the plasticity of the ISG is also evident from experiments in the exocrine pancreas 36 and the islet cells of the pancreas, 37 which suggest there is a pathway from the ISG to the plasma membrane whereby a small fraction of the regulated secretory proteins can be released rapidly, and independently of an external stimulus, in a constitutive fashion . The findings that the ISG can fuse with itself, or with the plasma membrane, have implications with respect to the sorting of granule-specific proteins during the formation of the ISG . Molecules involved in both the ISG-ISG, and in the ISG-plasma membrane fusion events, must be present on the ISG . Furthermore, one could imagine that proteins involved in formation and maturation of the ISG, for example the putative aggregate receptor and the proteins involved in ISG-ISG fusion are not required by the mature granule . Therefore, they may be removed from the ISG and recycled to the TGN and re-used in the next round of secretory granule budding and maturation .
Biogenesis of the granule membrane The newly forming granule obtains its membrane proteins from two sources ; recycling and re-use of the membranes of previous granules following exocytosis and de novo synthesis . The contribution of each source depends upon the cell type . In fully differentiated non-dividing secretory cells, for example freshly prepared primary chromaffin cells in culture 38 most of the MSG membrane is recycled . This is probably the mechanism by which newly forming granules receive their membranes in
3 62 secretory tissue . In established cell lines which maintain the capacity to divide, de novo synthesis predominates . This may also be the case during early stages of the development of secretory tissues which still show plasticity and have the capacity to divide . In addition, de novo synthesis may be used to replenish any membrane components that are degraded during the recycling process . The differences between fully differentiated tissue, and actively dividing cells are important to remember since many studies on granule formation have used established secretory cell lines . Recycling of granule membrane proteins The original idea that the membrane proteins of the MSG are re-used following exocytosis came from the observation that the turnover of membrane proteins in metabolically labelled adrenal medulla and exocrine pancreas glands was considerably slower than the secretory content (for review see ref 2) . Subsequently, by labelling the cell surface following exocytosis with either antibodies against luminally oriented membrane protein epitopes or by biotinylation, membrane recycling has been demonstrated in several secretory cell types (ref 38 and references therein) . The granule membrane components do not diffuse throughout the plasma membrane upon exocytosis but remain in discrete patches . These are rapidly removed from the plasma membrane, within 5 minutes of exocytosis, by a process that may involve the non-erythroid spectrin molecule fodrin, 39 returned to the TGN and ultimately detected in MSGs (reviewed by ref 2) . This suggests that once formed, the granule membrane retains its integrity and is re-used at the TGN to enwrap the newly forming granules . granule membrane protein synthesis
De novo
The situation for newly synthesized granule membrane proteins is different from that of recycled membrane components since here the individual proteins must both be segregated from the components of the other cellular membranes and incorporated into the new granule membrane . Two types of proteins are present in the granule membrane ; proteins that are found primarily as soluble proteins but also exist in membrane associated forms, and proteins found exclusively in
S .A . Tooze and J. C. Stinchcombe
the membrane . Little is known about either the sorting mechanism, or the intracellular route taken by granule membrane proteins from the TGN to the mature granule . The different properties of the two types of membrane proteins suggest that at least two different mechanisms and routes might exist .
Proteins present in both the granule content and membrane
Increasing evidence suggests that all the soluble granule proteins may also be present in the membrane . However, the amount which is granule membrane-associated appears to differ greatly, with values up to 507o for dopamine /3-hydroxylase (D(3H) 4° in the chromaffin cell . In addition, a variety of different mechanisms of membrane attachment are utilized the nature of which suggests that the proteins may be preferentially membraneassociated at different stages of the secretory pathway . D(3H may be anchored in the membrane by an uncleaved sequence ; 41 the newly synthesized protein is primarily found in the membrane but the proportion that is membrane-associated decreases with time . 42 Conversely, carboxypeptidase E (CPE), 43 in a variety of endocrine cell types, associates with the membrane via a low pH-dependent, amphipathic C-terminal helix . This suggests that the protein becomes membrane-associated as it moves into the more acidic environment of the TGN . 44 Additional mechanisms of membrane association involve PIanchors, as is found on GP2 from the exocrine pancreas, 45 which are added to the protein in the RER . The mechanistic and temporal differences in membrane association observed for different granule proteins may regulate their activity, and may also reflect the different function of each protein . However, in all cases membrane-associated forms of the proteins exist in the TGN . This suggests that, as discussed above, membrane-associated forms of soluble proteins may be important for the interaction of membranes with regulated protein aggregates and that they may be involved in the mechanism of soluble protein sorting . In the cases where the structure of the membrane and soluble forms of the protein has been determined, for example for Df3H, 41 CPE 44 and peptidyl-glycine a-amidating monooxygenase (PAM), 46 the two forms differ only in the small regions conferring membrane attatchment . Thus both forms of each protein are likely to contain the same sorting
Biogenesis
of secretory
granules
information, unless the presence of the protein in the membrane per se, or a resulting conformational change, alters its intracellular pathway to the granule . In support of this hypothesis is the differential intracellular localization of pc2 and pc3, the putative prohormone endopeptidases which are found in secretory granules (for review see ref 47), and Kex2 and furin, Golgi-localized endopeptidases that cleave at the di-basic residues of constitutively secreted proteins in yeast and mammalian cells, respectively (for review see ref 48) . Pc2 and pc3 are highly homologous to Kex2 and furin, also referred to as PACE, except that pc2 and pc3 do not have the serine/threonine rich region, the trans-membrane sequence or the cytoplasmic tail found in Kex2 and furin . Rather, pc2 and pc3 have an amphipathic domain at their C-terminus . Comparison of the domain structures of Kex2 and furin with that of pc2 and pc3 suggests that the intracellular localization is determined by the C-terminus of the molecule . In the case of Kex2 and furin, the trans-membrane and cytoplasmic domains may be responsible for the Golgi localization . In contrast, the amphipathic region at the C-terminus of pc2 and pc3 may mediate their association with the membrane, in a mechanism similar to that proposed for carboxypeptidase E (see above), and may direct these proteins to the secretory granule membrane .
Proteins exclusively located in membranes
Proteins in this group include the proteins involved in the transport of small molecules and the establishment of a chemiosmotic gradient, plus several characterized glycoproteins, and polypeptides with unidentified function (reviewed in ref 49, for the best characterized granule, the chromaffin granule) . All of the granule membrane proteins identified to date are also present in the membranes of other compartments of the same and other cells . For example, vacuolar proton pumps with subunits that are immunologically related to those of the chromaffin granule proton pump have been found in lysosomes, the Golgi complex, clathrin coated vesicles and synaptic vesicles (reviewed in ref 50) ; the chromaffin granule glycoprotein GPII has been found in lysosomes in the kidney 51 whilst the insulin granule proteins SM110 and SM80 are also in the liver ; 52 the proteins involved in catecholamine uptake and biosynthesis in chromaffin cells are also present in adrenergic synaptic vesicles ; and p65 and
363 SV2, which have been identified immunologically in a variety of neuroendocrine granules, were originally identified in neuronal synaptic vesicles and at least p65 may also be in the plasma membrane . 53,54 The identification of additional components of granule membranes has been further complicated by several other characteristics of the granule membranes : (1) each granule membrane protein constitutes only a minor percentage of the total cellular membrane protein . This is because the granule membrane has a low protein : lipid ratio compared to other cellular membranes and each individual protein is present at low abundance ; and (2) it has not proved possible to isolate significant quantities of membrane from most types of granules free of contaminating membranes from other organelles . The identification of any new granule membrane protein is further complicated by the slow turnover and possibility of recycling of these proteins . These points, combined with the fact that to date no unique secretory granule membrane protein from exocrine, endocrine or neuronal cells has been identified, means little information is available about the mechanism and location of their sorting .
Mechanisms of secretory granule membrane protein sorting Certain recent observations, however have provided some information on the sorting and routing of newly synthesized membrane proteins to secretory granules . Unlike the soluble proteins, granule membrane proteins may contain sorting signals . When human P-selectin, a membrane protein of platelet and endothelial cell granules, was expressed in the murine AtT-20 cell line the protein was very efficiently targeted to the storage granules of these cells .° 5 This indicates that the information for sorting to the regulated secretory pathway is conserved between both species and cell types . Furthermore, the cytoplasmic tail of P-selectin was sufficient to divert a normally constitutively secreted protein to the regulated secretory pathway . The cytoplasmic domain of P-selectin therefore probably contains a signal for sorting to the regulated secretory pathway . This is the first identification of a `positive' sorting signal in this pathway . Since the cytoplasmic domain of P-selectin shows no similarities in its primary structure to those of other known granule membrane proteins, for example cytochrome b561 and p65, the signal may be a conformational one .
364 The location of membrane protein sorting and intracellular pathway to the secretory granule The site at which the granule membrane proteins are segregated from other cellular membrane proteins and the route which they take from the TGN to the mature granule remains enigmatic . Sorting may have occurred already upon exit from the TGN, the granule membrane proteins leaving the TGN exclusively in the ISG . Alternatively, sorting might occur entirely or partially in a post-Golgi location ; the proteins may be randomly distributed throughout all the vesicles, including the ISG, leaving the TGN and subsequently sorted by a process of selective retention and exclusion, or they may travel first to the plasma membrane from which they would be selectively retrieved as appears to be the case for the lysosomal membrane proteins (reviewed by ref 56) and synaptic vesicle protein synaptophysisn . 57 Several observations are consistent with sorting of at least a subpopulation of granule membrane proteins at the TGN into the ISG . (1) Granule membranes retrieved from the plasma membrane following exocytosis are transported to the TGN before being found again in granules (reviewed in ref 2) . This suggests that the TGN may also be the site at which the composition of the granule membrane is defined . (2) P-selectin was observed by immunoelectron microscopy to be associated with dense cores in the TGN, 55 suggesting at least some granule membrane proteins are segregated with the granule content in the TGN . (3) Immature chromaffin granules are able to accumulate radiolabelled small molecules such as nucleotides and catecholamines from their extracellular environment49 and, as described above, ISGs of PC 12 cells exhibit calcium-dependent regulated exocytosis . 35 Thus the proteins required for some of the specific functions of the granule must already be present and active in the ISG membrane . This suggests that at least some membrane proteins present in the MSG leave the TGN in the ISG with the content proteins . Two observations provide indirect evidence in support of recycling granule membrane proteins from the plasma membrane . The first is the observation that granule membrane proteins are specifically retrieved from the plasma membrane following exocytosis . However, here complete membranes rather than individual proteins appear to be internalized . Secondly, the cytoplasmic tail of P-selectin contains two sequences believed to be signals for endocytosis at the plasma membrane 55 and
S .A . Tooze and ,J. C. Stinchcombe therefore possibly involved in membrane retrieval following exocytosis . However, there is no indication of whether these signals are part of the domain involved in targeting the newly synthesized protein to the ISG . Elucidating the mechanisms involved in sorting granule membrane proteins depends on the identification of a protein unique to the granule membrane which can be radiolabelled and whose transport can be followed after synthesis through the secretory pathway . To date such molecules have not been found . The difficulty encountered in identifying granule membrane proteins causes one to ask whether the mature secretory granule has any proteins in its membrane other than those molecules required for exocytosis of the secretory granule . All the other proteins required to form the granule, and those involved in the modification of the soluble content proteins, may be removed from the ISG during maturation . Conclusions Although the regulated pathway of secretion was originally the prototype secretory pathway, 8 today much less is known about it than is known about, for example, the constitutive secretory pathway, or the different pathways to the lysosome . The complexity of the sorting mechanism of the content proteins, combined with the major difficulties encountered in the identification of granule membrane protein, has left many questions unresolved about this enigmatic pathway . Acknowledgements We thank Dr John Tooze for critically reading the manuscript, and for helpful discussions . We also thank Dr Wieland Huttner for stimulating discussions and advice, and for support (J .C . S) . We also thank Dr Rodger McEver for communication of results before publication . References 1 . Pelham HR (1989) Control of protein exit from the endoplasmic reticulum . Annu Rev Cell Biol 5 :1-23 2 . Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of proteins . Annu Rev Cell Biol 3 :243-293 3 . Docherty K, Steiner DF (1982) Post-translational proteolysis in polypeptide hormone biosynthesis . Annu Rev Physiol 44 :625-638 4 . Hutton JC, Bailyes EM, Rhodes CJ, Rutherford NG, Arden SD, Guest PC (1990) Biosynthesis and storage of insulin . Biochem Soc Trans 18 :122-124
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5 . Vasiloudes P, Norbert P, Tooze J, Kern HF (1991) Acceleration of asynchronous enzyme transport in the rat exocrine pancreas following hormonal stimulation in siao . Eur J Cell Biol 54 :27-37 6 . Griffiths G, Simons K (1986) The trans Golgi network : sorting at the exit site of the Golgi complex . Science 234 :438-443 7 . Kornfeld S, Mellman 1 (1989) The biogenesis of lysosomes . Annu Rev Cell Biol 5 :483-526 8 . Palade GE (1975) Intracellular aspects of the process of protein synthesis . Science 189 :347-358 9 . Orci L, Ravazzola M, Amherdt M, Masden 0, Vassalli J-D, Perrelet A (1985) Direct identification of prohormone conversion site in insulin-secreting cells . Cell 42 :671-681 10 . Tooze J, Hollinshead M, Frank R, Burke B (1987) An antibody specific for an endoproteolytic cleavage site provides evidence that pro-opiomelanocorin is packaged into secretory granules in AtT20 cells before its cleavage . J Cell Biol 105 :155-162 11 . Huttner WB, Gerdes H-H, Rosa P (1991) The granin (chromogranin/secretogranin) family . Trends Biol Sci 16 :27-30 12 . Gerdes H-H, Rosa P, Phillips E, Baeuerle PA, Frank R, Argos P, Huttner WB (1989) The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calciuminduced aggregation . J Biol Chem 264 :12009-12015 13 . Gorr S-U, Shioi J, Cohn DV (1989) Interaction of calcium with porcine adrenal chromogranin A (secretory protein-I) and chromogranin B (secretogranin I) . Am J Physiol 257 :E247-E254 14, Chanat E, Huttner WB (1991) Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network . J Cell Biol 115 :1505-1519 15 . Tooze J, Kern HF, Fuller SD, Howell KE (1989) Condensation-sorting events in the rough endoplasmic reticulum of exocrine pancreatic cells . J Cell Biol 109 :35-50 16 . Kelly RB (1991) Secretory granule and synaptic vesicle formation . Curr Opin Cell Biol 3 :654-660 17 . Stoller TJ, Shields D (1989) The propeptide of presomatostatin mediates intracellular transport and secretion of a-globin from mammalian cells . J Cell Biol 108 : 1647-1655 18 . Rosa P, Weiss U, Pepperkok R, Ansorge W, Niehrs C, Stelzer EHK, Huttner WB (1989) An antibody against secretogranin I (chromogranin B) is packaged into secretory granules . J Cell Biol 109 :17-34 19 . Jung LJ, Scheller RH (1991) Peptide processing and targeting in the neuronal secretory pathway . Science 251 :1330-1335 20 . Sevarino KA, Stork P, Ventimiglia R, Mandel G, Goodman RH (1989) Amino-terminal sequences of prosomatostatin direct intracellular targeting but not processing specificity . Cell 57 :11-19 21 . Seethaler G, Chaminade M, Vlasak R, Ericsson M, Griffiths G, Toffoletto 0, Rossier J, Stunnenberg HG, Kreil G (1991) Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin . J Cell Biol 114 :1125-1133 22 . Kelly RB (1985) Pathways of protein secretion in eukaryotes . Science 230 :25-32 23 . Huttner WB, Friederich E, Gerdes H-H, Niehrs C, Rosa P (1988) Tyrosine sulfation and tyrosine-sulfated proteins common to endocrine secretory granules, in Progress in Endocrinology (Imura H, Shizume K, Yoshida S, eds), pp 325-328 . Excerpta Medica, Elsevier Science, Amsterdam 24 . Pimplikar SW, Huttner WB (1992) Chromogranin B (Secretogranin 1), a secretory protein of the regulated pathway, is also present in a tightly membrane-associated form in PC 12 cells . J Biol Chem 267 :4110-4118
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25 . Tooze J, Hollinshead M, Hensel G, Kern HF, Hoflack B (1991) Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells . Eur J Cell Biol 56 :187-200 26 . Orci L, Halban P, Amherdt M, Ravazzola M, Vassalli J-D, Perrelet A (1984) A clathrin-coated, Golgi-related compartment of the insulin secreting cell accumulates proinsulin in the presence of monensin . Cell 39 :39-47 27 . Tooze J, Tooze SA (1986) Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells . J Cell Biol 103 :839-850 28 . Tooze SA, Huttner WB (1990) Cell-free protein sorting to the regulated and constitutive secretory pathways . Cell 60 :837-847 29 . Melancon P, Franzusoff A, Howell KE (1991) Vesicle budding : insights from cell-free assays . Trends Cell Biol 1 :165-171 30 . Tooze SA, Weiss U, Huttner WB (1990) Requirement for GTP hydrolysis in the formation of secretory vesicles . Nature 347 :207-208 31 . Barr FA, Leyte A, Mollner S, Pfeuffer T, Tooze SA, Huttner WB (1991) Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules, FEBS 294 :239-243 32 . Farquhar MG, Palade GE (1981) The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage . J Cell Biol 91 :77s-103s 33 . Tooze SA (1991) Biogenesis of secretory granules . Implications arising from the immature secretory granule in the regulated pathway of secretion . FEBS 285 :220-224 34 . Salpeter MM, Farquhar MG (1981) High resolution analysis of the secretory pathway in mammotrophs of the rat anterior pituitary . J Cell Biol 91 :240-246 35 . Tooze SA, Flatmark T, ToozeJ, Huttner WB (1991) Characterization of the immature secretory granule, an intermediate in granule biogenesis . J Cell Biol 115 :1491-1503 36 . Aryan P, Castle JD (1987) Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas . J Cell Biol 104 :243-252 37 . Aryan P, Kuliawat R, Prabakaran D, Zavacki A-M, Elahi D, Wang S, Pilkey D (1991) Protein discharge from immature secretory granules displays both regulated and constitutive characteristics . J Biol Chem 266 :14171-14174 38 . Hunter A, Phillips JH (1989) The recycling of a secretory granule membrane protein . Exp Cell Res 182 :445-460 39 . Fugimoto T, Ogawa K (1989) Retrieving vesicles in secretion-induced rat chromaffin cells contain fodrin . J Histochem Cytochem 37 :1589-1599 40. Stewart LC, Klinman JP (1988) Dopamine beta-hydroxylase of adrenal chromaffin granules : structure and function . Annu Rev Biochem 57 :551-592 41, Taljanidisz J, Stewart L, Smith AJ, Klinman JP (1989) Structure of bovine dopamine 0-monooxygenase . a s deducted from cDNA and protein sequencing . Evidence that the membrane-bound form of the enzyme is anchored by an uncleaved signal peptide . Biochemistry 28 :10054-10061 42 . Sabban EL, Schwartz J, McMahon A (1990) Effect of compounds which disrupt proton gradients on secretion of neurosecretory proteins from PC12 pheochromocytoma cells . Neuroscience 38 :561-570 43 . Fricker LD (1988) Carboxypeptidase E . Annu Rev Physiol 50 :309-321 44 . Fricker LD, Das B, Angeletti RH (1990) Identification of the pH-dependent membrane anchor of carboxypeptidase E . J Biel Chem 265 :2476-2482 45 . LeBel D, Beattie M (1988) The major protein of pancreatic zymogen granule membranes (GP2) is anchored via covalent bonds to phosphatidylinositol . Biochem Biophys Res Common 154 :818-823
366 46 . Eipper BA, Park LP, Dickerson IM, Keutmann HT, Thiele EA, Rodriguez H, Schofield PR, Mains RE (1987) Structure of the precursor to an enzyme mediating COOHterminal amidation in peptide biosynthesis . Mol Endo 1 :777-790 47 . Steiner DF (1991) Prohormone convertases revealed at last . Curr Biol 1 :375-377 48 . Barr PJ (1991) Mammalian subtilisins : the long-sought dibasic processing endopeptidases . Cell 66 :1-3 49 . Winkler H, Fischer-Colbrie R (1990) Common membrane proteins of chromaffin granules, endocrine and synaptic vesicles : properties, tissue distribution, membrane topography and regulation of synthesis . Neurochem Int 17 :245-262 50 . Njus D, Kelley PM, Harnadek GJ (1986) Bioenergetics of secretory vesicles . Biochim Biophys Acta 853 :237-265 51 . Weiler R, Steiner HJ, Schmid KW, Obendorf D, Winkler H (1990) Glycoprotein II from adrenal chromaffin granules is also present in kidney lysosomes . Biochem j 272 :87-92 52 . Grimaldi KA, Hutton JC, Siddle K (1987) Production and
S.A . Tooze and J. C. Stinchcombe characterization of monoclonal antibodies to insulin secretory granule membranes . Biochem j 245 :557-566 53 . Buckley K, Kelly RB (1985) Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells . J Cell Biol 100 :1284-1294 54 . Matthew WD, Tsavaler L, Reichardt LF (1981) Identification of a synaptic-vesicle membrane protein with a wide distribution in neuronal and neurosecretory tissue . J Cell Biol 91 :257-269 55 . Disdier M, Morrissey JH, Fugate RD, Bainton D, McEver RP (1992) The cytoplasmic domain of P-selectin (CD62) contains the signal for sorting into the regulated secretory pathway . Mol Biol Cell 3 :309-321 56 . von Figura K (1991) Molecular recognition and targeting of lysosomal proteins . Curr Opin Cell Biol 3 : 642-646 57 . Regnier-Vigouroux A, Tooze SA, Huttner WB (1991) Newly synthesized synaptohysin is transported to synapticlike microvesicles via constitutive secretory vesicles and the plasma membrane . EMBO J 10 :3589-3601
CELL BIOLOGY, Vol 3, 1992 : pp 357-366
Biogenesis of secretory granules Sharon A . Tooze and Jane C . Stinchcombe of the secretory granule remain largely an enigma : the major proteins of the chromaffin granule have been biochemically identified (see below) but very little is known about the sorting of the membrane proteins to the secretory granule . All cells, including endocrine, exocrine, and neuronal cells also contain a second major secretory pathway referred to as the constitutive pathway . The specific proteins, referred to as constitutive secretory proteins, which follow this pathway are not stored or concentrated within the cell but are transported to the cell surface and secreted immediately independently of an extracellular stimulus . The constitutive secretory pathway is the major secretory pathway in most cell types and serves to allow the rapid and continuous secretion of the constitutive secretory proteins into the extracellular space . This review will focus on the biogenesis of secretory granules in neuroendocrine cells . In particular, the recent findings concerning the aggregation and sorting of both soluble and membrane-associated proteins to the regulated pathway, as well as the formation of secretory granules from the TGN will be discussed . The key steps believed to be involved in the formation of a new secretory granule are summarized in Figure 1 . These will be discussed below in the context of either the soluble secretory proteins or the granule membrane proteins . Much less is known about the biogenesis of the granule membrane than about how the soluble proteins reach the secretory granule, but the available data suggest that the mechanisms differ .
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 densecored 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 .
Key words : secretory granules / biogenesis of secretory granules / regulated secretion / aggregation of secretory granule proteins / secretory granule membrane proteins
THE SECRETORY GRANULE is a specialized cyto-
plasmic storage organelle typically found in endocrine, exocrine, and neuronal cells . Secretory granules have a unique composition of membrane proteins and contain a subset of the secretory proteins, hormones and small molecules synthesized by the cell . These secretory proteins are stored at high concentration, which may reach up to 200-fold of that in the ER, and gives the granules their characteristic electron dense core . The primary function of secretory granules is to store this subset of molecules within the cell, thereby allowing the controlled release of these molecules into the extracellular space . The granules exocytose their content only in response to a specific stimulation of the cell which causes the secretory granule to fuse with the plasma membrane . The membrane of the secretory granule has specific functions in the transport of the organelle, the accumulation of small molecules from the cytoplasm, acidification of the granule, and exocytosis . Consequently it has a specific lipid and protein composition that differs from that of the membranes of other organelles within the cell . The membrane proteins
Synthesis of secretory granule membrane proteins and content Soluble and membrane proteins destined for the secretory granule are synthesized by the RER in the same way as constitutive secretory proteins and proteins destined for the lysosomes or the plasma membrane (for review see ref 1) . All of these different classes of proteins are then vectorally transported through the intermediate compartment and the Golgi complex, to the trans-Golgi network (TGN) . Transport of all these different classes of proteins
From the EMBL, Meyerhofstrasse 1, W-6900 Heidelberg, Germany ©1992 Academic Press Ltd 1043-4682/92/050357 + 10$8 .00/0
357
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358 . .
.
. .
I
Figure 1 . Regulated secretory proteins are synthesized at the RER and transported through the Golgi complex to the trans-Golgi network (TGN) (1) . The initial milieu of the TGN induces the proteins to form aggregates (2) which associate with the membrane via a membrane-bound receptor protein (3) . The aggregate becomes progressively enwrapped with membrane (4 and 5) and finally buds from the TGN to form immature secretory granules (ISG) partially coated with clathrin (6) . The clathrin patches are rapidly lost (7) and may possibly be recycled back to the TGN with proteins involved only in ISG formation and not required in the mature granule (8) . The ISG can either exocytose its contents in response to an extracellular signal (9) or fuse with other ISGs to form a dense-cored mature secretory granule (MSG) (10) . The MSG is stored at the periphery of the cell until the cell receives an external signal which triggers it to fuse with the plasma membrane and release its secretory content (11) . Following exocytosis, the membrane is rapidly internalized (12) and returned to the TGN (13) to be used in the next round of granule formation . The aggregate receptor may be returned to the TGN from the plasma membrane with other membrane proteins present in the MSG or alternatively it might be recycled from the ISG . • , regulated secretory protein ; A, aggregate receptor protein ; T, Clathrin coat ; r °s , ISG membrane ; -, MSG membrane .
Biogenesis of secretory granules
is probably by bulk flow until the TGN, where their sorting is thought to occur (for review see ref 2) . Two aspects of the biosynthesis of regulated secretory proteins are of particular physiological importance . First, many regulated secretory proteins are synthesized as large prohormones (for review see ref 3) . This allows the precursor prohormone to be processed to a variety of different bioactive peptides in a cell-specific manner . The transport of these prohormone molecules through the secretory pathway to their site of processing is indistinguishable from the transport of other regulated secretory molecules . Secondly, in some cell types, the biosynthetic rate of certain regulated secretory proteins may be altered by the physiological state of the cell or organism . For example, an alteration of the glucose concentration can change the biosynthetic rate of proinsulin synthesis by 20-fold in less than an hour . 4 It is not clear if pertubations in the synthetic rate of proinsulin effects the sorting and the efficiency of its packaging into secretory granules . In the rat exocrine pancreas, after a 5-fold increase in synthesis induced by hormonal stimulation, it was found that the zymogens are correctly sorted and packaged into secretory granule .'
Segregation of regulated secretory proteins from other soluble proteins in the TGN
In cells with a regulated secretory pathway, a tripartite sorting event occurs in the TGN . 6 The constitutive secretory proteins, lysosomal enzymes and regulated secretory proteins are segregated from each other and directed to their respective destinations . The constitutive secretory proteins are passively segregated by bulk flow into COP-coated vesicles, while the lysosomal enzymes and regulated secretory proteins are actively sorted to pre-lysosomes and immature secretory granules (ISG), respectively . The mechanism for the sorting of lysosomal enzymes involves the mannose-6-phosphate recognition signal and the mannose-6-phosphate receptor (for review see ref 7) . As discussed below, the sorting of the regulated secretory proteins in the TGN appears to be a consequence of the unique milieu of the TGN, and may also depend on the presence of membrane proteins in the TGN .
359 Sorting of regulated and constitutive secretory proteins in the TGN
In cells with a regulated secretory pathway the first indication that a specialized sorting event had occurred in the TGN, was the morphological identification of dense-cored aggregates in the TGN of exocrine pancreatic cells . These dense-cored aggregates were shown to contain regulated secretory proteins, the zymogens (for review see ref 8) . Likewise in endocrine cells, aggregates have also been found and were shown to contain prohormones, such as proinsulin 9 or POMC . 10 It has been suggested that this precipitation of dense-core aggregates in the TGN could segregate the regulated secretory proteins from the other soluble proteins present in the TGN, and that the precipitation results from two factors working coordinately : the milieu of the TGN and the inherent solubility properties of the regulated secretory proteins .
Aggregation of regulated secretory proteins
The aggregation of regulated secretory proteins in the TGN has been studied using the granin family of proteins . These proteins are found in virtually all secretory granules in neuronal or endocrine cells (for review see ref 11) . The granins are acidic proteins which can precipitate in vitro at an acidic pH in the presence of Cat + , conditions similar to those believed to exist in the lumen of the TGN . 12,13 Recent studies have directly demonstrated that in the lumen of the TGN the granins are in an aggregated state, and this aggregation segregates them from constitutively secreted proteins . 14 In line with earlier reports, where it was demonstrated that in the exocrine pancreas, intracisternal granules found in the RER contain the complete set of zymogens, and exclude soluble RER resident proteins such as BIP, 15 it was also shown that the granins, 14 but not soluble resident ER proteins, can be induced to aggregate in the RER by altering the milieu of this compartment to one similar to the TGN . These results show that the aggregation of the regulated secretory proteins is itself a sorting step and could indeed function to segregate regulated secretory proteins from constitutive secretory proteins .
3 60 The sorting of regulated secretory proteins to the secretory granule has also been investigated in experiments which involve transfection of tissue culture cells with cDNAs encoding foreign prohormones, or hybrid prohormone proteins, followed by assays to determine if the protein is targeted to the regulated secretory pathway . It has been shown that heterologous mammalian prohormones from both endocrine and exocrine sources can be sorted into neuroendocrine secretory granules . 16 This was also found to be the case for a cytoplasmic protien, a-globin, when it was fused to a prohormone domain . 17 Similarly, a constitutive secretory protein, an antibody molecule, can be diverted to secretory granules when it is specific for a granin . 18 One could postulate that these foreign molecules are sorted into secretory granules exclusively by co-aggregation with the endogenous molecules, or as in one case as an antibody-antigen complex . Therefore their correct sorting would require no defined sorting signal in the polypeptide, but rather would be a result of an inherent property of these molecules . This hypothesis is confirmed by experiments which involved transfection of the cDNA encoding a prohormone precursor, ELH, from a marine mollusc into AtT20 cells . The mature, processed ELH was stored in dense-core granules in the AtT20 cells, 19 suggesting that the aggregation of both the mammalian and molluscan regulated secretory proteins requires a similar milieu in the TGN, and utilizes the same mechanism, therefore allowing co-aggregation and correct targeting of the ELH hormone in AtT20 cells . On the other hand, it has also been shown that prohormones from other species, such as anglerfish preprosomatostatin 20 or prodermorphin from frogs, 21 when introduced into mammalian cells, are not sorted into the mammalian secretory granule unless they are fused to a mammalian prohormone . These results suggest that the mechanism for sorting regulated secretory proteins to the secretory granule is more complex than can be explained simply by co-aggregation, and may require either as yet unidentified sorting sequences or additional components, such as molecules in the membrane of the TGN . Association of regulated secretory proteins with the TGN membrane
One unanswered question is whether the densecored aggregate interacts with the membrane of the
S .A . Tooze and J. C. Stinchcombe
TGN, and initiates the budding of a secretory granule . Previous models (see refs 22, 23) have envisaged that the aggregate interacts with a protein, or proteins, in the TGN membrane . It has long been known that a small population of the normally soluble regulated secretory proteins exists as membrane bound molecules (see further discussion below) . These membrane-bound forms of the regulated secretory proteins may link the dense-core aggregate to the membrane by interacting in a homophilic manner . 24 Alternatively, there may be heterophilic interactions between unidentified membrane proteins and the aggregate, similar to a receptor-ligand interaction . This latter hypothesis remains attractive, and is supported by some experimental evidence (reviewed in ref 2) ; to date however, no receptor molecule has been found . The major difficulty in identifying such a receptor molecule may be its low abundance : one would expect that a single receptor molecule would bind to an aggregate containing many secretory proteins . In addition, after budding the receptor molecule may recycle from the ISG to the TGN, and be reused . It may not therefore be present in the dense mature secretory granules which can be isolated by cell fractionation .
Sorting of regulated secretory proteins and lysosomal enzymes in the TGN
In the lumen of the TGN the newly-synthesized lysosomal enzymes bearing the mannose-6-phosphate recognition signal interact with the mannose-6phosphate receptor . These receptor-ligand complexes are then sorted to the pre-lysosome by a transport step which involves clathrin coated vesicles . 7 However, in exocrine pancreatic cells which are highly specialized for the secretion of molecules by the regulated pathway, it appears that the sorting of lysosomal enzymes to the lysosome is perturbed : cathepsin B, a lysosomal enzyme, is found in the dense-core of zymogen granules, and in the pancreatic juice secreted from the cells after an appropriate stimulus . 25 This raises the possibility that in other regulated secretory cells a small percentage of the newly-synthesized lysosomal enzymes may be missorted into the secretory granule . To avoid proteolytic degradation of the regulated secretory proteins by the lysosomal enzymes in the secretory granule, the lysosomal enzymes must
Biogenesis of secretory granules be removed or inactivated . One could postulate that these missorted molecules are removed from the secretory granule by the same mechanism that is utilized in the TGN, that is, clathrin coated vesicles . This could also explain the function of the clathrincoated patches seen not only on regions of the TGN containing dense-cored aggregates but also on the newly formed ISG (see below) . In short, sorting may continue after the ISG has detached from the TGN .
Formation of secretory granules from the TGN After the aggregation and sorting of the regulated secretory proteins from the constitutive secretory proteins, the dense-core is enveloped by membrane and buds from the TGN to form an ISG . Likewise, constitutive secretory vesicles, containing constitutive secretory proteins, also bud from the TGN . This is aided by a cytoplasmic coat, which fully encompasses the budding vesicle while it is still attached to the TGN . The formation of ISGs does not appear to involve a continuous cytoplasmic coat, although patches of clathrin can be detected on the membrane of the TGN, often in regions juxtaposed to the densecored aggregates in the lumen . 26,27 To investigate the requirements for vesicle formation from the TGN, cell-free systems have been developed which reconstitute the budding of vesicles from the TGN . 28,29 One such system monitored the budding of both constitutive and regulated secretory vesicles . 28 Here, . the ISG formed from the TGN contained only regulated secretory proteins, while the constitutive secretory vesicles formed from the TGN contained only constitutive secretory proteins, showing that the regulated secretory proteins are sorted from the constitutive secretory proteins before their exit from the TGN . This cell-free system was further exploited to demonstrate first that the formation of both regulated and constitutive secretory vesicles from the TGN is inhibited by the addition of non-hydrolyzable analogues of GTP, i .e . GTP-yS, 30 and second that budding of both the regulated and constitutive secretory vesicles involves a member of the family of heterotrimeric G-proteins . 31 Properties of immature secretory granules and their maturation The first vesicle of the regulated secretory pathway formed from the TGN has been alternatively referred
361 to as a condensing vacuole, 32 a coated granule, 26 and an ISG (for review see ref 33) . In the neuroendocrine cells, it has been shown that the ISG is an intermediate in the formation of mature secretory granule (MSG) . The process by which an ISG matures to an MSG involves an increase in size of the ISG, perhaps as a result of fusion of multiple ISGs to form one MSG . 34,35 It has also been shown recently that the ISG can be translocated to the plasma membrane, in a microtubule-dependent fashion, and can undergo regulated exocytosis after an appropriate stimulus . 35 The morphological evidence that the ISG has clathrin-coated regions, 26,27 and the biochemical evidence suggesting that ISGs undergo a fusion process during their maturation, imply that the ISG is a dynamic intermediate . Indeed, the plasticity of the ISG is also evident from experiments in the exocrine pancreas 36 and the islet cells of the pancreas, 37 which suggest there is a pathway from the ISG to the plasma membrane whereby a small fraction of the regulated secretory proteins can be released rapidly, and independently of an external stimulus, in a constitutive fashion . The findings that the ISG can fuse with itself, or with the plasma membrane, have implications with respect to the sorting of granule-specific proteins during the formation of the ISG . Molecules involved in both the ISG-ISG, and in the ISG-plasma membrane fusion events, must be present on the ISG . Furthermore, one could imagine that proteins involved in formation and maturation of the ISG, for example the putative aggregate receptor and the proteins involved in ISG-ISG fusion are not required by the mature granule . Therefore, they may be removed from the ISG and recycled to the TGN and re-used in the next round of secretory granule budding and maturation .
Biogenesis of the granule membrane The newly forming granule obtains its membrane proteins from two sources ; recycling and re-use of the membranes of previous granules following exocytosis and de novo synthesis . The contribution of each source depends upon the cell type . In fully differentiated non-dividing secretory cells, for example freshly prepared primary chromaffin cells in culture 38 most of the MSG membrane is recycled . This is probably the mechanism by which newly forming granules receive their membranes in
3 62 secretory tissue . In established cell lines which maintain the capacity to divide, de novo synthesis predominates . This may also be the case during early stages of the development of secretory tissues which still show plasticity and have the capacity to divide . In addition, de novo synthesis may be used to replenish any membrane components that are degraded during the recycling process . The differences between fully differentiated tissue, and actively dividing cells are important to remember since many studies on granule formation have used established secretory cell lines . Recycling of granule membrane proteins The original idea that the membrane proteins of the MSG are re-used following exocytosis came from the observation that the turnover of membrane proteins in metabolically labelled adrenal medulla and exocrine pancreas glands was considerably slower than the secretory content (for review see ref 2) . Subsequently, by labelling the cell surface following exocytosis with either antibodies against luminally oriented membrane protein epitopes or by biotinylation, membrane recycling has been demonstrated in several secretory cell types (ref 38 and references therein) . The granule membrane components do not diffuse throughout the plasma membrane upon exocytosis but remain in discrete patches . These are rapidly removed from the plasma membrane, within 5 minutes of exocytosis, by a process that may involve the non-erythroid spectrin molecule fodrin, 39 returned to the TGN and ultimately detected in MSGs (reviewed by ref 2) . This suggests that once formed, the granule membrane retains its integrity and is re-used at the TGN to enwrap the newly forming granules . granule membrane protein synthesis
De novo
The situation for newly synthesized granule membrane proteins is different from that of recycled membrane components since here the individual proteins must both be segregated from the components of the other cellular membranes and incorporated into the new granule membrane . Two types of proteins are present in the granule membrane ; proteins that are found primarily as soluble proteins but also exist in membrane associated forms, and proteins found exclusively in
S .A . Tooze and J. C. Stinchcombe
the membrane . Little is known about either the sorting mechanism, or the intracellular route taken by granule membrane proteins from the TGN to the mature granule . The different properties of the two types of membrane proteins suggest that at least two different mechanisms and routes might exist .
Proteins present in both the granule content and membrane
Increasing evidence suggests that all the soluble granule proteins may also be present in the membrane . However, the amount which is granule membrane-associated appears to differ greatly, with values up to 507o for dopamine /3-hydroxylase (D(3H) 4° in the chromaffin cell . In addition, a variety of different mechanisms of membrane attachment are utilized the nature of which suggests that the proteins may be preferentially membraneassociated at different stages of the secretory pathway . D(3H may be anchored in the membrane by an uncleaved sequence ; 41 the newly synthesized protein is primarily found in the membrane but the proportion that is membrane-associated decreases with time . 42 Conversely, carboxypeptidase E (CPE), 43 in a variety of endocrine cell types, associates with the membrane via a low pH-dependent, amphipathic C-terminal helix . This suggests that the protein becomes membrane-associated as it moves into the more acidic environment of the TGN . 44 Additional mechanisms of membrane association involve PIanchors, as is found on GP2 from the exocrine pancreas, 45 which are added to the protein in the RER . The mechanistic and temporal differences in membrane association observed for different granule proteins may regulate their activity, and may also reflect the different function of each protein . However, in all cases membrane-associated forms of the proteins exist in the TGN . This suggests that, as discussed above, membrane-associated forms of soluble proteins may be important for the interaction of membranes with regulated protein aggregates and that they may be involved in the mechanism of soluble protein sorting . In the cases where the structure of the membrane and soluble forms of the protein has been determined, for example for Df3H, 41 CPE 44 and peptidyl-glycine a-amidating monooxygenase (PAM), 46 the two forms differ only in the small regions conferring membrane attatchment . Thus both forms of each protein are likely to contain the same sorting
Biogenesis
of secretory
granules
information, unless the presence of the protein in the membrane per se, or a resulting conformational change, alters its intracellular pathway to the granule . In support of this hypothesis is the differential intracellular localization of pc2 and pc3, the putative prohormone endopeptidases which are found in secretory granules (for review see ref 47), and Kex2 and furin, Golgi-localized endopeptidases that cleave at the di-basic residues of constitutively secreted proteins in yeast and mammalian cells, respectively (for review see ref 48) . Pc2 and pc3 are highly homologous to Kex2 and furin, also referred to as PACE, except that pc2 and pc3 do not have the serine/threonine rich region, the trans-membrane sequence or the cytoplasmic tail found in Kex2 and furin . Rather, pc2 and pc3 have an amphipathic domain at their C-terminus . Comparison of the domain structures of Kex2 and furin with that of pc2 and pc3 suggests that the intracellular localization is determined by the C-terminus of the molecule . In the case of Kex2 and furin, the trans-membrane and cytoplasmic domains may be responsible for the Golgi localization . In contrast, the amphipathic region at the C-terminus of pc2 and pc3 may mediate their association with the membrane, in a mechanism similar to that proposed for carboxypeptidase E (see above), and may direct these proteins to the secretory granule membrane .
Proteins exclusively located in membranes
Proteins in this group include the proteins involved in the transport of small molecules and the establishment of a chemiosmotic gradient, plus several characterized glycoproteins, and polypeptides with unidentified function (reviewed in ref 49, for the best characterized granule, the chromaffin granule) . All of the granule membrane proteins identified to date are also present in the membranes of other compartments of the same and other cells . For example, vacuolar proton pumps with subunits that are immunologically related to those of the chromaffin granule proton pump have been found in lysosomes, the Golgi complex, clathrin coated vesicles and synaptic vesicles (reviewed in ref 50) ; the chromaffin granule glycoprotein GPII has been found in lysosomes in the kidney 51 whilst the insulin granule proteins SM110 and SM80 are also in the liver ; 52 the proteins involved in catecholamine uptake and biosynthesis in chromaffin cells are also present in adrenergic synaptic vesicles ; and p65 and
363 SV2, which have been identified immunologically in a variety of neuroendocrine granules, were originally identified in neuronal synaptic vesicles and at least p65 may also be in the plasma membrane . 53,54 The identification of additional components of granule membranes has been further complicated by several other characteristics of the granule membranes : (1) each granule membrane protein constitutes only a minor percentage of the total cellular membrane protein . This is because the granule membrane has a low protein : lipid ratio compared to other cellular membranes and each individual protein is present at low abundance ; and (2) it has not proved possible to isolate significant quantities of membrane from most types of granules free of contaminating membranes from other organelles . The identification of any new granule membrane protein is further complicated by the slow turnover and possibility of recycling of these proteins . These points, combined with the fact that to date no unique secretory granule membrane protein from exocrine, endocrine or neuronal cells has been identified, means little information is available about the mechanism and location of their sorting .
Mechanisms of secretory granule membrane protein sorting Certain recent observations, however have provided some information on the sorting and routing of newly synthesized membrane proteins to secretory granules . Unlike the soluble proteins, granule membrane proteins may contain sorting signals . When human P-selectin, a membrane protein of platelet and endothelial cell granules, was expressed in the murine AtT-20 cell line the protein was very efficiently targeted to the storage granules of these cells .° 5 This indicates that the information for sorting to the regulated secretory pathway is conserved between both species and cell types . Furthermore, the cytoplasmic tail of P-selectin was sufficient to divert a normally constitutively secreted protein to the regulated secretory pathway . The cytoplasmic domain of P-selectin therefore probably contains a signal for sorting to the regulated secretory pathway . This is the first identification of a `positive' sorting signal in this pathway . Since the cytoplasmic domain of P-selectin shows no similarities in its primary structure to those of other known granule membrane proteins, for example cytochrome b561 and p65, the signal may be a conformational one .
364 The location of membrane protein sorting and intracellular pathway to the secretory granule The site at which the granule membrane proteins are segregated from other cellular membrane proteins and the route which they take from the TGN to the mature granule remains enigmatic . Sorting may have occurred already upon exit from the TGN, the granule membrane proteins leaving the TGN exclusively in the ISG . Alternatively, sorting might occur entirely or partially in a post-Golgi location ; the proteins may be randomly distributed throughout all the vesicles, including the ISG, leaving the TGN and subsequently sorted by a process of selective retention and exclusion, or they may travel first to the plasma membrane from which they would be selectively retrieved as appears to be the case for the lysosomal membrane proteins (reviewed by ref 56) and synaptic vesicle protein synaptophysisn . 57 Several observations are consistent with sorting of at least a subpopulation of granule membrane proteins at the TGN into the ISG . (1) Granule membranes retrieved from the plasma membrane following exocytosis are transported to the TGN before being found again in granules (reviewed in ref 2) . This suggests that the TGN may also be the site at which the composition of the granule membrane is defined . (2) P-selectin was observed by immunoelectron microscopy to be associated with dense cores in the TGN, 55 suggesting at least some granule membrane proteins are segregated with the granule content in the TGN . (3) Immature chromaffin granules are able to accumulate radiolabelled small molecules such as nucleotides and catecholamines from their extracellular environment49 and, as described above, ISGs of PC 12 cells exhibit calcium-dependent regulated exocytosis . 35 Thus the proteins required for some of the specific functions of the granule must already be present and active in the ISG membrane . This suggests that at least some membrane proteins present in the MSG leave the TGN in the ISG with the content proteins . Two observations provide indirect evidence in support of recycling granule membrane proteins from the plasma membrane . The first is the observation that granule membrane proteins are specifically retrieved from the plasma membrane following exocytosis . However, here complete membranes rather than individual proteins appear to be internalized . Secondly, the cytoplasmic tail of P-selectin contains two sequences believed to be signals for endocytosis at the plasma membrane 55 and
S .A . Tooze and ,J. C. Stinchcombe therefore possibly involved in membrane retrieval following exocytosis . However, there is no indication of whether these signals are part of the domain involved in targeting the newly synthesized protein to the ISG . Elucidating the mechanisms involved in sorting granule membrane proteins depends on the identification of a protein unique to the granule membrane which can be radiolabelled and whose transport can be followed after synthesis through the secretory pathway . To date such molecules have not been found . The difficulty encountered in identifying granule membrane proteins causes one to ask whether the mature secretory granule has any proteins in its membrane other than those molecules required for exocytosis of the secretory granule . All the other proteins required to form the granule, and those involved in the modification of the soluble content proteins, may be removed from the ISG during maturation . Conclusions Although the regulated pathway of secretion was originally the prototype secretory pathway, 8 today much less is known about it than is known about, for example, the constitutive secretory pathway, or the different pathways to the lysosome . The complexity of the sorting mechanism of the content proteins, combined with the major difficulties encountered in the identification of granule membrane protein, has left many questions unresolved about this enigmatic pathway . Acknowledgements We thank Dr John Tooze for critically reading the manuscript, and for helpful discussions . We also thank Dr Wieland Huttner for stimulating discussions and advice, and for support (J .C . S) . We also thank Dr Rodger McEver for communication of results before publication . References 1 . Pelham HR (1989) Control of protein exit from the endoplasmic reticulum . Annu Rev Cell Biol 5 :1-23 2 . Burgess TL, Kelly RB (1987) Constitutive and regulated secretion of proteins . Annu Rev Cell Biol 3 :243-293 3 . Docherty K, Steiner DF (1982) Post-translational proteolysis in polypeptide hormone biosynthesis . Annu Rev Physiol 44 :625-638 4 . Hutton JC, Bailyes EM, Rhodes CJ, Rutherford NG, Arden SD, Guest PC (1990) Biosynthesis and storage of insulin . Biochem Soc Trans 18 :122-124
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