Biogenesis, Trafficking & Exocytosis of ZGs: still a lot to Sort Out
A first and crucial step in ZG biogenesis is the selection/sorting of proteins to be packaged in the ZG. This process takes place at the trans-Golgi network (TGN) and is thought to involve selective protein aggregation and protein–membrane interactions. Some of the ZG enzymes form protein complexes already within the lumen of the endoplasmic reticulum.[8,9] These complexes exhibit distinct protein compositions and contain certain zymogens in association with nonenzyme proteins, which have been proposed to act as 'helper' proteins in complex formation, aggregation and/or sorting to the membrane.[8,10] The protein complexes progressively aggregate at the mildly acidic pH and high Ca2+ levels within the TGN and form dense core aggregates that are supposed to interact with the TGN membrane.[11–14] Whereas the selective aggregation of ZG proteins has been well documented (Figure 1D & E), their interaction with the TGN/ZG membrane is poorly understood at the molecular level, and neither a common sorting signal nor a sorting receptor has been identified so far. Figure 1D & E highlights the pH-dependent aggregation of ZG proteins in an in vitro condensation-sorting assay after processing for electron microscopy.
In an active model for sorting ('sorting by entry'), membrane binding of secretory proteins is assumed to depend on a 'sorting receptor' within the TGN (e.g., a transmembrane protein), and entry into forming granules is restricted to receptor-mediated trafficking. In an alternative, passive model ('sorting by retention'), aggregation of regulated secretory proteins is the primary sorting event. In this model, a receptor is not required and entry into the forming granule is not solely restricted to regulated secretory proteins. The nonsecretory proteins fail to aggregate and are removed from the maturing granules in a clathrin-dependent process (reviewed in [15,16]). In this case, the immature secretory granule can also be a site for sorting. However, the retention of the granule-specific membrane components is likely to involve their interaction with regulated secretory proteins.
Sorting during ZG biogenesis is also supposed to take place after the TGN, at the level of the ZG itself. Ultrastructural studies indicate that parts of the Golgi cisternae become dilated and filled with electron-opaque material. These condensing vacuoles (CVs), the initial stage of ZGs, then pinch off as immature granules, which mature into ZGs by the selective removal of nonsecretory granule proteins (via a clathrin-mediated process called 'constitutive-like secretion') and further condensation of the aggregated proteins occurs, as well as a reduction in granule size.[15,17,18] In addition, the proper assembly of lipid microdomains as well as cholesterol biosynthesis has been shown to be crucial for granule formation at the TGN.[19,20]
Trafficking of ZGs towards the apical plasma membrane occurs in a microtubule- and actin-dependent manner, also requiring dynein, dynactin and myosin motors. Despite extensive study, the mechanism underlying ZG exocytosis (e.g., the identification of the Ca2+ sensor) is still far from clear; however, some information is beginning to emerge regarding the proteins that mediate the exocytotic membrane fusion event.[6,21] Several soluble N-ethylmaleimide-sensitive factor-activating protein receptor (SNARE) proteins, Rab GTPases and aquaporins are suggested to be involved in granule docking/priming, granule swelling and exocytosis. In addition, the recent development of atomic-force microscopy (AFM) has allowed the investigation of the role of fusion pores in living pancreatic acinar cells.[22,23] The membrane material of the granule, which is transiently incorporated into the apical plasma membrane is removed and recycled via endocytosis by clathrin-coated vesicles. Upon exocytosis of the ZG, the digestive enzymes are exposed to the alkaline pH of the acinar/ductal lumen, and the proteins are solubilized for their transit to the duodenum. Once in the intestinal tract, granule proteins are also supposed to fulfil regulatory and protective functions, for example, in host defence.[25,26]
The exocrine pancreas served as the model that first established the role of intracellular compartments in the secretory pathway, and ZGs have been used as a model system to study secretory-granule biogenesis and regulated secretion in general. As briefly outlined earlier, the molecular mechanisms required for ZG formation at the TGN and for the packaging and sorting of cargo proteins, as well as for granule fusion and exocytosis, are still poorly defined (reviewed in[2,4,21,28]). According to recent models, part of the molecular machinery required for digestive enzyme sorting, granule trafficking and exocytosis is supposed to be associated with the ZG membrane (ZGM). In addition to basic research interests, ZGs also play important roles in pancreatic injury and disease.[29,30] It is well documented that the mis-sorting of zymogens to lysosomes or the basolateral membrane domain can lead to acinar cell injury and pancreatic disease. Unravelling the molecular machinery that mediates the proper trafficking of potentially damaging proteases to distinct membrane domains by specific coat, adapter and Rab proteins is therefore of great biological and clinical importance. Thus, there is currently great interest in the identification and molecular characterization of ZG and ZGM components by conducting antibody screens and raft analyses, as well as by proteomics.[19,31–35]
Expert Rev Proteomics. 2010;7(5):735-747. © 2010
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