Porosome: Isolation, Composition, and Reconstitution
In the last decade, a number of studies demonstrate the involvement of cytoskeletal proteins in secretion, and some studies implicate direct interaction of cytoskeleton protein with SNAREs (3, 33–37). Furthermore, actin and micro-tubule-based cytoskeleton have been implicated in intra-cellular vesicle traffic (3, 34). Fodrin, which was previously implicated in exocytosis (33), has recently been shown to directly interact with SNAREs (35). Studies demonstrate -fodrin to regulate exocytosis via its interaction with t-SNARE syntaxin family of proteins (37). The c-terminal coiled coil region of syntaxin interacts with -fodrin, a major component of the submembranous cytoskeleton. Similarly, vimentin filaments interact with SNAP23/25 and hence are able to control the availability of free SNAP23/25 for assembly of the SNARE complex (34). All these findings suggest that vimentin, -fodrin, actin, and SNAREs may be part of the porosome complex. Additional proteins, such as v-SNARE (VAMP or synaptobrevin), synaptophysin, and myosin, may associate when the porosome establishes continuity with the secretory vesicle membrane. The globular tail domain of myosin V contains a binding site for VAMP, which is bound in a calcium-independent manner (36). Further interaction of myosin V with syntaxin requires both calcium and calmodulin. It has been suggested that VAMP acts as a myosin V receptor on secretory vesicles and regulates formation of the SNARE complex (36). Interaction of VAMP with synaptophysin and myosin V has also been observed (37). In agreement with these earlier findings, recent studies demonstrate the association of SNAP-23, syntaxin 2, cytoskeletal proteins actin, -fodrin, vimentin, and calcium channels ß3 and 1c, together with the SNARE regulatory protein NSF, in porosomes (7, 8). Additionally, chloride ion channels ClC2 and ClC3 are identified as part of the porosome complex (7, 8). Isoforms of the various proteins identified in the porosome complex have also been demonstrated using 2D-BAC gel electrophoresis (8, 14). Three isoforms each of the calcium ion channel and vimentin have been found in porosomes (8). Using yeast two-hybrid analysis, recent studies confirm the presence and interaction of some of these proteins with t-SNAREs within the porosome complex (14).
The size and shape of the immunoisolated porosome complex was determined in greater detail when examined using both negative staining EM and by AFM (8). The images of the immunoisolated porosome obtained by both EM and AFM were superimposable (8). The immunoisolated supramolecular porosome complex has also been reconstituted into liposomes and in lipid bilayers (8). Transmission electron micrographs of porosome-reconstituted liposomes reveal a 150–200-nm cup-shaped, basket-like structure as observed of the porosome when coisolated with ZGs. To test the functionality of reconstituted porosome complexes, purified porosomes were reconstituted into lipid membranes in an electrophysiological bilayer setup (EPC9) and challenged with isolated ZGs (in reconstituted porosomes from exocrine pancreas) or synaptic vesicles (in reconstituted neuronal porosomes). Both the electrical activity of the reconstituted membrane and the transport of vesicular contents from the cis to the trans compartment were monitored. Results of these experiments demonstrate that the lipid membrane-reconstituted porosomes are functional supramolecular complexes (Fig. 4; Ref. 8). The ZGs fuse at the porosome-reconstituted bilayer, which is demonstrated by an increase in capacitance and conductance and a time-dependent release of the ZG enzyme amylase from cis to the trans compartment of the bilayer chamber (Fig. 4). Amylase is detected using immunoblot analysis of the buffer in the cis and trans compartment, using a previously characterized amylase-specific antibody (4). As observed in immunoblot assays of isolated porosomes, chloride channel activity is also detected in the reconstituted porosome complex, and the chloride channel inhibitor DIDS inhibits current activity in the reconstituted porosome. Similarly, the structure (Figs. 2 and 5) and biochemical composition of the neuronal porosome has also been determined (15). In summary, these studies demonstrate that the porosome in the exocrine pancreas and in neurons are 100–180 nm and 8–12 nm in diameter, respectively. The porosomes are supramolecular cup-shaped lipoprotein baskets at the cell PM, where membrane-bound secretory vesicles transiently dock and fuse to release vesicular contents to the outside.