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Molecular mechanism of membrane fusion
- Discovery of the Porosome: revealing the molecular mechanism of secretion and membrane fusion in cells

As eluded earlier, membrane fusion is mediated via a specialized set of proteins in the secretory vesicles and the plasma membrane. Three soluble N-ethylmaleimide- sensitive factor (NSF)-attachment protein receptors (SNAREs) have been implicated in membrane fusion [10]. Target membrane proteins, SNAP-25 and syntaxin (t-SNARE) and secretory vesicle-associated membrane protein (v-SNARE), are part of the conserved protein complex involved in fusion of opposing bilayers [9, 10]. Although the structure of SNARE complex formed by interacting native [19] or recombinant [20, 21] t- and v- SNAREs was known from studies using electron microscopy [19, 20] and x-ray crystallography [21], the molecular mechanism of the involvement of SNAREs to bring about membrane fusion remained unknown until two years ago [12]. To determine the molecular mechanism of SNARE-induced membrane fusion, the structure and arrangement of SNAREs associated with lipid bilayers were examined using atomic force microscopy. The bilayer electrophysiological setup (EPC9) allowed measurements of membrane conductance and capacitance, prior to and after t- SNARE or v-SNARE reconstitution, and following exposure of v-SNARE or t-SNARE reconstituted vesicles. These studies demonstrate that the interaction of t-/v-SNARE proteins to form a fusion pore is dependent on the presence of t-SNAREs and v- SNARE in opposing bilayers. Addition of purified recombinant v-SNARE to a t-SNARE-reconstituted lipid membrane increased only the size of the globular t-SNARE oligomer without influencing the electrical properties of the membrane (Fig. 14). However when t-SNARE vesicles were added to a v-SNARE membrane, SNAREs assembles in a ring pattern (Fig. 15) and a stepwise increase in capacitance, and increase in conductance were observed (Fig. 16). Thus, t- and v-SNAREs are required to reside in opposing bilayers to allow appropriate t-/v-SNARE interactions leading to membrane fusion in the presence of calcium [12]. Fusion of membrane-bounded secretory vesicles with the target membrane of the porosome is a highly regulated event, where a large number of proteins participate [8, 11]. Among them the SNAREs, which were suggested to be the minimal fusion machinery [10]. Studies however report calcium (Ca2+) to be a major fusogen, whereas SNAREs promote Ca2+ sensitivity to the fusion process [22-24]. Studies further reveal that micro domains of high Ca2+ concentrations co-localize at the fusion site [25,26]. Ca2+ ion channels have been found to associate with the SNARE complex [27] and with the porosome complex [8, 11]. Furthermore, in the presence of Ca2+, t- SNAREs and v-SNARE in opposing bilayers interact in a circular array to form conducting pores [12]. Finally, several Ca2+-binding proteins such as synaptotagmin and syncollin, which interact with SNAREs in a Ca2+-dependent manner, have also been identified [28, 29], further supporting the involvement of Ca2+ in membrane fusion. Hence, there was growing evidence supporting Ca2+ as a key player in membrane fusion. To determine the role of Ca2+ in SNARE-induced membrane fusion, the fusion of t- /v-SNARE-reconstituted liposomes was investigated using various approaches [30]. Results from these studies lead to the same conclusion i.e., (i) a low fusion rate (t=16 min) between t- and v-SNAREreconstituted liposomes in the absence of Ca2+; and (ii) exposure of t-/v-SNARE liposomes to Ca2+, drives vesicle fusion on a near physiological relevant time-scale (t ~10s), demonstrating an essential role of Ca2+ in membrane fusion (Fig. 17, 18). This study also supports earlier findings on the role of Ca2+ and SNAREs in cortical vesicle fusion in sea urchin eggs [22, 24], where Ca2+ was found to acts downstream of SNAREs. Since the Ca2+ effect on membrane fusion in SNARE-reconstituted liposomes is downstream of SNAREs, suggests a regulatory role for Ca2+-binding proteins in membrane fusion in the physiological state [30]. These studies further demonstrate that in the physiological state in cells, both SNAREs and Ca2+ operate as the minimal fusion machinery [30]. Native and synthetic vesicles exhibit a significant negative surface charge primarily due to the polar phosphate head groups. These polar head groups produce a repulsive force, preventing aggregation and fusion of apposing vesicles. SNAREs bring opposing bilayers closer to within a distance of 2-3 Å, allowing Ca2+ to bridge them [30]. The bound Ca2+ would then lead to expulsion of water between the bilayers at the bridging site, allowing lipid mixing and promote membrane fusion. Hence SNAREs, besides bringing opposing bilayers closer, dictate the site and size of the fusion area (fusion occurs within the circular array [12] formed by the t-/v-SNARE complex) during secretion. The size of the t-/v-SNARE complex forming the pore is found dependent on the size of t-SNARE or v-SNARE reconstituted vesicles used. The smaller the vesicles, the smaller the pores formed (unpublished observation).

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