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Molecular Mechanisms of Membrane Trafficking and Exocytosis
Summary: Richard Scheller's research focuses on the mechanisms of membrane trafficking, neurotransmitter release, and synapse development.
Eukaryotic cells contain a series of membrane-bound organelles responsible for compartmentalizing biochemical activities and regulating transport into and out of the cell. While there is continuous flux of protein and lipid through membrane compartments, each organelle maintains a distinct identity. This is accomplished through the process of vesicular trafficking. The events that underlie a vesicular transport step include the packaging of cargo into a vesicle as it buds from a donor membrane, transport of the vesicle to a target membrane, fusion of the vesicle with a target membrane, and recycling of the vesicular trafficking machinery.
We are interested in understanding the molecular mechanisms that underlie this process, particularly as they function in nerve cell synaptic transmission as well as neuronal development and plasticity. However, every cell makes use of these processes, and studies in neurons shed light on membrane-trafficking mechanisms common to all cell types. We are particularly focused on understanding the mechanism of intracellular membrane fusion, including its regulation and specificity.
Membrane Fusion
Characterization of the proteins associated with synaptic vesicles, biochemical studies of transport in vitro, and genetic analyses of membrane trafficking in yeast led to the definition of a set of proteins that appear to be at the heart of the membrane fusion process. Biochemical studies of these proteins allowed specific and testable models to be proposed for the mechanisms of membrane fusion. Sets of proteins on acceptor and donor membranes of the VAMP (vesicle-associated membrane protein, later also named synaptobrevin), syntaxin, and SNAP-25 (synaptosome-associated 25-kDa protein) families (later also collectively referred to as SNAREs, for SNAP receptors) were proposed to associate into a complex spanning the vesicle and target membranes.
Structural predictions, deletion mutagenesis, point mutagenesis, and circular dichroism studies showed that the SNARE complex forms from the association of four α-helical regions—one from VAMP, one from syntaxin, and two from SNAP-25. If the syntaxin and VAMP helical domains are parallel, the vesicle and target membranes should be brought into direct apposition; however, if the helical domains form in an antiparallel orientation, the vesicle and target membranes should be left up to 10 nm apart. Resonance energy transfer studies showed that the helical domains of VAMP and syntaxin are parallel. Thus formation of the complex between the SNAREs, VAMP, syntaxin, and SNAP-25 was proposed to bring the vesicle and target membranes together, possibly even leading to their fusion. Then after the fusion, ATP hydrolysis by N-ethylmaleimide–sensitive factor would dissociate the complex, allowing the proteins to recycle for another round of vesicle transport.
We have developed a system to investigate membrane fusion during transmitter release that makes use of PC12 cells in culture. The cells are loaded with radioactive catecholamine that is concentrated in vesicles. The cells are then passed through a 1-μm-clearance dounce, which results in a single large tear in each cell. While cytosolic components leak from the cells, vesicles, cytoskeleton, and other components remain associated with the cell ghost. About 200–300 vesicles remain associated with the plasma membrane, and their exocytosis can be achieved in a two-step process. Addition of cytosol and ATP in a “priming” step, followed by cytosol and Ca2+ in a “triggering” step, efficiently releases the neurotransmitter.
This system allows ready access to the fusion protein machinery due to the large hole in the membrane. Studies with the system suggest that Ca2+ triggers SNARE complex formation and that formation of the complex drives the membrane fusion event. Further studies with these cracked cells are aimed at gaining a more fundamental understanding of the membrane fusion process.
The Organization of Membrane Compartments
If SNARE complex formation is indeed the driving force behind membrane fusion, perhaps the specificity of vesicle trafficking is determined, at least in part, by the selective pairing of SNAREs localized to particular membranes. If this hypothesis is true, several criteria should be met. First, there should be a large number of SNARE proteins expressed in cells. Second, they should be specifically localized to the various membrane compartments within cells. We have clearly shown this to be the case. Third, the various SNARE proteins should form specific sets of complexes consistent with the known membrane-trafficking pathways within cells. Initial studies demonstrated that SNARE complexes interact promiscuously, suggesting that the specificity needed for membrane compartment organization is not determined by the ability of SNAREs to form core complexes.
The Mammalian sec6/8 Complex
Many of the genes defined through analyses of synaptic vesicles and nerve terminals have been found through an independent genetic analysis of secretion in yeast. Also, many of the late-acting sec, or secretion, genes, are the orthologues of proteins mentioned above. However, a distinct class of proteins—sec3, -5, -6, -8, -10, and -15 and exo70—are components of a large complex thought to be involved in determining the sites of exocytosis along the plasma membrane. To understand the mechanisms important in defining the sites of exocytosis, we have characterized this complex in mammalian cells. We found an expressed sequence tag encoding one of the subunits (sec8), raised antibodies to this subunit, and then purified the complex from brain tissue. The eight proteins of the complex were microsequenced, and cDNAs encoding the proteins were characterized.
The function of the mammalian sec6/8 complex has been studied in polarized epithelial cells, Madin-Darby canine kidney (MDCK) cells, and neurons. In MDCK cells, the complex is cytosolic prior to cell contact and polarization. After cell contact, the complex accumulates at the site of cell-cell interactions, and as the cell becomes polarized and develops tight junctions, the complex becomes further restricted in its localization to the tight-junction region. Interestingly, antibodies directed against the complex inhibit trafficking to the basal and lateral membranes but not to the apical aspect of the cell.
In neurons, we observe an intriguing localization of the sec6/8 complex. As expected, the complex is localized at tips of the growth cones of both axons and dendrites. However, in axons the complex localizes at a periodic spacing of ~2–4 μm. The complex demarcates sites along axons that have the capacity to become synapses, and as they become mature synapses, the complex is down-regulated. Thus the sec6/8 complex is the earliest axonal marker of synapses that we know. All of the studies from yeast to mammalian neurons suggest that the sec6/8 complex is an important determinant of the sites of membrane fusion, although the mechanism of its function remains totally unknown. (These projects are funded in part by a grant from the National Institutes of Health.)
Last updated October 20, 2000
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