There exist in eucaryotic cells two unique macromolecular assemblies which mediated pore-selective transport of molecules across double membrane systems: the gap junction array and the nuclear pore complex. Both organelles are involved in communication processes between or within cells. Their functions are essential for maintenance of metabolic synchronization, growth and development of cells within specific tissues. The structure, function and regulation of these 2 transmembrane complexes will be investigated utilizing various biochemical and diffraction methods. The quaternary transitions which occur in liver and brain gap junctions in response to H+, at physiological ionic strengths, will be investigated using low angle x-ray diffraction, electron microscopy and 3-dimensional image reconstruction. A study of the detergent solubilization of gap junctions will be carried out for 3 reasons. First, reconstitution studies will be carried out with the goal of obtaining a direct assay of pore-mediated transport and regulation by H+ and Ca+2 in large liposomes. Second, solubilized connexin hexamers and lipid will be used in attempts to reconstitute single-layered junction membranes with improved structural order. Third, solubilized connexin will be used in 3-dimensional crystallization trials. A complete understanding of the biology of the nuclear pore complex will require a combination of x-ray crystallographic studies of the constituent proteins, coupled with a knowledge of the disposition of subunits in the complex obtained by electron microscopy. Structural studies will be aimed at comparing the positions of subunits within pore complexes isolated from either transport-active or inactive nuclei of frog oocytes. Three-dimensional structures will be determined using electron microscopy of membranes embedded in a thin ice film at various fixed tilts. Maps of active and inactive nuclear pore complexes will be obtained at 2-4 nm resolution utilizing 3-dimensional image reconstruction methods and the proposed 822 symmetry of the pore complex will be evaluated. Furthermore, differences in the 2 putative states of the complex should allow possible transport mechanisms to be deduced.
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