Nuclear pore complexes are large molecular assemblies that mediate bidirectional transport between the nucleus and the cytoplasm. A novel family of yeast nuclear pore proteins has recently been identified (Wente et al., 1992). Three members of this family, NUP49, NUP100, and NUP116, have been isolated and characterized by a combination of molecular genetics and immunolocaliztion. We are further characterizing the structural and functional defects in strains with deletions in the genes encoding the nuclear pore proteins. Up to now, structural information on strains with mutations in these genes has been limited, due to artefactual changes in nuclear organization and nuclear pore structure introduced during conventional chemical fixation. We are therefore using high pressure freezing and freeze substitution to maximally preserve the ultrastructure of the nucleus and nuclear pores. Serial images through the nucleus of the yeast are digitized and modeled using the 3D reconstruction programs developed in our lab. From these reconstructions, we have obtained information about the 3D organization of nuclear pores as well as quantitative information on the surface area and volume of the nuclear membrane and pores in these defective strains. We have found that one strain, a temperature-sensitive null allele at NUP116, forms a nuclear envelope seal over the yeast nuclear pore complex; this presumably results in a blockage of nucleocytoplasmic traffic. By using a combination of molecular and structural biology, we hope to gain insight into the mechanisms of nuclear pore assembly and nucleocytoplasmic trafficking in yeast.
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