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We have isolated and characterized the gene encoding a fourth yeast glycine-leucine-phenylalanine-glycine (GLFG) repeat nucleoporin with a calculated molecular mass of 145.3 kD, and therefore termed NUP145. The amino-terminal half of Nup145p is similar to two previously identified GLFG nucleoporins, Nup116p and Nup100p (Wente, S. R., M. P. Rout, and G. Blobel. 1992. J. Cell Biol. 119:705-723). A deletion/disruption in the amino-terminal half of NUP145 (nup145 delta N) had only a slight effect on cell growth at temperatures between 17 and 37 degrees C. However, immunofluorescence microscopy of nup145 delta N cells with antinucleoporin antibodies showed that the characteristic punctate nuclear staining normally seen in wild-type yeast cells was reduced, with the majority of the signal located in one or two intense spots at the nuclear periphery. Thin section electron microscopy analysis revealed the presence of what appeared to be successive herniations of the nuclear envelope forming grape-like structures at primarily one site on the nup145 delta N nuclei. These successive herniations contained numerous NPC-like structures, correlating to the limited bright patches of anti-nucleoporin immunofluorescence signal. In some cases the successive herniations were small. Occasionally, however, multi-lobulated nuclei were seen. We suggest that the ultrastructural phenotype of nup145 delta N cells is due to a defective interaction of nup145 delta N NPCs with the surrounding pore membrane domain of the nuclear envelope. We have also analyzed the synthetic lethal phenotypes among GLFG nucleoporin mutant alleles, and found that strains harboring nup116 and either nup100 or nup145 mutations were not viable. This, in combination with the morphological analysis, may reflect overlapping yet distinct roles for these three GLFG nucleoporins in NPC-nuclear envelope interactions.