Abstract. In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RA77 gene (~bonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive ratT-1 allele from 23°C to 37°C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23°C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37°C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.

Mouse C3H 1OT1/2 cells and the established rat embryo fibroblast cell line REF-52 are two cell lines widely used in studies of viral transformation. Studies have shown that transformation of 10T1/2 cells requires only the amino-terminal 121 amino acids of simian virus 40 (SV40) large T antigen, while transformation of REF-52 cells requires considerably more of large T antigen, extending from near the N terminus to beyond residue 600. The ability of a large set of linker insertion, small deletion, and point mutants of SV40 T antigen to transform these two cell lines and to bind p105Rb was determined. Transformation of 1OT1/2 cells was greatly reduced by mutations within the first exon of the gene for large T antigen but was only modestly affected by mutations affecting the p105Rb binding site or the p53 binding region. All mutants defective for transformation of 1OT1/2 cells were also defective for transformation of REF-52 cells. In addition, mutants whose T antigens had alterations in the Rb binding site showed a substantial reduction in transformation of REF-52 cells, and the degree of this reduction could be correlated with the ability of the mutant T antigens to bind p105Rb. There was a tight correlation between the ability of mutants to transform REF-52 cells and the ability of their T antigens to bind p53. These results demonstrate that multiple regions of large T antigen are required for full transformation by SV40.

Rat7p/Nup159p is an essential nucleoporin of Sac-charomyces cerevisiae originally isolated in a genetic screen designed to identify yeast temperature-sensitive mutants defective in mRNA export. Here we describe a detailed structural-functional analysis of Rat7p/Nup159p. The mutation in the rat7-1 ts allele, isolated in the original genetic screen, was found to be a single base pair change that created a stop codon approximately 100 amino acids upstream of the actual stop codon of this 1,460 amino acid polypeptide, thus eliminating one of the two predicted coiled-coil regions located near the carboxyl terminus of the protein. These coiled-coil regions are essential since an allele lacking both coiled-coil regions was unable to support growth under any conditions. In contrast, no other region of the protein was absolutely required. The SAFG/PSFG repeat region in the central third of the protein was completely dispensable for growth at temperatures between 16 degrees C and 37 degrees C and cells expressing this mutant allele were indistinguishable from wild type. Deletion of the amino-terminal third of the protein, upstream from the repeat region, or the portion between the repeat region and the coiled-coils resulted in temperature-sensitivity, but the two alleles showed distinct phenotypes with respect to the behavior of nuclear pore complexes (NPCs). Taken together, our data suggest that Rat7p/Nup159p is anchored within the NPC through its coiled-coil region and adjacent sequences. In addition, we postulate that the N-terminal third of Rat7p/Nup159p plays an important role in mRNA export.