In a screen for mutants defective in nucleocytoplasmic export of mRNA, we have identified a new component of the Saccharomyces cerevisiae nuclear pore complex (NPC). The RAT9/NUP85 (ribonucleic acid trafficking) gene encodes an 84.9-kDa protein that we have localized to NPCs by tagging the RAT9/NUP85 gene with the in vivo molecular marker Green Fluorescent Protein. In cells containinf either the rat9-1 allele or a complete deletion of the RAT9/NUP85 gene, poly(A) RNA accumulates rapidly in nuclei after a shift from 23°C to 37°C. Under these same conditions, rapid fragmentation of the nucleolus was also observed. At the permissive growth temperature in rat9-1 or RAT9 deletion strains, the nuclear envelope (NE) becomes detached from the main body of the nucleus, forming long thin double sheets of NE. NPCs within these sheets are clustered and some appear to be locked together between opposing sheets of NE such that their nucleoplasmic faces are in contact. The Rat9/Nup85 protein could not be detected in cells carrying a mutation of RAT2/NUP120, suggesting that Rat9p/Nup85p cannot be assembled into NPCs in the absence of Rat2p/Nupl2Op. In contrast, Rat9/ Nup85 protein was readily incorporated into NPCs in strains carrying mutant alleles of other nucleoporin genes. The possible role of Rat9p/Nup85p in NE integrity and the loss of nucleoporins when another nucleoporin is mutant or absent are discussed.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rsslp encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rsslp partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37°C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37°C, the mutant Rat7-lp/Nupl59-lp is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rsslp did not result in retention of the mutant Rat7-lp/Nupl59-lp in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rsslp by placing the RSS1 open reading frame (ORF) under control of the GALl promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rsslp are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rsslp overexpression suppresses the growth defect of rat7-1 cells at 37°C by acting to maintain mRNA export.

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)؉ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. ؉ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37؇C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23؇C and did not grow at 30؇C, efficient growth at 23 or 30؇C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37؇C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A) ؉ RNA following a shift to 37؇C. Surprisingly, these strains grew efficiently at 37؇C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.Macromolecules move between the nucleus and the cytoplasm through nuclear pore complexes (NPCs) (see references 9, 11, 13, 15, and 25 for recent reviews). NPCs are supramolecular structures that perforate the nuclear envelope, forming channels between the nucleus and the cytoplasm. Characterization of NPCs at the ultrastructural level has produced a model wherein two rings, one nuclear and one cytoplasmic, are joined by "spokes" that are arrayed to produce an eightfold symmetry around an axis perpendicular to the plane of the nuclear envelope (2, 23,

The ability of DNA tumor virus proteins to trigger apoptosis in mammalian cells is well established. For example, transgenic expression of a simian virus 40 (SV40) T-antigen N-terminal fragment (N-termTag) is known to induce apoptosis in choroid plexus epithelial cells. SV40 T-antigen-induced apoptosis has generally been considered to be a p53-dependent event because cell death in the brain is greatly diminished in a p53 ؊/؊ background strain and is abrogated by expression of wild-type (p53-binding) SV40 T antigen. We now show that while N-termTags triggered apoptosis in rat embryo fibroblasts cultured in low serum, expression of full-length T antigens unable to bind p53 [mut (p53؊) Tags] protected against apoptosis without causing transformation. One domain essential for blocking apoptosis by T antigen was mapped to amino acids 525 to 541. This domain has >60% homology with a domain of adenovirus type 5 E1B 19K required to prevent E1A-induced apoptosis. In the context of both wild-type T antigen and mut (p53؊) Tags, mutation of two conserved amino acids in this region eliminated T antigen's antiapoptotic activity in REF-52 cells. These data suggest that SV40 T antigen contains a novel functional domain involved in preventing apoptosis independently of inactivation of p53.

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.