Pleiotropic nuclear defects associated with a conditional allele of the novel nucleoporin Rat9p/Nup85p.

Abstract

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.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

“…In nup159 cells, the phenomenon is actually the opposite of that in gle2 cells: nup159 NPCs are clustered at 23°C, whereas at 37°C their distribution is over the entire nuclear surface (Gorsch et al, 1995). The herniated clustered NPCs in gle2 cells are distinct from the successive interconnected NPCs found in grape-like aggregates of constitutively clustered NPCs in nup145, nup120, nup84, and nup85 mutant cells (Wente and Blobel, 1994;Aitchison et al, 1995a;Heath et al, 1995;Goldstein et al, 1996;Siniossoglou et al, 1996). Cells deleted for NUP116 form herniations when shifted to the restrictive temperature (Wente and Blobel, 1993 (Wente and Blobel, 1993).…”

“…Thus, protein import was not noticeably diminished. This suggested that Gle2p is specifically required for a NPC export func- (Wente and Blobel, 1993;Bogerd et al, 1994;Doye et al, 1994;Aitchison et al, 1995a;Gorsch et al, 1995;Heath et al, 1995;Li et al, 1995;Goldstein et al, 1996;Siniossoglou et al, 1996).…”

“…Mutations in NUP145 (Wente and Blobel, 1994), NUP133 (Doye et al, 1994;Li et al, 1995;Pemberton et al, 1995), NUP120 (Aitchison et al, 1995a;Heath et al, 1995), NUP159 (Gorsch et al, 1995), NUP84 (Siniossoglou et al, 1996), and NUP85 (Goldstein et al, 1996;Siniossoglou et al, 1996) (Figure 6, right). The same was true for gle2 cells grown at 23°C (igure 6, top left).…”

“…Collections of temperaturesensitive mutant yeast strains have been directly screened for blocks in polyadenylated poly(A)+ RNA export (Amberg et al, 1992;Kadowaki et al, 1992Kadowaki et al, , 1994. Interestingly, numerous rat mutants isolated by Cole and coworkers encode for nucleoporins (rat2/ nup120, rat3/nupl33, rat7/nupl59, and rat9/nup85; Gorsch et al, 1995;Heath et al, 1995;Li et al, 1995;Goldstein et al, 1996) that may play a direct role in export.…”

Deciphering Networks of Protein Interactions at the Nuclear Pore Complex

“…In yeast, the Nup84p complex performs essential roles in NPC biogenesis and mutants in Nup84p, Nup85p, Nup120p, and Nup145p display defects in the export of mRNA as well as drastic structural abnormalities of the nuclear envelope (20,30,31). Nup133p mutants display the same phenotypes and are lethal when combined with mutant members of the Nup84p complex (32)(33)(34)(35). Despite all the evidence, it has not been possible to formally demonstrate that Nup133p forms part of the endogenous Nup84p complex in S. cerevisiae.…”