Identification of genes encoding putative nucleoporins and transport factors in the fission yeast Schizosaccharomyces pombe: a deletion analysis

Abstract

In a systematic approach to study genes that are related to nucleocytoplasmic trafficking in the fission yeast Schizosaccharomyces pombe, the open reading frames (ORFs) of 26 putative nucleoporins and transport factors were deleted. Here we report the initial characterization of these deletion mutants. Of the 26 putative genes deleted, 14 were found to be essential for viability. Null mutations of essential genes resulted in failure to either complete one round or to sustain cell division. Four of the 14 essential genes, SPBC582.11c, SPBC17G9.04c, SPBC3B9.16c and SPCC162.08c, encode putative nucleoporins and a myosin-like protein with homologues NUP84, NUP85, NUP120 and MLP1, respectively, that are not required for viability in Saccharomyces cerevisiae, suggesting that their gene products perform critical functions in Sz. pombe. On the basis of combined drug sensitivity assays and genetic analysis we have identified five non-essential null mutants that were hypersensitive to the microtubule depolymerizing drug thiabendazole (TBZ) and exhibited a cut phenotype upon TBZ treatment, suggesting possible involvement in microtubule function. Three of the corresponding ORFs, SPCC18B5.07c, nup40 and SPAC1805.04, encode putative nucleoporins with low similarity to the S. cerevisiae nucleoporins NUP2p, NUP53p and NUP133p, respectively. Further genetic analysis revealed that one of the nucleoporin genes, nup40, and another gene, SPCC1322.06, encoding a putative importin-β/Cse1p superfamily protein may have a spindle checkpoint function.

The Fission Yeast Schizosaccharomyces pombe Has Two Importin-α Proteins, Imp1p and Cut15p, Which Have Common and Unique Functions in Nucleocytoplasmic Transport and Cell Cycle Progression

“…Imp1p is also an authentic importin-a, which can support nuclear protein import both in vivo and in vitro (Figures 2 and 4). In contrast to our results showing that imp1D cells, derived from a heterozygous null diploid strain, are viable ( Figure 3D-1), imp1 was recently reported to be essential for viability (Chen et al 2004). However, in our hands the haploid imp1D strain reported in this article is viable and has the same telophase delay and growth characteristics as the imp1D strain described in this article.…”

“…Because the SV40 NLS is functional in fission yeast, an SV40 NLS-GFP-b-Gal fusion protein was used to assess nuclear protein import competence (Pasion and Forsburg 1999;Fleig et al 2000;Salus et al 2002;Chen et al 2004;reviewed in Yoshida and Sazer 2004). The protein accumulates exclusively in the nucleus of wild-type cells, but mislocalizes to the cytoplasm when the NLS is inactivated by mutation (Pasion and Forsburg 1999;Yoshida and Sazer 2004).…”

“…Some groups have attempted to overcome this problem by inducing expression of the reporter and then shifting cells to the restrictive temperature before it accumulates in the nucleus (Matsusaka et al 1998;Chen et al 2004). However, even using this strategy, cut15-85 was competent to import an SV40 NLS cargo (Matsusaka et al 1998).…”

“…The high degree of structural and functional conservation of components of the Ran GTPase system (Sazer 1996) make it likely that Ran directly participates in transport in S. pombe as well. However, aside from a mutation in the nuclear export receptor crm1 (Fukuda et al 1997), no previously characterized S. pombe mutants are defective in nucleocytoplasmic transport, including null mutations of six transport factors and six nucleoporins (Chen et al 2004). Even cut15, an essential gene that encodes a nuclear import receptor of the importin-a type, has been reported to be competent for the transport of an SV40 NLS fusion protein in vivo (Matsusaka et al 1998).…”

Systematic Deletion and Mitotic Localization of the Nuclear Pore Complex Proteins ofAspergillus nidulans

“…Subsequent to these analysis, the Nup genes of several species, including Caenorhabditis elegans (Galy et al, 2003) and Schizosaccharomyces pombe (Chen et al, 2004) have been identified based on sequence conservation.…”