Isolated Mammalian andSchizosaccharomyces pombeRan-binding Domains RescueS. pombe sbp1(RanBP1) Genomic Mutants

Abstract

Mammalian Ran-binding protein-1 (RanBP1) and its fission yeast homologue, sbp1p, are cytosolic proteins that interact with the GTP-charged form of Ran GTPase through a conserved Ran-binding domain (RBD). In vitro, this interaction can accelerate the Ran GTPase-activating protein-mediated hydrolysis of GTP on Ran and the turnover of nuclear import and export complexes. To analyze RanBP1 function in vivo, we expressed exogenous RanBP1, sbp1p, and the RBD of each in mammalian cells, in wild-type fission yeast, and in yeast whose endogenous sbp1 gene was disrupted. Mammalian cells and wild-type yeast expressing moderate levels of each protein were viable and displayed normal nuclear protein import. sbp1 Ϫ yeast were inviable but could be rescued by all four exogenous proteins. Two RBDs of the mammalian nucleoporin RanBP2 also rescued sbp1 Ϫ yeast. In mammalian cells, wild-type yeast, and rescued mutant yeast, exogenous full-length RanBP1 and sbp1p localized predominantly to the cytosol, whereas exogenous RBDs localized predominantly to the cell nucleus. These results suggest that only the RBD of sbp1p is required for its function in fission yeast, and that this function may not require confinement of the RBD to the cytosol. The results also indicate that the polar amino-terminal portion of sbp1p mediates cytosolic localization of the protein in both yeast and mammalian cells. INTRODUCTIONProteins to be transported across the eukaryotic nuclear membrane often contain sequence motifs that function as nuclear import or export signals (Nigg, 1997). Transport requires both insoluble components, associated with the nuclear pore, and soluble components that interact with the transport cargo and/or pore. Among the soluble factors are members of the karyopherin ␤ family, the small GTPase Ran, Ranbinding protein-1 (RanBP1), and nuclear transport factor 2 (Mattaj and Englmeier, 1998). Components of the nuclear pore complex, termed nucleoporins, have been identified by biochemical analysis of partially purified pore complexes and by genetic analysis of nuclear transport in budding yeast (Fabre and Hurt, 1997;Ohno et al., 1998). A pore component that appears to play a central role in interactions with the soluble factors is RanBP2 (also known as Nup358) (Wu et al., 1995;Yokoyama et al., 1995).Many models of nuclear transport have been proposed, and central to them all is the control exerted by Ran in regulating the association and dissociation of transport complexes, linked to the hydrolysis and turnover of Ran-bound guanine nucleotides (Koepp and Silver, 1996;Panté and Aebi, 1996;Rush et al., 1996;Gö rlich, 1997;Nakielny and Dreyfuss, 1997;Nigg, 1997;Yoneda, 1997;Izaurralde and Adam, 1998;Mattaj and Englmeier, 1998;Melchior and Gerace, 1998;Moore, 1998). The intrinsic rates of GTP hydrolysis and guanine nucleotide release by Ran are slow ‡ Corresponding author. E-mail address: deustp01@mcrcr0.med. nyu.edu. Abbreviations used: GFP, green fluorescent protein; HA, hemagglutinin; NES, nuclear export signal; NLS, nuclear localizatio...

Subcellular localization and role of Ran1 in Tetrahymena thermophila amitotic macronucleus

“…Consistent with the localization of HA‐BP1 binding domain of Ran1, HA‐tagged RanBP1 localized mostly to the cytoplasm in interphase of Tetrahymena , despite the lack of any discernible NES motif (D. Zhao & W. Wang, unpublished results). The same subcellular distribution has been also observed for yeast RanBP1 homologue: sbp1p without the NES motif [37]. Isolated RBD motif which localizes predominantly to the nucleus in mammalian and yeast cells [37] may mediate the macronuclear localization of RanBP1 during amitosis of Tetrahymena .…”

“…The same subcellular distribution has been also observed for yeast RanBP1 homologue: sbp1p without the NES motif [37]. Isolated RBD motif which localizes predominantly to the nucleus in mammalian and yeast cells [37] may mediate the macronuclear localization of RanBP1 during amitosis of Tetrahymena . Tetrahymena contains 16 proteins containing two to eight RCC1 repeats of a 30‐ to 70‐amino acid domain.…”

Human Raf‐1 proteins associate with Rad24 and Cdc25 in cell‐cycle checkpoint pathway of fission yeast, Schizosaccharomyces pombe

“…Many laboratories used complementation of yeast mutants to detect human homolog genes with similar functions [Freire et al, 1998;Novoa et al, 1999]. Thus, it is of great significance that Raf-1-interacting proteins are firstly identified in S. pombe, which is a relatively simple and easy-handling eukaryote compared to human, and then their homolog in mammalian cells are screened using S. pombe cDNA.…”

Yeast Ran-Binding Protein 1 (Yrb1) Shuttles between the Nucleus and Cytoplasm and Is Exported from the Nucleus via a CRM1(XPO1)-Dependent Pathway

“…Yeast RanBP1 sequences exhibit an N-terminal extension outside of the RBD which shares no sequence similarity to the C-terminal extension of metazoan RanBP1 and also lacks the leucine-rich NES. Nevertheless, recent reports of nuclear pools of RanBP1 in yeast (36,57,69) indicated that the dynamic localization of RanBP1 might be conserved throughout the eukaryotic kingdom.…”