Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-β, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-β, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominantlethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC.

Ran, a Ras‐like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione‐S‐transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP‐bound state. A major protein with the apparent size of 34 kDa co‐purifies with the GTP‐bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature‐sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.

Ran/TC4, a Ras-like GTP-binding protein, and its nucleotide exchanger, RCC1, have been implicated in control of protein movement into the nucleus and cytoplasmic accumulation of mRNA. Saccharomyces cerevisiae contains two homologues of the mammalian Ran/TC4, encoded by the GSPI and GSP2 genes. We have constructed yeast strains that overproduce either wild-type Gspl or a form of Gspl with glycine-21 converted to valine (Gspl-G21V), which we show stabilizes the GTP-bound form. Cells producing Gspl-G21V have defects in localization of nuclear proteins; nuclear proteins accumulate in the cytoplasm following galactose induction of Gspl-G21V. Similarly, cells producing Gspl-G21V retain poly(A)+ RNA in their nuclei. These findings suggest that hydrolysis of GTP by Ran/TC4 is necessary for proper import of proteins into the nucleus and appearance of poly(A)+ RNA in the cytoplasm.Movement of proteins and RNAs across the nuclear envelope via the nuclear pores is rapid, specific, and highly regulated. Proteins destined for the nucleoplasm often contain short stretches of amino acids, termed nuclear localization sequences (NLSs), that are sufficient to direct them to the nucleus (reviewed in ref.

The nuclear import system is highly conserved among eukaryotes. Here we report the effects of a conditional mutation in SRPI, which encodes a Saccharomyces cerevisiae homolog of the vertebrate nuclear import receptor importin. Importin was isolated as a factor required for the initial targeting step of a nuclear import substrate to the nuclear envelope in a mammalian in vitro assay. We show that yeast Srpl is similarly required for protein import. In addition, Srpl is also required for the execution of mitosis: we demonstrate that cells containing a conditional mutation of SRPI arrest with a G2/M phenotype in a manner analogous to classic cdc mutants. This defect may be due to the failure of the mutant to degrade the mitotic cyclin Clb2 and other proteins required for mitosis. The requirement of a nuclear import receptor for cell cycle-regulated proteolysis implies that import of cell cycle regulators into the nucleus is critical for cell cycle progression.The import of proteins into eukaryotic nuclei consists of two separable steps: the binding of import substrate to the nuclear envelope and the subsequent translocation of the substrate across the nuclear pore complex (NPC) into the nucleoplasm (1, 2). The recognition of proteins for nuclear localization is mediated by the interaction of short signal sequences (nuclear localization sequence; NLS) within the targeted protein (3) with specific receptors. One approach to identify receptors for nuclear import has been to purify proteins that bind to NLS peptides. The major NLS binding protein in animals, fungi, and plants is an -55-to 70-kDa protein that has been identified by chemical cross-linking and blot-overlay assays (4, 5). The activity of these proteins is necessary for the binding step of the import reaction as determined by specific antibody inhibition of binding in vitro (6) and by reconstitution of a partially fractionated import reaction based on permeabilized mammalian cells with addition of purified NLS-binding protein (7). Furthermore, these proteins are highly phosphorylated, and their phosphorylation is required for NLS binding in vitro (8).Another approach to understanding the import machinery has been to purify the cytosolic factors required for protein import in permeabilized cells. A protein required for targeting a nuclear import substrate to the nuclear envelope in vitro has recently been isolated and cloned by this strategy (9). This 60-kDa protein, importin, is absolutely required for the accumulation of import substrate on the nuclear envelope and functions as a NLS-binding protein in vitro (10,11). Importin was shown to be 44% identical to Srpl, a previously identified protein from Saccharomyces cerevisiae (12). Mutations in SRP1 have pleiotropic effects, including suppression of conditional mutations in RNA polymerase I (12), defects in nucleolar structure (13), and synthetic lethality with mutations in the nuclear pore component gene, NUP1 (14). Furthermore, Srpl is physically associated with Nupl and Nup2, a Nupl-related NPC c...

Abstract. The Saccharomyces cerevisiae gene, RNA1, encodes a protein with extensive homology to the mammalian Ran/TC4 GTPase activating protein. Using indirect immunofluorescence microscopy, we have demonstrated that rnal-1 mutant cells are defective in nuclear import of several proteins. The same result is obtained when nuclear import is examined in living cells using a nuclear protein fused to the naturally green fluorescent protein. These findings suggest a role for the Rnalp in trafficking of proteins across the nuclear membrane. To investigate this role more directly, an in vitro import assay that monitors the import of a fluorescently labeled substrate into the nuclei of semiintact yeast cells was used. Import to the nucleus requires the addition of exogenous cytosol. Results indicate that, in contrast to wild-type cytosols, extracts made from rnal-1 mutant cells are unable to support import of the fluorescently labeled substrate into competent nuclei. Immunoblotting demonstrates that these mutant-derived extracts are depleted of Rnalp. However, when purified Rnalp is added back to these extracts the import activity is restored in a dose-dependent manner. These results demonstrate that Rnalp plays a direct role in the import of proteins into the nucleus.

Abstract. Eukaryotic cells contain multiple Hsp70 proteins and DnaJ homologues. The partnership between a given Hsp70 and its interacting DnaJ could, in principle, be determined by their cellular colocalization or by specific protein-protein interactions. The yeast SCJ1 gene encodes one of several homologues of the bacterial chaperone DnaJ. We show that Scjlp is located in the lumen of the endoplasmic reticulum (ER), where it can function with Kar2p (the ERlumenal BiP/Hsp70 of yeast). The region common to all DnaJ homologues (termed the J domain) from Scjlp can be swapped for a similar region in Sec63p, which is known to interact with Kar2p in the ER lumen, to form a functional transmembrane protein component of the secretory machinery. Thus, Kar2p can interact with two different DnaJ proteins. On the other hand, J domains from two other non-ER DnaJs, Sislp and Mdjlp, do not function when swapped into Sec63p. However, only three amino acid changes in the Sislp J domain render the Sec63 fusion protein fully functional in the ER lumen. These results indicate that the choice of an Hsp70 partner by a given DnaJ homologue is specified by the J domain.

In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the ␥-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p inf luences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p͞Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.In the yeast Saccharomyces cerevisiae, microtubule-organizing functions are provided by the spindle pole body (SPB) (for review, see ref. 1). The SPB is a multilayered structure that duplicates once per cell cycle and is embedded in the nuclear envelope through the central plaque structure (ref. 2; see Fig. 6). The outer plaque is located on the cytoplasmic side of the SPB, whereas the inner plaque extends into the nucleoplasm. An additional SPB substructure is the half bridge, a one-sided extension of the central plaque.Some insight into the SPB duplication process came from electron microscopic studies and from the phenotype of conditional-lethal mutants. In early G 1 phase of the cell cycle, the half bridge elongates and develops an appendix toward the cytoplasmic side, which was named the satellite. The satellite is probably a precursor of the newly formed SPB. After start of the cell cycle, the SPB fully duplicates, resulting in two SPBs side by side (3). CDC31 (4, 5) and KAR1 (6, 7) code for components of the SPB half bridge required early in SPB duplication. In mps2-1 and ndc1-1 cells, a fully functional SPB is associated with a partially duplicated SPB containing an outer and a central plaque, sitting on top of the cytoplasmic side of the nuclear envelope (8, 9). The mps2/ndc1 phenotype suggests that the outer plaque and some part of the central plaque are built from cytoplasmic components, whereas the inner plaque is probably assembled in the nucleus. This view is further supported by the fact that SPB components of the inner plaque such as Spc110p (10) are imported into the nucleus, whereas the outer plaque component Spc72p is not (11). Finally, the cytoplasmic and nuclear assembled ...

Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin ␣ binds to the NLS and to importin ␤, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin ␤. The importin subunits are exported separately. We investigated the role of Cse1p, the Saccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin ␣). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin ␤ accumulated in nuclei of cse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.Transport of proteins and RNAs between the cytoplasm and the nucleus across the nuclear pore complex (NPC) is mediated by shuttling transport receptors. All transporters so far identified belong to the importin ␤ superfamily. Substrate binding and release of these transporters are modulated by the small GTPase Ran, the key regulator of nucleocytoplasmic transport. Several specific transport pathways for different import and export substrates have recently been identified (reviewed in references 52 and 73).The best understood transport pathway is the import of proteins carrying a classical nuclear localization sequence (NLS), which is characterized by a short segment of basic amino acid residues (20). The NLS is recognized by importin ␣ in the cytoplasm, which in turn binds to importin ␤. Importin ␤ mediates docking at the NPC and subsequent transport into the nucleus. Docking of NLS substrates to the nuclear envelope and subsequent energy-dependent transfer into the nucleus can be reconstituted with recombinant factors and permeabilized cells in vitro. Two soluble factors, Ran and p10, are sufficient to promote the translocation step (for reviews, see references 16, 32 and 50).Ran is an abundant, mostly nuclear protein that switches between two conformational states, i.e., bound to GDP (RanGDP) and to GTP (RanGTP). The specific regulators of the Ran GTPase cycle, the cytoplasmic GTPase-activating protein RanGAP1/Rna1p and the nuclear nucleotide exchange factor RCC1/Prp20p, generate RanGDP in the cytoplasm and RanGTP in the nucleus (reviewed in references 32 and 61). RanGTP binds to importin ␤, which is thereby released from the NLS-importin complex (...