We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.Transport through nuclear pores requires concerted action between the structural components of the nuclear pore complex (NPC) and the soluble transport factors that bind to the transport substrates and shuttle between the nuclear and cytoplasmic compartments (for reviews, see references 2 and 31). Substantial progress toward an understanding of nuclear protein import has been achieved in the past few years, but very little is known about how RNA is exported from the nucleus into the cytoplasm. Among the factors required for nuclear protein import are the classical nuclear localization signalreceptor complex, consisting of importin/karyopherin ␣ and ␤, the small GTPase Ran, and several Ran-binding proteins, as well as repeat sequences containing nucleoporins (for reviews, see references 5 and 11). Recently, additional routes of import into the nucleus were discovered, suggesting that major classes of transport substrates use different import pathways. Transportin and Kap123p were identified as novel transport factors that bind directly to their transport substrates, hnRNP protein A1 and ribosomal protein L25, respectively (30, 38). Transportin and Kap123p belong to a growing family of importin ␤-like proteins which have a Ran GTP-binding domain in their amino-terminal portions (5, 10). Recently, Mtr10p, which is also a member of this protein family, was shown to be the importin for yeast Np13p (34,41). An essential role for Ran in energydependent nuclear protein import has been firmly established, but how Ran and the many Ran activity-modulating proteins participate in the actual translocation process is still controversial.The Ran system is also involved in transport from the nucleus (9,14,18,36). It has been firmly established that nuclear export sequences (NES), first identified in viral proteins such as human immunodeficiency virus Rev and protein kinase inhibitor, mediate the exit of proteins from the nucleus (for a review, see reference 8). For the Rev protein, which is an RNA-binding protein with a specificity for unspliced or partially spliced viral transcripts, viral mRNA is coexported through association with Rev (3). Initially, it was found that Rev NES interact with Rip (6,47), which resembles repeat sequence-containing nucleoporins and accordingly was suggested to be a NES receptor at the nuclear pores. Recen...

Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-␤-like proteins on the basis of its sequence similarity. In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p. Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p. By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran. Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways. Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay. These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.In eukaryotic cells, all transport between the nuclear interior and the cytoplasm occurs through the nuclear pore complexes (NPCs) (reviewed in reference 16). According to the data that have accumulated during the last few years, proteins destined to enter the nucleus associate in the cytoplasm with receptors that recognize and bind specific sequences, termed nuclear localization signals (NLSs). These complexes are targeted to the NPC and are translocated into the nucleoplasm, where the import cargo is released and the receptor is recycled to the cytoplasm (reviewed in references 13, 31, 33, 65, and 68). In the case of the basic-type (classical) NLS, the receptor consists of importin ␣ (karyopherin ␣), the NLS-binding component, and importin ␤ (karyopherin ␤), which can interact with repeatcontaining nucleoporins and is responsible for docking to the NPC. Importin ␤ belongs to a large protein family whose members are characterized by the presence of an amino-terminally located Ran-GTP binding domain (23,32). Other members of this family include transportin and Kap123p (Yrb4p), which respectively directly bind to some hnRNP proteins and ribosomal proteins, and mediate their nuclear import (24,72,79,83,96). Similar functions have also been proposed for their homologues Kap104p (1) and karyopherin ␤3 (105). Recently two more importin ␤ homologues, Mtr10p and Sxm1p, have been shown to function as import receptors for Npl3p (a yeast hnRNP protein) and Lhp1p (the yeast La homologue), respectively (71,78,86).The principles of active nuclear protein import may also apply to active nuclear export of proteins and RNA. Indeed, two members of the importin ␤ family have been shown to be involved in nuclear export processes and were therefore termed exportins (reviewed in reference 102). Export of importin ␣ from the nucleus is mediated by CAS (57), while CRM1 functions as an export receptor for the leucine-rich nuclear export signal (NES) (22,26,56,67,69,98). This type of NES can mediate n...

Los1p and Pus1p, which are involved in tRNA biogenesis, were found in a genetic screen for components interacting with the nuclear pore protein Nsp1p. LOS1, PUS1 and NSP1 interact functionally, since the combination of mutations in the three genes causes synthetic lethality. Pus1p is an intranuclear protein which exhibits a nucleotide‐specific and intron‐dependent tRNA pseudouridine synthase activity. Los1p was shown previously to be required for efficient pre‐tRNA splicing; we report here that Los1p localizes to the nuclear pores and is linked functionally to several components of the tRNA biogenesis machinery including Pus1p and Tfc4p. When the formation of functional tRNA was analyzed by an in vivo assay, the los1(‐) pus1(‐) double mutant, as well as several thermosensitive nucleoporin mutants including nsp1, nup116, nup133 and nup85, exhibited loss of suppressor tRNA activity even at permissive temperatures. These data suggest that nuclear pore proteins are required for the biogenesis of functional tRNA.

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1␣, involves several post-translational modifications. To study HIF-1␣ phosphorylation, we have used human full-length recombinant HIF-1␣ as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1␣. Analysis of in vitro phosphorylated HIF-1␣ by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1␣. When these mutant forms of HIF-1␣ were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1␣ mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1␣. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1␣ mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1␣. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1␣ by blocking its CRM1-dependent nuclear export.

MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive Cterminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA.