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.

Tankyrase is an ankyrin repeat-containing poly(ADPribose) polymerase originally isolated as a binding partner for the telomeric protein TRF1, but recently identified as a mitogen-activated protein kinase substrate implicated in regulation of Golgi vesicle trafficking. In this study, a novel human tankyrase, designated tankyrase 2, was isolated in a yeast two-hybrid screen as a binding partner for the Src homology 2 domain-containing adaptor protein Grb14. Tankyrase 2 is a 130-kDa protein, which lacks the N-terminal histidine/proline/ serine-rich region of tankyrase, but contains a corresponding ankyrin repeat region, sterile ␣ motif module, and poly(ADP-ribose) polymerase homology domain. The TANKYRASE 2 gene localizes to chromosome 10q23.2 and is widely expressed, with mRNA transcripts particularly abundant in skeletal muscle and placenta. Upon subcellular fractionation, both Grb14 and tankyrase 2 associate with the low density microsome fraction, and association of these proteins in vivo can be detected by co-immunoprecipitation analysis. Deletion analyses implicate the N-terminal 110 amino acids of Grb14 and ankyrin repeats 10 -19 of tankyrase 2 in mediating this interaction. This study supports a role for the tankyrases in cytoplasmic signal transduction pathways and suggests that vesicle trafficking may be involved in the subcellular localization or signaling function of Grb14.

We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-␤, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-␤, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-␣-dependent import of proteins with a classical NLS.Ran/TC4 is a highly abundant, small GTP-binding and -hydrolyzing protein that is located predominantly in the nucleus (8,12). Ran has the biochemical properties of a GTPase switch cycling between two conformational states, the GTP-bound state and the GDP-bound state. The intrinsic rates of nucleotide exchange and GTP hydrolysis are very low and can be increased up to five orders of magnitude by the regulatory proteins RCC1 and RanGAP1, respectively (4,8,22). The first indications concerning the functions of Ran came from the analysis of mutants of these Ran regulators in both mammalian and yeast cells. These studies implicated Ran in a variety of processes, including the onset of mitosis, initiation of S phase, exit from mitosis, maintenance of nuclear structure, and premRNA processing and mRNA export into the cytoplasm (for reviews, see references 41 and 43).Furthermore, in vitro studies with permeabilized cells showed that Ran is an essential factor for the nuclear localization signal (NLS)-dependent nuclear protein import (30, 31). Macromolecular transport across the nuclear envelope occurs at the nuclear pore complexes (NPCs) and involves the import of proteins into the cell nucleus and the export of RNAs and proteins. Four soluble cytosolic factors are required to reconstitute nuclear import of NLS substrates in vitro. In addition to Ran, these are importin-␣ and importin-␤ (also known as karyopherin-␣ and karyopherin-␤) (17,19,36) and NTF2 (alternatively known as pp15 or p10) (32, 34). Together, importin-␣ and importin-␤ comprise the import receptor complex, where importin-␣ binds proteins bearing an NLS and importin-␤ mediates the interaction with the NPC. Ran appears to be required for at least two steps in nuclear import. First, translocation through the nuclear pore requires GTP hydrolysis by Ran, probably even as the sole source of energy (47). Second, the disassembly of the importin-␣/-␤-NLS protein complex following translocation ...