Ran-Binding Protein 5 (RanBP5) Is Related to the Nuclear Transport Factor Importin-β but Interacts Differently with RanBP1

Abstract

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 ...

Karyopherin β3: A new cellular target for the HPV-16 E5 oncoprotein

“…KNβ3 shows a typical cytoplasmic and perinuclear staining pattern [18,22] in both cell types (Fig. 3, A and B, left column), while 16E5 exhibits staining typical for ER-localized proteins in HECs and staining typical for ER-and Golgi-localized proteins in COS cells [10,11] (Fig.…”

“…These proteins regulate the nuclear import of various cargo, including ribosomal proteins, histones and viral proteins; however, they may also act as cytoplasmic chaperones and as microtubule motor adaptors [20]. KNβ3 localizes mainly to the cytoplasm, but also binds to some nucleoporins and can therefore be visualized in the nuclear envelope by immunostaining [18,22].…”

Random Mutagenesis and Functional Analysis of the Ran-binding Protein, RanBP1

“…Only in the presence of RanBP1, or the RanBDs of Nup358 or importin-␣, can RanGAP catalyze GTP hydrolysis and release of importin-␣ (11,23). Likewise, RanBP1 is able to promote the release of Yrb4 or importin-5 from Ran⅐GTP (44,46). Similarly, RanBP1 can release the exportin, Cse1 (and CAS, its mammalian homolog) from nuclear pores, Ran⅐GTP and its cargo, importin-␣ (18,48,53).…”

Importin α binds to an unusual bipartite nuclear localization signal in the heterogeneous ribonucleoprotein type I

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