The Nup358-RanGAP Complex Is Required for Efficient Importin α/β-dependent Nuclear Import

Abstract

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin ␣/␤-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin ␤, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin ␤ strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin ␤ recycling and reformation of novel import complexes. INTRODUCTIONThe giant nucleoporin Nup358/RanBP2 (Wu et al., 1995;Yokoyama et al., 1995) is a major component of the filaments that emanate from the cytoplasmic ring of the nuclear pore complex (NPC) into the cytoplasm (Walther et al., 2002). As many other nucleoporins, Nup358 interacts via phenylalanine-glycine repeats (FG repeats) with karyopherins, transport receptors that mediate import and export across the NPC (for review see Tran and Wente, 2006). In recent nuclear transport models, a hydrophobic milieu that is established by FG repeats derived from several nucleoporins is suggested to allow selective translocation of karyopherins, with or without cargo molecules, across the NPC (for review see Weis, 2007). Individual nucleoporins do not play a major role in these models. Nevertheless, several nucleoporins have been shown to affect specific transport pathways. Nup153, for example, interacts with various import receptors, regulating late steps in nuclear import (Shah and Forbes, 1998). Very recently, specific FG nucleoporins were shown to be required for mRNA export (Terry and Wente, 2007). In this study, we investigate the role of Nup358 in nuclear protein transport in detail.Ran is a small GTP-binding protein that binds to karyopherins and plays an essential role in the majority of nucleocytoplasmic transport pathways. RanGTP is generated in the nucleus, resulting from the activity of the chromatinbound guanosine nucleotide exchange factor RCC1. In the cytoplasm, vertebrate RanGAP1 (RanGAP for short), together with the RanGTP-bind...

Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment

“…One can imagine importin-␤ recognizing and binding to incoming capsids via VP1/2's nuclear localization sequence or another region on the capsid and thus carrying the capsid cargo to the nucleus. Further, Nup358 has recently been shown to play an important role in importin ␣/␤-dependent nuclear import (24). Once at the nucleus, capsid-bound importin-␤ could interact with Nup358 to anchor capsids to the NPC.…”

Nuclear pore complex tethers to the cytoskeleton

“…Interestingly, SUMO isopeptidases, such as SENP2, which cleave SUMO from the substrate are located at the nucleoplasmic face of the NPC (Zhang et al, 2002), suggesting a role for SUMOylation in the directionality of nucleo-cytoplasmic transport. Therefore, Nup358 is important for the translocation of molecules through the NPC (Singh et al, 1999;Hutten et al, 2008;Wälde et al, 2012;Hamada et al, 2011;Forler et al, 2004;Mahadevan et al, 2013).…”

RANBP2 (RAN binding protein 2)

“…RANBP2 itself does not directly participate in import, but facilitates it. In RANBP2-depleted HeLa cells, in vivo nuclear import by either Importin alpha/beta (Hutten et al, 2008) or transportin (Hutten et al, 2009) still occurs, but at substantially reduced rates. In a screening for nuclear proteins that accumulate in the cytoplasm upon RANBP2 depletion, Wälde and coworkers (2012) have also identified direct RANBP2 interactors: they found that an N-terminal fragment of RANBP2, harboring the NPC-binding domain, three FG motifs and RBD1, was sufficient to promote protein import, while neither the interaction with RANGAP1 nor the SUMO E3 ligase activity were required (Wälde et al, 2012).…”