Identification and Characterization of the Arabidopsis Orthologs of Nuclear Transport Factor 2, the Nuclear Import Factor of Ran

Abstract

Ran is a multifunctional small GTPase that is involved in nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. Nuclear import of Ran relies on a small RanGDP-binding protein, Nuclear Transport Factor 2 (NTF2). Three proteins are expressed in Arabidopsis (Arabidopsis thaliana) that show significant sequence similarity to human and yeast (Saccharomyces cerevisiae) NTF2. Here, we demonstrate that two of them, AtNTF2a and AtNTF2b, can functionally replace the essential NTF2 gene in yeast. Consistent with this finding, both AtNTF2a and AtNTF2b interact with yeast and Arabidopsis Ran. The third NTF2-related protein, AtNTL, does not functionally replace NTF2 in yeast. Similar to yeast NTF2-green fluorescent protein (GFP), AtNTF2a-GFP and AtNTF2b-GFP accumulate at the nuclear rim. The AtNTF2a E38K and E91K mutants, which fail to bind Ran, are not functional in yeast, indicating conservation of the requirement for these key amino acids in plants and yeast. AtNTF2a overexpression, but not AtNTF2aE38K overexpression, blocks nuclear import of a plant transcription factor in Nicotiana benthamiana leaves, indicating that excess AtNTF2a disrupts nuclear import in a Ran-bindingdependent manner. On the basis of these results, we propose that AtNTF2a and AtNTF2b function in Ran import in Arabidopsis and that nuclear import of Ran is functionally conserved in plants.

RanGAP2 Mediates Nucleocytoplasmic Partitioning of the NB-LRR Immune Receptor Rx in the Solanaceae, Thereby Dictating Rx Function

“…NTF2A specifically imports Ran-GDP into the nucleus, which is essential for proper functioning of the nucleocytoplasmic trafficking machinery (Merkle, 2003). NTF2A overexpression in N. benthamiana was previously shown to block nuclear import of R-GFP (Zhao et al, 2006). Indeed, NUK6-GFP primarily localized to the cytoplasm when coexpressed with NTF2A, whereas coexpression with the loss-of-function mutant NTF2A(E38K) (Zhao et al, 2006) did not have this effect ( Figure 4A, top panel).…”

“…For this we used the maize (Zea mays) transcription factor R (Shieh et al, 1993;Zhao et al, 2006) and the Phytophthora infestans effector NUK6, fused to GFP (Kanneganti et al, 2007). Both proteins contain NLS motifs and are imported via the classical nuclear import mechanism (Meier, 2007).…”

Two Distinct Interacting Classes of Nuclear Envelope–Associated Coiled-Coil Proteins Are Required for the Tissue-Specific Nuclear Envelope Targeting ofArabidopsisRanGAP

“…The Agrobacterium strains transformed with GFP-WIP1, GFP-WIP2a GFP-WIP3, RanGAP1-GFP, RanGAP1(WPP/AAP), and GFP-RanGAP2 were described previously (Jeong et al, 2005;Xu et al, 2007). Agrobacterium cultures containing different plasmids were either infiltrated or coinfiltrated transiently into N. benthamiana leaves as described previously (Zhao et al, 2006).…”

The Plant TPX2 Protein Regulates Prospindle Assembly before Nuclear Envelope Breakdown

“…Plants have a Ran GTPase and most factors that associate with it (Jeong et al, 2005;Zhao et al, 2006;Meier, 2007). Three Arabidopsis thaliana Aurora-like kinases (At AURORA1 to -3) were picked up in a large-scale green fluorescent protein (GFP)-tagging approach (van Damme et al, 2004;Demidov et al, 2005) and independently by homology with the animal and yeast Aurora kinases (Kawabe et al, 2005).…”