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-binding-dependent 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.

Figure 1.

The Arabidopsis genome encodes three NTF2-like proteins. A, Alignment of the three Arabidopsis NTF2-like proteins (AtNTF2a, AtNTF2b, and AtNTL) with human NTF2 (hNTF2) and yeast NTF2 (yNTF2) is shown. Black shading indicates amino acids that match the majority (at least three out of five). Gray shading indicates amino acids similar to the majority. Asterisks indicate the position of two highly conserved Glu residues (E38 and E91 in Arabidopsis). B, Phylogenetic tree of the alignment shown in A. C, Percentage of similarity between the three Arabidopsis NTF2-like proteins and human and yeast NTF2.

Figure 2.

AtNTF2a and AtNTF2b, but not AtNTL, can functionally replace yeast NTF2. Yeast cells deleted for the genomic copy of the essential NTF2 gene but maintained by an NTF2 genomic plasmid (see “Materials and Methods”) were transformed with plasmids expressing AtNTF2a, AtNTF2b, or AtNTL under the control of a Gal-inducible promoter. Cultures were grown to saturation, serially diluted, and spotted on control (left) or 5-FOA (right) plates to eliminate the NTF2 maintenance plasmid. The control plate indicates that similar numbers of cells were spotted for each sample. The 5-FOA plate, where each of the indicated plasmids constitutes the sole source of NTF2, shows that both AtNTF2a and AtNTF2b can function in vivo, as cells expressing these proteins grow as well as the control cells that express yNTF2. In contrast, AtNTL cannot function in place of yeast NTF2, as cells that express this plasmid show no growth (similar to the vector-only control).

Figure 3.

AtNTF2a and AtNTF2b bind to Arabidopsis, human, and yeast Ran. A yeast two-hybrid assay was used to examine the interaction between AtNTF2a, AtNTF2b, and Ran. Each of the AtNTF proteins was expressed as an activation domain fusion as described in “Materials and Methods.” Ran proteins were expressed as DNA-binding domain fusion proteins. Interactions with Arabidopsis (AtRAN1), human (hRAN), and yeast (yRAN) Ran were examined for AtNTF2a (top) and AtNTF2b (bottom). As a negative control, interaction with vector alone was also examined. In this two-hybrid system, activation domain fusion proteins are expressed under a Gal-inducible promoter; thus, interactions should only occur in the presence of Gal. As indicated by the blue color indicative of β-galactosidase expression, both AtNTF2a and AtNTF2b interact with Ran from all species tested.

Figure 4.

E38K and E91K mutations in AtNTF2a disrupt Ran binding and AtNTF2a function in vivo. A, Yeast two hybrid was used to assay the interaction between wild-type and mutant AtNTF2a and Arabidopsis Ran (AtRAN1) or a vector control (Vec). Neither AtNTF2aE38K nor AtNTF2aE91K interacted with AtRAN, as indicated by loss of the blue color indicative of β-galactosidase expression in these spots. As controls, wild-type AtNTF2a and AtNTF2b both interacted with AtRAN in this experiment. No interaction was observed with the vector control. B, AtNTF2a mutants interact with AtNTF2b. The interaction of AtNTF2b with itself and with both wild-type and mutant AtNTF2a was examined by two-hybrid assay. The blue color indicative of β-galactosidase expression reveals that AtNTF2b interacts with all versions of NTF1/2 tested, indicating that the mutant AtNTF2a proteins are expressed and capable of interacting in the two-hybrid assay. C, The function of AtNTF2aE38K and AtNTF2aE91K was examined by a plasmid shuffle assay. Yeast cells deleted for the genomic copy of the essential NTF2 gene but maintained by an NTF2 genomic plasmid (see “Materials and Methods”) were transformed with plasmids expressing AtNTF2a, AtNTF2aE38K, AtNTF2aE91K, or as controls, vector, yNTF2, or AtNTF2b under the control of a Gal-inducible promoter. Cultures were grown to saturation, serially diluted, and spotted on control (left) or 5-FOA (right) plates to eliminate the NTF2 maintenance plasmid. Only wild-type AtNTF2a and AtNTF2b supported cell growth, as did the control (yNTF2).

Figure 5.

AtNTF2a and AtNTF2b are ubiquitously expressed in Arabidopsis. RT-PCR was performed on total RNA isolated from flowers, leaves, roots, stems, and siliques of 30-d-old Arabidopsis plants. As a control, the amount of Actin-related protein 6 (At3g33520; actin) is also shown for each sample.

Figure 6.

AtNTF2a and AtNTF2b accumulate at the nuclear envelope. AtNTF2a-GFP (A and B), AtNTF2b-GFP (C and D), and free GFP (E and F) were transiently expressed in tobacco BY-2 protoplasts, and GFP fluorescence was imaged by confocal laser scanning microscopy. A, C, and E, GFP fluorescence; B, D, and F, transmitted light images of protoplasts. Cellular compartments are indicated in E: n, nucleus; no, nucleolus; c, cytoplasm; and v, vacuole. Size bars = 10 μm.

Figure 7.

Overexpression of AtNTF2a in plants disrupts nuclear import in a Ran-binding-dependent manner. A, GFP fluorescence of an R-GFP fusion protein expressed in N. benthamiana leaf epidermis cells; B, corresponding transmitted light image; C, GFP fluorescence of R-GFP coexpressed with AtNTF2a; D, corresponding transmitted light image; E, GFP fluorescence of R-GFP coexpressed with AtNTF2aE38K; and F, corresponding transmitted light image. Size bars = 10 μm.