A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim

Abstract

Ran is a small signaling GTPase that is involved in nucleocytoplasmic transport. Two additional functions of animal Ran in the formation of spindle asters and the reassembly of the nuclear envelope in mitotic cells have been recently reported. In contrast to Ras or Rho, Ran is not associated with membranes. Instead, the spatial sequestering of its accessory proteins, the Ran GTPase-activating protein RanGAP and the nucleotide exchange factor RCC1, appears to define the local concentration of RanGTP vs. RanGDP involved in signaling. Mammalian RanGAP is bound to the nuclear pore by a mechanism involving the attachment of small ubiquitin-related modifier protein (SUMO) to its C terminus and the subsequent binding of the SUMOylated domain to the nucleoporin Nup358. Here we show that plant RanGAP utilizes a different mechanism for nuclear envelope association, involving a novel targeting domain that appears to be unique to plants. The N-terminal WPP domain is highly conserved among plant RanGAPs and the small, plant-specific nuclear envelope-associated protein MAF1, but not present in yeast or animal RanGAP. Confocal laser scanning microscopy of green fluorescent protein (GFP) fusion proteins showed that it is necessary for RanGAP targeting and sufficient to target the heterologous protein GFP to the plant nuclear rim. The highly conserved tryptophan and proline residues of the WPP motif are necessary for its function. The 110-aa WPP domain is the first nuclear-envelope targeting domain identified in plants. Its fundamental difference to its mammalian counterpart implies that different mechanisms have evolved in plants and animals to anchor RanGAP at the nuclear surface.

Figure 1

Plant RanGAPs contain a unique N-terminal domain. (A) Schematic comparison of the domain structure of yeast, vertebrate, and plant RanGAPs. LRR, red; acidic domain, yellow; SUMO-attachment domain (SUMO), green; MAF1-like WPP domain, cyan; and unique C-terminal domain of OsRanGAP, blue. The S. pombe (SpRna1p), S. cerevisiae (ScRna1p), Homo sapiens (HsRanGAP1), Mus musculus (MsRanGAP1), and Xenopus laevis (XlRanGAP1) RanGAPs are grouped. The plant sequences are derived from A. thaliana (AtRanGAP1 and AtRanGAP2), M. sativa (MsRanGAP), and rice (OsRanGAP). Arabidopsis MAF1 (AtMAF1) is included for size comparison. The number of total residues for each protein is indicated. (B) Graphic depiction of the consensus sequence of the WPP domain derived from an alignment of the N-terminal 120 aa of AtRanGAP1, AtRanGAP2, MsRanGAP, OsRanGAP, and full-length MAF1 from Arabidopsis (AtMAF1), tomato (LeMAF1), soybean (GmMAF1), maize (ZmMAF1), and Canna edulis (CeMAF1; ref. 19). Amino acid residues of the consensus are indicated in one letter code. Bar colors represent consensus strength: red, nine of nine; orange, eight of nine; green, six or seven of nine; light blue, four or five of nine; dark blue, two or three of nine. Dashes, no consensus, including gaps in the alignment.

Figure 2

Plant RanGAPs share a core domain with the animal and yeast proteins. (A) Amino acid-sequence alignment of human, yeast, and plant RanGAPs after trimming the kingdom-specific domains. Amino acid identities and similarities in at least five sequences are highlighted in black and gray, respectively. The arginine residue necessary for RanGAP activity is marked by a red asterisk; the acidic domains are underlined in red. (B) Molecular modeling of AtRanGAP1 and OsRanGAP onto the crystal structure of SpRna1p. Bar diagrams show the domains in the same color code as in Fig. 1A. The first and last amino acids represented in the structure are indicated above the bars. (B) Ribbon representation of the crystal structure of SpRna1p (Center), the predicted structure of amino acid 116 to 447 of AtRanGAP1 (Left), and of amino acid 122 to 453 of OsRanGAP (Right). In SpRna1p, α helices are labeled in blue and β sheets in yellow. In the two predicted structures, successful fit onto the SpRna1p structure is indicated in green, nonfitting areas in red.

Figure 3

AtRanGAP1 is located at the nuclear envelope. (A) Expression cassettes for transient transformation of AtRanGAP1-GFP and LeMAF1-GFP-LeMAF1 fusions. The LeMAF1-GFP-LeMAF1 sandwich expression cassette, designed to prevent passive diffusion into the nucleus, has been described previously (20). 35S P, cauliflower mosaic virus 35S promoter; L, tobacco etch virus translational leader; GFP, mGFPS65T from pRTL2-mGFPS65T (25); T, 35S terminator. (B–E) GFP fluorescence of BY-2 cells transiently transformed with AtRanGAP1-GFP (B and C) and LeMAF1-GFP-LeMAF1 (D and E). In B and D, SYTO 82 orange was used to counterstain for DNA, labeling the nucleus, as well as mitochondria and plastids in the cytoplasm. (In B–E, bars equal 10 μm.) See Movie 1, which is published as supporting information on the PNAS web site, www.pnas.org. Sequence 1 shows a scan through a cell expressing AtRanGAP1-GFP, sequence 2 a scan through a cell expressing LeMAF1-GFP-LeMAF1.

Figure 4

The WPP domain is necessary and sufficient for nuclear-envelope targeting of AtRanGAP1. (A) Fusion constructs in pRTL2-mGFPS65T. Color code of AtRanGAP1 domains is as in Fig. 1. (B) GFP fluorescence of BY-2 cells transiently transformed with AtRanGAP1-GFP (1), AtRanGAP1ΔN-GFP (2), and AtRanGAP1ΔC-GFP (3). (Left) GFP fluorescence. (Right) Transmitted light images of cells. (Bars equal 10 μm.)

Figure 5

Site-directed mutagenesis of the WPP motif. (A) Constructs in pRTL2-mGFPS65T used for transient transformation of BY-2 cells. The wild-type sequence of the WPP motif (see Fig. 1B) and the introduced base pair and amino acid changes are indicated above the bar diagram of AtRanGAP1mut-GFP. The asterisk indicates the position of the identical mutation in AtRanGAP1ΔCmut-GFP. Color code of the AtRanGAP1 domains is as in Fig. 1. (B) GFP fluorescence of BY-2 cells transiently transformed with the constructs shown in A. (Left) GFP fluorescence. (Right) Transmitted light images. (Bar equals 10 μm.)