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A Domain Unique to Plant Rangap Is Responsible for Its Targeting to the Plant Nuclear Rim

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A Domain Unique to Plant Rangap Is Responsible for Its Targeting to the Plant Nuclear Rim A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim Annkatrin Rose and Iris Meier* Plant Biotechnology Center and Department of Plant Biology, Ohio State University, Columbus, OH 43210 Edited by Maarten J. Chrispeels, University of California at San Diego, La Jolla, CA, and approved October 24, 2001 (received for review August 30, 2001) Ran is a small signaling GTPase that is involved in nucleocytoplas- chromatin (4). Vertebrate RanGAP contains a C-terminal mic transport. Two additional functions of animal Ran in the Nup358-binding domain. Modification of this domain by the formation of spindle asters and the reassembly of the nuclear small ubiquitin-like protein SUMO causes a conformational envelope in mitotic cells have been recently reported. In contrast change of RanGAP that allows Nup358 binding (5). The to Ras or Rho, Ran is not associated with membranes. Instead, the Nup358-binding domain is not present in Rna1p, the Saccharo- spatial sequestering of its accessory proteins, the Ran GTPase- myces cerevisiae and Schizosaccharomyces pombe RanGAP ho- activating protein RanGAP and the nucleotide exchange factor molog (6), which appears to be localized in the cytoplasm (7, 8). RCC1, appears to define the local concentration of RanGTP vs. Besides the well-established role of Ran in animal and yeast RanGDP involved in signaling. Mammalian RanGAP is bound to the nucleocytoplasmic transport, several exciting recent findings nuclear pore by a mechanism involving the attachment of small point toward a wider function of Ran in cellular signaling in ubiquitin-related modifier protein (SUMO) to its C terminus and the animals (9, 10). RanGTP can induce microtubule self- subsequent binding of the SUMOylated domain to the nucleoporin organization and spindle assembly by releasing microtubule- Nup358. Here we show that plant RanGAP utilizes a different assembly factors from inhibition by importin ␣ and ␤ (3, 11–13). mechanism for nuclear envelope association, involving a novel At the end of mitosis, GTP hydrolysis by Ran and a high local targeting domain that appears to be unique to plants. The N- concentration of RanGDP are required for the association of terminal WPP domain is highly conserved among plant RanGAPs nuclear envelope material with the decondensing chromatin and the small, plant-specific nuclear envelope-associated protein (14, 15). MAF1, but not present in yeast or animal RanGAP. Confocal laser Ran has been identified in plants and has been shown to scanning microscopy of green fluorescent protein (GFP) fusion complement the respective S. pombe mutant (16, 17). Plant proteins showed that it is necessary for RanGAP targeting and Ran-binding proteins with high similarity to mammalian͞yeast sufficient to target the heterologous protein GFP to the plant RanBP1 have been identified (18). No plant homolog for RCC1 nuclear rim. The highly conserved tryptophan and proline residues is presently known, but several sequences for putative plant of the WPP motif are necessary for its function. The 110-aa WPP RanGAPs have been found. At present, nothing is known about domain is the first nuclear-envelope targeting domain identified in the potential additional functions of Ran and its access- plants. Its fundamental difference to its mammalian counterpart ory proteins in plant spindle formation and nuclear envelope implies that different mechanisms have evolved in plants and assembly. animals to anchor RanGAP at the nuclear surface. We have shown previously that plant RanGAP sequences contain a unique N-terminal domain (WPP domain) not present n emerging theme in signal transduction research is how the in yeast or animal RanGAPs (19). The WPP domain shows Adifferent pathways of signaling events are both separated strong similarity to the small nuclear envelope-associated pro- and coordinated in a temporal and spatial manner in the living tein MAF1, which also appears to be unique to plants (20). Here, cell. It is becoming increasingly evident that discrete spatial we demonstrate that Arabidopsis RanGAP1 is localized at the positioning within the cell is a major aspect of this coordination. nuclear envelope of plant interphase cells, similar to mammalian However, how this positioning is achieved for individual signal- RanGAP and different from yeast Rna1p. We show that the ing molecules remains a fundamental question of molecular cell WPP domain constitutes a targeting domain that is necessary biology. and sufficient for anchoring AtRanGAP1 to the nuclear enve- The small GTP-binding protein Ran is required for the lope and that the WPP motif is involved in its function. These trafficking of proteins and RNA in and out of the nucleus (1). findings indicate that the mechanism for subcellular anchoring of It forms a complex with nuclear transport receptors and their RanGAP differs fundamentally between plants and animals. cargoes and is involved in their directional passage through the nuclear pores. Like all small GTP-binding proteins, RanGTP has Materials and Methods a very low intrinsic GTPase activity that requires stimulation by Sequence Comparison and Structural Modeling. GenBank and the Ran GTPase activating protein (RanGAP) and its accessory Arabidopsis genome database were accessed through the Na- factor RanBP1 (2). Replacement of GDP from RanGDP with tional Center for Biotechnology Information (http:͞͞ GTP is accomplished by the Ran guanine nucleotide exchange www.ncbi.nlm.nih.gov͞) and The Arabidopsis Information Re- factor RCC1, which closes the cycle. RanGTP and RanGDP source (http:͞͞www.arabidopsis.org͞), respectively. Sequence have different roles in nuclear transport, and their respective abundance is regulated by a spatial separation of RanGAP and RanBP1 outside and RCC1 inside the nucleus (1). The nucle- This paper was submitted directly (Track II) to the PNAS office. otide binding state of Ran thus serves as a marker for compart- Abbreviations: GFP, green fluorescent protein; RanGAP, Ran GTPase activating protein; ment identity. In contrast to other small signaling GTPases like LRR, leucine-rich repeat; SUMO, small ubiquitin-related modifier protein; RT, reverse Ras and Rho, Ran itself is not membrane bound. It is unique in transcription. that the sequestering of its accessory proteins provides the *To whom reprint requests should be addressed at: Plant Biotechnology Center and Department of Plant Biology, Ohio State University, 210 Rightmire Hall, 1060 Carmack spatial information for its respective activities (3). Road, Columbus, OH 43210. E-mail: [email protected]. In animal cells, RanGAP is anchored to the outer basket of the The publication costs of this article were defrayed in part by page charge payment. This nuclear pore by interaction with the nucleoporin Nup358, article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. PLANT BIOLOGY whereas RCC1 is sequestered in the nucleus through binding to §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.261459698 PNAS ͉ December 18, 2001 ͉ vol. 98 ͉ no. 26 ͉ 15377–15382 similarity searches were performed by using BLAST (21). Multiple were screened by using the destruction of a MscI site in the sequence alignments were performed with the DNASTAR protein WPP motive, and the RanGAP inserts were sequenced for alignment protocol (DNASTAR, Madison, WI) by using the confirmation. CLUSTAL algorithm. Parameters for CLUSTAL alignments were as follows. Pairwise alignment was: Ktuple, 1; gap Penalty, 3; Ballistic Transient Transformation of Tobacco BY-2 Cells. Tobacco windows, 5; and diagonals saved, 5. Multiple alignment was: gap BY-2 cells were cultured in Murashige and Skoog medium (MS penalty, 10; and gap length penalty, 10. The PAM250 weight salts; Life Technologies, Gaithersburg, MD) supplemented with ͞ ͞ table was used. 0.3% (wt vol) sucrose, 1.87 mM KH2PO4, 100 mg liter myo- Structural fitting data were acquired by accessing Swiss-Model inositol, 1 mg͞liter thiamine, and 0.2 mg͞liter 2,4-D, with a final through the interfaced SWISS PDB VIEWER (version 3.7b2) soft- pH of 5.0 adjusted with KOH. Cells were maintained by shaking ware (22, 23). The Ϸ10,000 protein structures deposited in the at 200 rpm in constant light at 24°C and subcultured weekly by Protein Data Bank (PDB) were searched through Swiss-Model 1:50 dilution. Transient transformation of BY-2 cells was per- for a template to model AtRanGAP1 and OsRanGAP with the formed essentially as described for NT-1 cells (20). For DNA full-length amino acid sequences of the two proteins. The staining, cells were incubated with SYTO 82 orange fluorescent structure files 1YRGA and 1YRGB, representing the crystal nucleic acid stain (Molecular Probes) at a final concentration of structures of the two identical subunits of the SpRna1p crystal 500 nM for 45 min at room temperature before microscopic dimer, were identified with both sequences. Sequence identity of imaging. the regions identified for threading was 28.3% for AtRanGAP1 and 27.7% for OsRanGAP. 1YRGB was used for modeling. Confocal Laser Scanning Microscopy. Digitized confocal images Preliminary threading was performed with MAGIC FIT in SWISS were acquired at 1024 ϫ 1024 pixel resolution with a Nikon Plan PDB VIEWER, and the models were subsequently optimized on the Fluor ϫ40͞0.75 air objective (1 pixel ϭ 0.3 ␮m) or Nikon Plan Swiss-Model server (22, 23). The quality of the received models Fluor ϫ100͞1.30 oil objective (1 pixel ϭ 0.12 ␮m) on a PCM was confirmed with the WHAT࿝CHECK verification routine (24). 2000͞Nikon Eclipse E600 confocal laser scanning microscope Of the 48 parameters checked by the program, all parameters (Nikon Bioscience Confocal Systems, Melville, NY). For the with impact on modeling by homology scored acceptable. Im- detection of GFP in the green channel, the 488-nm excitation portantly, the Ramachandran Z score (measure for backbone line of an Argon laser was used in combination with a 515͞30-nm structure) and the ␹-1͞␹-2 Z score (measure for side-chain bandpass emission filter (EM515͞30HQ).
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