A domain unique to plant RanGAP is responsible for its targeting to the plant nuclear rim.
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.
Rose
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A
Plant Biotechnology Center and Department of Plant Biology, Ohio State University, Columbus, OH 43210, USA.
Meier
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I
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009685:Nuclear Envelope / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D020931:ran GTP-Binding Protein