CHARACTERIZATION OF PROTEINS THAT INTERACT WITH THE GTP-BOUND FORM OFTHE REGULATORY GTPASE RAN IN ARABIDOPSIS

Ran, a small soluble GTP-binding protein, has been shown to be essenti al for the nuclear translocation of proteins and it is also thought to be involved in regulating cell cycle progression in mammalian and yea st cells. Genes encoding Ran-like proteins have been isolated from dif ferent higher plant species. Overexpression of plant Ran cDNAs, simila rly to their mammalian/yeast homologues, suppresses the phenotype of t he pim46-1 cell cycle mutant in yeast cells. The mammalian/yeast Ran p roteins have been shown to interact with a battery of Ran-binding prot eins, including the guanidine nucleotide exchange factor RCC1, the GTP ase-activating Ran-GAP, nucleoporins and other Ran-binding proteins (R anBPs) specific for Ran-GTP. Here, the characterization of the first R an-binding proteins from higher plants is reported. The yeast two-hybr id system was used to isolate cDNA clones encoding proteins of approxi mately 28 kDa (At-RanBP1a, At-RanBP1b) that interact with the GTP-boun d forms of the Ran1, Ran2 and Ran3 proteins of Arabidopsis thaliana. T he deduced amino acid sequences of the At-RanBP1s display high similar ity (60%) to mammalian/yeast RanBP1 proteins and contain the character istic Ran-binding domains. Furthermore, interaction of the plant Ran a nd RanBP1 proteins, is shown to require the acidic C-terminal domain ( -DEDDDL) of Ran proteins in addition to the presence of an intact Ran- binding domain. In whole cell extracts, the GST-RanBP1a fusion protein binds specifically to GTP-Ran and will not interact with Rab/Ypt-type small GTP-binding proteins. Finally, in good agreement with their pro posed biological function, the At-Ran and the At-RanBP genes are expre ssed coordinately and show the highest level of expression in meristem atic tissues.