Molecular, biochemical, and histochemical characterization of a plant Ran/TC4-like protein and its binding proteins.

摘要

Ran/TC4 (Ras-related nuclear protein) is the only known member of the family of small GTP-binding proteins primarily localized inside of the nucleus. In an effort to understand the functions of plant Ran, I cloned a cDNA encoding a pea Ran (PsRan1) and two Arabidopsis PsRan1-binding proteins (AtRanBP1b,-1c), characterized the differential expression of PsRan1 and the role of AtRanBP1c in modulating the GTP-bound state of PsRan1, examined the role of the regions flanking the N- and C-terminal sides of the Ran-binding domain (RanBD) of AtRanBP1c in determining the cytoplasmic localization of the protein, and analyzed the phenotypes of transgenic Arabidopsis expressing antisense-orientation of AtRanBP1c. PsRan1 is a member of a multigene family, although there is only one specific gene encoding PsRan1. The gene is expressed in all pea tissues, with a relatively high expression in radicles. The mRNA expression of psRan1 was not affected by red light treatment, but there was an increased abundance of multiple pea Ran mRNAs in response to very low fluences of red light. AtRanBP1c is highly homologous in its amino acid sequence to other known plant Ran-binding proteins (RanBPs) and has a conserved RanBD motif. However, it also has a sequence at its C-terminus that distinguishes it from other RanBPs. AtRanBP1c binds preferentially to the GTP-bound conformation of PsRan1, and the RanBD of AtRanBP1c is sufficient for the protein to bind to PsRan1-GTP. AtRanBP1c stabilizes PsRan1 in its GTP-bound conformation by binding to the same conformation. AtRanBP1c itself is not a GTPase activating protein, but can act as a coactivator of RanGAP activity. Transiently expressed, GUS-fused AtRanBP1c localizes primarily to the cytosol, with some localization also in the periphery of the nucleus. Truncation of the AtRanBP1c protein at its C-terminus, N-terminus, or at both termini revealed that the N- and C-termini of AtRanBP1c are both necessary for the localization of AtRanBP1c primarily to the cytosol, even though these regions do not contribute much to the Ran-binding activity of the protein. Transgenic expression of antisense-oriented AtRanBP1c generated a plant that has a long primary root. This implies that AtRanBP1c is involved in root development of plants.