Functional analysis of rice proteins which bind to the α subunit of nuclear transport complex.

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The purpose of this research is to identify a novel nuclear protein which might be involved in the light-response in rice plants, by isolating proteins that bind to rice importin α 1a (IMP α 1a) whose expression is down-regulated by light. Below are the results.1. Analysis of an IMP α 1a-binding protein, IABP4, which has been isolated by cDNA screening by Far western method.(1) Determination of the full cDNA sequence : The IABP4 cDNA contained the entire coding sequence for a novel protein of predicted molecular mass of 122 kDa, and the homologous gene was found on the chromosome 2 of Arabidopsis thaliana.. In the N-terminal region containing TPR motifs, IABP4 protein shows high homology to a mouse nuclear phosphoprotein, TSP, whereas the similarity is low in the C-terminal region which contains the SH2-binding domain in mouse TSP, suggesting that the IABP4 protein is not necessarily homologous in function to TSP.The C-terminal part contains a putative nuclear localization signal.(2) Analysis of gene expression : The result of RT-PCR indicated that the IABP4 transcript level decreases by illuminating dark-grown seedlings of rice.(3) Analysis of binding to IMP α 1a : Usins purified recombinant proteins expressed in E.coil, the binding assay with native polyacrylamide gel electrophoresis demonstrated the complex formation between the C-terminal part of IABP4 and GST-IMP α 1a.Further experiments are required for determination of nuclear localization and the in planta function of the IABP4 protein.2. By another approach using affinity chromatography, several IMP α 1a-binding proteins were isolated from etiolated rice seedlings and the N-terminal sequences of three of them were determined. For one protein with homology to proline-rich protein, the corresponding cDNA was isolated and its expression was found to be down-regulated by light in dark-grown seedlings.