A yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay based on amber suppression was used to identify proteins participating in the nuclear tRNA export process in Saccharomyces cerevisiae. This strategy identified a novel 85-kDa protein designated Cex1p. Cex1p is a non-essential cytoplasmic protein homologous to an uncharacterized ORF found in every eukaryotic genome sequenced to date. Like the S. cerevisiae Los1p and Utp8p and mammalian exportin-t proteins, which are known to facilitate nuclear tRNA export, overexpression of Cex1p restored export of tRNATyr am mutants defective in nuclear export. A double mutant strain of Cex1p and Arc1p, a cytoplasmic tRNA chaperone that delivers certain species of non-aminoacylated tRNAs exiting the nucleus to their cognate aminoacyl-tRNA synthetases, grows very slowly. This double mutant strain is being used to test if putative mammalian homologues of Cex1p are involved in nuclear tRNA export. Using colocalization analyses, we also found that a small amount of Cex1p associates with the NPC. Sequence comparisons and structural modeling suggest that the N-terminal half of Cex1p forms a structure similar to serine (threonine) protein kinase related to p38 MAP kinase and human Cdk2 kinase of the Hanks CMGC kinase family. However, Cex1p does not contain signature sequences or the catalytic residues of kinases. Proteins with a kinase structure but lacking kinase activity are called kinase accessory proteins and are responsible for delivering substrates to the appropriate kinases. Cex1p also contains a HEAT repeat motif. Cex1p binds tRNA saturably, indicating that it has a tRNAbinding site. Furthermore, TAP purification using extract from cells with a chromosomal Cex1–TAP fusion gene, followed by Western blot analysis with an anti-Los1p antibody, suggests that Los1p copurifies with Cex1p. Based on these findings, we suggest that Cex1p may be a kinase accessory protein that binds the export receptor – tRNA complex at the NPC and recruits a kinase, which then phosphorylates the receptor, allowing Cex1p to remove the aminoacylated tRNA cargo. It is also possible that the kinase domain of Cex1p allows the protein to interact with the receptor, thereby facilitating removal of the aminoacylated tRNA cargo by Cex1p.

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