Studies On The Functions Of Arabidopsis Thaliana Transportin 1 Protein

Transportin 1(TRN1)is evolutionary conserved protein nuclear import receptor which is widely distributed in diverse eukaryotes.TRN1 recognizes and binds its substrates in the cytoplasm,gets through the nuclear pore by interaction with certain nucleoporin(s),and unloads its cargo by binding with Ran GTPase in the nucleus.Although many exciting achievements have been obtained from the research works on human and yeast TRN1 homologues,our knowledge about plant TRN1 is scarce.Protein is synthesized in ribosomes,and ribosomes are assembled in the nucleolus through a highly sophisticated biological process including pre-r RNA transcription,processing and the recruitment of ribosomal proteins.Owing to the nuclear envelope between the nucleoplasm and cytoplasm,ribosomal proteins synthesized in the cytoplasm are imported into the nucleus for ribosome assembly.In human cells,TRN1 is responsible for the nuclear import of several ribosomal proteins.However,this process in plants is barely known.During an Arabidopsis mutant pool screen,we isolated a temperature-sensitive mutant that was termed stunted in cool 1(sic1-1).This mutant has shorter roots,smaller leaves,dwarf style and accelerated flowering phenotype under normal temperature(22 °C),but shows a significantly enhanced phenotype including severely shortened internodes and the resultant clustering of siliques under a lower temperature(17 °C).However,all of these morphological alterations of sic1-1 will be greatly recovered under a higher temperature(27 °C).We found a T-DNA insertion in a gene of sic1-1 encoding an Arabidopsis TRN1 homologue,At TRN1,by TAIL-PCR,mapping and Southern blotting.We also performed gene expression assay,alleles assay,RNA interference and complementation experiment to show that sic1-1 mutant is genetically linked to the knockdown of At TRN1 gene expression resulted from T-DNA insertion.AtTRN1 protein shows high homology to its homologues in human,yeast and rice,and has the ability to partially rescue the lethal phenotype of yeast Kap104 p knockout mutant.At TRN1 gene is globally expressed in various organs and tissues.At TRN1 protein is localized in both the nucleus and cytoplasm.Using yeast two hybrid and bimolecular fluorescence complementation,we isolated several candidate cargoes of At TRN1,including RNA-binding proteins and ribosomal proteins.Then we separated ribosomes of sic1-1 via sucrose gradient ultracentrifugation and found that sic1-1 contains fewer ribosomes than the WT.We also found that the protein synthesis efficiency in sic1-1 is decreased by isotope-labeled methionine incorporation.Therefore it is possible that At TRN1 is responsible for the nuclear import of ribosomal proteins in Arabidopsis.In addition,we found that the auxin level in sic1-1 is reduced,and the phenotype of sic1-1 could be partially rescued by the overexpression of an Arabidopsis IAA synthase.Based on these results,we concluded that At TRN1 has similar protein import function as its homologues in human and yeast.The deficiency of At TRN1 affects plant growth and induces temperature-sensitive phenotype by the reduced protein and auxin biosynthesis.sic1-1 is the first TRN1 mutant found in multicellular organisms,and our results are also the first report about the function of plant TRN1 and the effect of TRN1 deficiency on multicellular organism growth and development.Moreover,our results provide evidences for At TRN1 participating in ribosomal protein nuclear import and ribosome assembly,which supporting the idea that plant TRN1 s may share similar function and substrates with their homologues in other organisms,and developing a novel direction on understanding how plant response to ambient temperature change.