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United States Department of Agriculture

Agricultural Research Service

Title: Regulation of Nucleocytoplasmic Localization of the Atdjc6 Chaperone Protein

Authors
item Suo, Yuying - BIOCHEMISTRY DEPT-UMC
item Miernyk, Jan

Submitted to: Annual Missouri Symposium
Publication Type: Proceedings
Publication Acceptance Date: January 18, 2004
Publication Date: October 1, 2004
Citation: Suo, Y., Miernyk, J.A. 2004. Regulation of nucleocytoplasmic localization of the atdjc6 chaperone protein [abstract]. Annual Missouri Symposium. 224:79-89.

Technical Abstract: The atDjC6 protein is a member of the Arabidopsis thaliana J-domain molecular chaperone family that does not have any well-studied homolog in mammalian or microbial cells. To start our analysis of the specific function of the atDjC6, we need to know the subcellular location. The atDjC6 protein includes three potential nuclear localization signal (NLS) sequences (A-C), and three potential nuclear export signal (NES) sequences (X-Z). The results from in silico analyses predict that atDjC6 is a nuclear protein. The subcellular localization of atDjC6 was studied by scanning laser confocal microscopy of chimera with the green fluorescent protein (GFP) transiently expressed in tobacco BY-2 cells. Localization of the atDjC6:GFP chimera was entirely coincident with the nuclear stain propidium iodide. Site-directed mutagenesis was used to verify the predicted NLS sequences. Each was individually fused to GFP and tested for protein localization. The individual NLS sequences were sufficient to direct partial nuclear localization of GFP, although the targeting information within NLS-B is apparently conformation-sensitive. Site-directed mutagenesis of the NES sequences shifted the chimera that were partially nuclear localized to entirely nuclear localized. When any pair of NLS sequences was appended to GFP, the chimera was entirely nuclear localized. Quantitative two-hybrid analysis was used to verify that decoding of NLS sequence information involved interaction with the receptor/usher protein importin-alpha. The NLS sequences that were inactivated by site-directed mutagenesis did not interact with importin-alpha in the two-hybrid assays. Furthermore, we observed selectivity in recognition of NLS sequences by the different importin-alpha proteins. Each of the NLS sequences is flanked by a potential Ser phosphorylation site. Mutagenesis of these Ser residues to the P-Ser mimic Asp interfered with nuclear targeting, apparently by preventing recognition or binding by importin-alpha. When recombinant atDjC6 was incubated in vitro with gamma-32P-ATP plus a cytosol fraction, there was phosphorylation of Ser residues. In toto, our results are consistant with multi-component regulated nucleocytoplasmic localization of the atDjC6 chaperone protein. The need for this unusually complex control of subcellular localization is the subject of ongoing research.

Last Modified: 12/25/2014
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