Subcellular localization of the Arabidopsis thaliana atDjC37 molecular chaperone protein

Abstract

There are 94 genes encoding J-domain molecular chaperone proteins in the Arabidopsis thaliana genome. These genes have been grouped into 51 families (Miernyk 2001 Cell Stress Chaperones 6: 209-218). Family 4 consists of two proteins, atDjC6 and atDjC37. It has been previously determined that atDjC6 is nuclear localized (Suo & Miernyk 2004 Protoplasma 224: 79-89). We now wish to determine the subcellular localization of atDjC37. In silico analysis of the atDjC37 deduced amino acid sequence (http://maple.bioc.columbia.edu/predictNLS/) yielded the prediction that residues -R253RSSKKS- comprise a nuclear localization signal (NLS) sequence. Our experimental strategy has been to construct plasmids that encode full-length atDjC37 protein and a C-terminal truncated version that lacks the NLS sequence, fused to the red fluorescent protein. These proteins will be transiently expressed in biolistically-transformed tobacco BY2 cells, and localized using laser-scanning confocal microscopy. The transformed cells will be simultaneously incubated with a fluorescent nuclear stain to test for signal coincidence. Four nuclear stains are being evaluated for their utility; propidium iodide (PI), DAPI, SYTO Green, and Hoechst 33342. The SYTO and Hoechst 33342 stains are considered cell-permeant, while DAPI is "semi-permeant" and PI is impermeant. The PI and DAPI stains are UV blue-fluorescent, while PI is red and SYTO Green is, naturally, green.