Quantification of the Nucleocytoplasmic Distribution of Wild Type and Modified Proteins Using Confocal Microscopy: Interaction between 90-kDa Heat Shock Protein (Hsp90α) and Glucocorticosteroid Receptor (GR)

The investigation of molecular interactions in whole cells by immunofluorescence was developed recently, based on the targeting of the protein partners to different cellular compartments and analysis of the modifications in their subcellular distribution resulting from their interaction. This paper describes the adaptation of the confocal microscopy to the quantification of the partitioning of transiently coexpressed proteins between nucleus and cytoplasm. We defined a nucleocytoplasmic ratio R, corresponding to the difference between nuclear and cytoplasmic fluorescence intensities divided by their sum (N − C/N + C), which does not refer to absolute fluorescence intensities. Interaction was detected by statistically comparing the distribution of Rvalue frequencies in cell populations expressing one or both proteins. The convenience of this whole cell method was demonstrated by detecting and analyzing interaction between the human glucocorticosteroid receptor (GR) and the chick 90-kDa heat shock protein (Hsp90), using various combinations of wild-type and nuclear- or cytoplasmic-targeted GR and Hsp90. In addition, three Hsp90 deletion/truncation mutants were tested: the C-terminal truncated mutant NC4 interacted slightly, indicating the contribution of this part of the molecule to the interaction with GR, while the shorter truncated mutant NC6 did not interact with GR, likely resulting from an incorrect folding of the molecule. No role for the first charged region (ΔA') was found as shown by the strong interaction detected for the ΔA'Hsp90. This method can fruitfully be applied to the delimitation of the amino-acid sequences involved in protein–protein interaction by mutational analysis, especially to seek confirmation of other methods or when other approaches have failed.