QUANTIFICATION OF THE NUCLEOCYTOPLASMIC DISTRIBUTION OF WILD-TYPE ANDMODIFIED PROTEINS USING CONFOCAL MICROSCOPY - INTERACTION BETWEEN 90-KDA HEAT-SHOCK-PROTEIN (HSP90-ALPHA) AND GLUCOCORTICOSTEROID RECEPTOR (GR)

The investigation of molecular interactions in whole cells by immunofl uorescence was developed recently, based on the targeting of the prote in partners to different cellular compartments and analysis of the mod ifications in their subcellular distribution resulting from their inte raction. This paper describes the adaptation of the confocal microscop y to the quantification of the partitioning of transiently coexpressed proteins between nucleus and cytoplasm. We defined a nucleocytoplasmi c ratio R, corresponding to the difference between nuclear and cytopla smic fluorescence intensities divided by their sum (N - C/N + C), whic h does not refer to absolute fluorescence intensities. Interaction was detected by statistically comparing the distribution of R value frequ encies in cell populations expressing one or both proteins. The conven ience of this whole cell method was demonstrated by detecting and anal yzing interaction between the human glucocorticosteroid receptor (GR) and the chick 90-kDa heat shock protein (Hsp90), using various combina tions 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 co ntribution of this part of the molecule to the interaction with GR, wh ile the shorter truncated mutant NC6 did not interact with GR, likely resulting from an incorrect folding of the molecule. No role for the f irst charged region (Delta A') was found as shown by the strong intera ction detected for the Delta A'Hsp90. This method can fruitfully be ap plied to the delimitation of the amino-acid sequences involved in prot ein-protein interaction by mutational analysis, especially to seek con firmation of other methods or when other approaches have failed. (C) 1 998 Academic Press.