2-color photobleaching experiments reveal distinct intracellular dynamics of two components of the Hsp90 complex.
The abundant molecular chaperone Hsp90 functions in association with co-chaperones including p23 to promote the folding and maturation of a subset of cytosolic proteins. "Fluorescence recovery after photobleaching" (FRAP) experiments showed that the dynamics of p23 in live cells is dictated by Hsp90. Since Hsp90 is present in large excess over p23, the mobility of Hsp90 could conceivably be quite different. To facilitate the analysis and to allow a direct comparison with p23, we developed a 2-color FRAP technique. Two test proteins are expressed as fusion proteins with the two spectrally separable fluorescent proteins mCherry and enhanced green fluorescent protein (EGFP). The 2-color FRAP technique is powerful for the concomitant recording of two proteins located in the same area of a cell, two components of the same protein complex, or mutant and wild-type versions of the same protein under identical experimental conditions. 2-color FRAP of Hsp90 and p23 is virtually indistinguishable, consistent with the notion that they are both engaged in a multitude of large protein complexes. However, when Hsp90-p23 complexes are disrupted by the Hsp90 inhibitor geldanamycin, p23 moves by free diffusion while Hsp90 maintains its low mobility because it remains bound in remodeled multicomponent complexes.
Picard
Didier
D
Département de Biologie Cellulaire, Université de Genève, Sciences III, 30 quai Ernest-Ansermet, CH-1211 Genève 4, Switzerland.
Suslova
Elena
E
Briand
Pierre-André
PA
D003600:Cytosol / Q000378:metabolism; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046912:Multiprotein Complexes; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071876:Prostaglandin-E Synthases; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D005456:Fluorescent Dyes; D018841:HSP90 Heat-Shock Proteins; D018832:Molecular Chaperones; D046912:Multiprotein Complexes; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C490849:PTGES3 protein, human; D000071876:Prostaglandin-E Synthases