title: Localization of GroEL determined by in vivo incorporation of a fluorescent amino acid.
abstract: The molecular chaperone GroEL is required for bacterial growth under all conditions, mediating folding assistance, via its central cavity, to a diverse set of cytosolic proteins; yet the subcellular localization of GroEL remains unresolved. An earlier study, using antibody probing of fixed Escherichia coli cells, indicated colocalization with the cell division protein FtsZ at the cleavage furrow, while a second E. coli study of fixed cells indicated more even distribution throughout the cytoplasm. Here, for the first time, we have examined the spatial distribution of GroEL in living cells using incorporation of a fluorescent unnatural amino acid into the chaperone. Fluorescence microscopy indicated that GroEL is diffusely distributed, both under normal and stress conditions. Importantly, the present procedure uses a small, fluorescent unnatural amino acid to visualize GroEL in vivo, avoiding the steric demands of a fluorescent protein fusion, which compromises proper GroEL assembly. Further, this unnatural amino acid incorporation avoids artifacts that can occur with fixation and antibody staining.
authors: Department of Molecular, Cellular, and Developmental Biology, KBT 1032, Yale University, New Haven, CT 06520, USA.
authors: Godefroid
authors: G
authors: Charbon
authors: Jiangyun
authors: J
authors: Wang
authors: Eric
authors: E
authors: Brustad
authors: Peter G
authors: PG
authors: Schultz
authors: Arthur L
authors: AL
authors: Horwich
authors: Christine
authors: C
authors: Jacobs-Wagner
authors: Eli
authors: E
authors: Chapman
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D018834:Chaperonin 60 / Q000032:analysis*; D004926:Escherichia coli / Q000166:cytology*; D005456:Fluorescent Dyes / Q000032:analysis*; D008856:Microscopy, Fluorescence
chemical_list: D000596:Amino Acids; D018834:Chaperonin 60; D005456:Fluorescent Dyes