Open AccessEvaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging
Author(s) -
Jun J. Yang,
Hongtao Chen,
Iontcho R. Vlahov,
Ji Cheng,
Philip S. Low
Publication year - 2006
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.0601455103
Subject(s) - endosome , endocytosis , endocytic cycle , förster resonance energy transfer , chemistry , microbiology and biotechnology , biophysics , golgi apparatus , biochemistry , receptor mediated endocytosis , receptor , fluorescence , biology , cell , physics , quantum mechanics
Despite functional evidence for disulfide bond-reducing activity in endosomal compartments, the mechanistic details pertaining to such process (e.g., kinetics and sites of disulfide reduction) remain largely controversial. To address these questions directly, we have synthesized a previously uncharacterized fluorescent folate conjugate, folate-(BODIPY FL)-SS-rhodamine (folate-FRET), that changes fluorescence from red to green upon disulfide bond reduction. Using this construct, we have observed that disulfide reduction: (i ) occurs with a half-time of 6 h after folate-FRET endocytosis, (ii ) begins in endosomes and does not depend significantly on redox machinery located on the cell surface or within the lysosome or the Golgi apparatus, (iii ) occurs independently of endocytic vesicle trafficking along microtubules, and (iv ) yields products that are subsequently sorted into distinct endosomes and trafficked in different directions. Finally, colocalization of folate and transferrin receptors suggest that conclusions derived from this study may apply to other endocytic pathways.