Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging.
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.
Yang
Jun
J
Department of Chemistry and Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN 47907, USA.
Chen
Hongtao
H
Vlahov
Iontcho R
IR
Cheng
Ji-Xin
JX
Low
Philip S
PS
D001896:Boron Compounds / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D004220:Disulfides / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005492:Folic Acid / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D006801:Humans; D010084:Oxidation-Reduction; D012235:Rhodamines / Q000032:analysis / Q000378:metabolism; D022161:Transport Vesicles
D001896:Boron Compounds; D004220:Disulfides; D012235:Rhodamines; C514387:folate-(BODIPY FL)-SS-rhodamine; D005492:Folic Acid