TY - JOUR
T1 - Evaluation of disulfide reduction during receptor-mediated endocytosis by using FRET imaging
AU - Yang, Jun
AU - Chen, Hongtao
AU - Vlahov, Iontcho R.
AU - Cheng, Ji Xin
AU - Low, Philip S.
PY - 2006/9/12
Y1 - 2006/9/12
N2 - 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.
AB - 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.
KW - Disulfide bond reduction
KW - Drug targeting
KW - Endosome
KW - Folate receptor
UR - http://www.scopus.com/inward/record.url?scp=33748792549&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33748792549&partnerID=8YFLogxK
U2 - 10.1073/pnas.0601455103
DO - 10.1073/pnas.0601455103
M3 - Article
C2 - 16950881
AN - SCOPUS:33748792549
VL - 103
SP - 13872
EP - 13877
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
SN - 0027-8424
IS - 37
ER -