Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling.
Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.
Yin
Jun
J
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Lin
Alison J
AJ
Buckett
Peter D
PD
Wessling-Resnick
Marianne
M
Golan
David E
DE
Walsh
Christopher T
CT
D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D004705:Endocytosis; D031541:Fluorescence Resonance Energy Transfer; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000378:metabolism*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism
D001426:Bacterial Proteins; D011990:Receptors, Transferrin; D014168:Transferrin; C082662:phosphopantetheinyl transferase; D017855:Transferases (Other Substituted Phosphate Groups)