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MBC in Press, published online ahead of print November 5, 2008
Mol. Biol. Cell 10.1091/mbc.E08-06-0635

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Submitted on June 23, 2008
Revised on October 3, 2008
Accepted on October 29, 2008

Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of APP and Prion Proteins

Yuzuru Taguchi,* Zhen-Dan Shi,{dagger} Brian Ruddy,{dagger} David W. Dorward,{ddagger} Lois Greene,{sect} and Gerald S. Baron*

*Laboratory of Persistent Viral Diseases, and {ddagger}Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, NIH, Hamilton, MT 59840; {dagger}Imaging Probe Development Center, National Heart Lung and Blood Institute (NHLBI), NIH, Rockville, MD 20850; {sect}Laboratory of Cell Biology, NHLBI, NIH, Bethesda, MD 20892-8017

Monitoring Editor: Jennifer Lippincott-Schwartz

Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, IDEAL-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL-labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.

Address correspondence to: Gerald S. Baron (gbaron{at}niaid.nih.gov)






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