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Vol. 20, Issue 1, 233-244, January 1, 2009

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Specific Biarsenical Labeling of Cell Surface Proteins Allows Fluorescent- and Biotin-tagging of Amyloid Precursor Protein and Prion Proteins

Yuzuru Taguchi^*, Zhen-Dan Shi, Brian Ruddy, David W. Dorward, Lois Greene, and Gerald S. Baron^*

*Laboratory of Persistent Viral Diseases and Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840; Imaging Probe Development Center, National Heart Lung and Blood Institute, National Institutes of Health, Rockville, MD 20892-3372; and Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8017

Submitted June 23, 2008; Revised October 3, 2008; Accepted October 29, 2008
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, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by 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 by 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).

Abbreviations used: IDEAL-labeling, instant with DTT, EDT, and low temperature-labeling; PK, proteinase K; PrP, prion protein; PrPsen, normal protease-sensitive prion protein; PrPres, prion disease-associated protease-resistant prion protein; PrP(90TC), prion protein with tetracysteine motif at residue 90; PrP(230TC), prion protein with tetracysteine motif at residue 230; PTA, phosphotungstate; TC, tetracysteine.

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