Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins

• Published in: Molecular biology of the cell Vol. 20; no. 1; pp. 233 - 244
• Main Authors: Taguchi, Yuzuru, Shi, Zhen-Dan, Ruddy, Brian, Dorward, David W, Greene, Lois, Baron, Gerald S
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01-01-2009
• Subjects: Amino Acid Sequence; Amyloid beta-Protein Precursor - chemistry; Amyloid beta-Protein Precursor - metabolism; Animals; Arsenicals - chemistry; Arsenicals - metabolism; Biotin - chemistry; Biotin - metabolism; Cell Line; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; Membrane Proteins - chemistry; Membrane Proteins - metabolism; Mice; Molecular Sequence Data; Molecular Structure; Prions - chemistry; Prions - metabolism; Protein Isoforms - chemistry; Protein Isoforms - metabolism; Staining and Labeling - methods
• ISSN: 1059-1524; 1939-4586; 1939-4586
• DOI: 10.1091/mbc.E08-06-0635

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.