Specific biarsenical labeling of cell surface proteins allows fluorescent- and biotin-tagging of amyloid precursor protein and prion proteins.
Mol Biol Cell
; 20(1): 233-44, 2009 Jan.
Article
in En
| MEDLINE
| ID: mdl-18987338
ABSTRACT
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Arsenicals
/
Staining and Labeling
/
Prions
/
Amyloid beta-Protein Precursor
/
Fluorescent Dyes
/
Membrane Proteins
Limits:
Animals
Language:
En
Journal:
Mol Biol Cell
Journal subject:
BIOLOGIA MOLECULAR
Year:
2009
Document type:
Article
Affiliation country:
United States