Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the (Ctm)PrP transmembrane form. Selective detection of (Ctm)PrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized (Ctm)PrP, revealing a previously unanticipated mechanism of (Ctm)PrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.
Emerman
Amy B
AB
Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Zhang
Zai-Rong
ZR
Chakrabarti
Oishee
O
Hegde
Ramanujan S
RS
D000818:Animals; D001483:Base Sequence; D019921:Biotinylation / Q000379:methods*; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D009154:Mutation; D011328:Prions / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000235:genetics; D015854:Up-Regulation
D011328:Prions; D020033:Protein Isoforms