α-cleavage of the prion protein occurs in a late compartment of the secretory pathway and is independent of lipid rafts

Endoproteolysis of the cellular prion protein (PrPC) modulates both the normal function of the protein and the pathogenesis of the neurodegenerative prion diseases. PrPC undergoes α-cleavage to generate the N-terminally truncated fragment C1. Utilizing various constructs of PrPC expressed in human neuroblastoma cells we investigated the subcellular compartment where α-cleavage occurs. C1 was detected at the cell surface and the generation of C1 occurred in mutants of PrPC incapable of Cu2+-mediated endocytosis. A transmembrane-anchored form that is not lipid raft-localised, as well as a secreted construct lacking the glycosyl-phosphatidylinositol membrane anchor, were also subject to α-cleavage. However, when this transmembrane-anchored form was modified with an endoplasmic reticulum retention motif, C1 was not formed. Inhibition of protein export from the Golgi by temperature block increased the amount of C1. Our data thus demonstrate that the α-cleavage of PrPC occurs predominantly in a raft-independent manner in a late compartment of the secretory pathway.