Lysine residues are not required for proteasome-mediated proteolysis of cellular prion protein

Cellular prion protein (PrP^C) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrP^C is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrP^C degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrP^C are involved in the ubiquitination and subsequent degradation of PrP^C. We generated a plasmid vector expressing a mutant PrP^C (called lysine-null PrP^C) in which all lysine residues were replaced with arginine residues. Subsequently, we established stably transformed cell lines (designated HpL2-1 PrP-WT and HpL2-1 PrP-K/R, respectively) using the mouse PrP^C-deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrP^C (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrP^C which translocated efficiently to the plasma membrane. Subsequently, using immunoblotting and confocal microscopy, we found that treatment with cycloheximide (CHX; a protein synthesis inhibitor) significantly reduced PrP^C expression in both these transformed cell lines, indicating that WT and lysine-null PrP^C are degraded similarly. Taken together, these results indicate that the lysine residues of PrP^C do not regulate its degradation.