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

Cellular prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein. The mature cell-surface PrP is internalized and subsequently degraded by lysosomes. Although, proteasomes are proposed to be involved, the precise mechanism of PrP degradation remains uncertain. Given that proteins are ubiquitinated primarily on lysine residues, we sought to determine whether lysine residues within PrP are involved in the ubiquitination and subsequent degradation of PrP. We generated a plasmid vector expressing a mutant PrP (called lysine-null PrP) 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-deficient neuronal cell line (HpL2-1) and plasmids expressing wild-type (WT) or lysine-null PrP (PrP-K/R). We found that HpL2-1 PrP-WT and HpL2-1 PrP-K/R cells correctly expressed their respective PrP 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 expression in both these transformed cell lines, indicating that WT and lysine-null PrP are degraded similarly. Taken together, these results indicate that the lysine residues of PrP do not regulate its degradation.