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.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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