Abstract
Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types. We have investigated this poorly understood phenomenon by expressing fusion proteins of sheep prion protein and green fluorescent protein ((GFP)PrP) in N2a cells, with variable sequence context surrounding the start codon Met(1). (GFP)PrP expressed with the wild-type sequence was transported normally through the secretory pathway to the cell surface with acquisition of N-glycan groups, but two N-terminal fragments of (GFP)PrP were detected intracellularly, starting in frame from Met(17). When (GFP)PrP was expressed with a compromised Kozak sequence ((GFP)PrP*), dispersed intracellular fluorescence was observed. A similar switch from pericellular to intracellular PrP localization was seen when analogous constructs of sheep PrP, without inserted GFP, were expressed, showing that this phenomenon is not caused by the GFP tag. Western blotting revealed a reduction in glycosylated forms of (GFP)PrP*, whereas the N-terminal fragments starting from Met(17) were still present. Formation of these N-terminal fragments was completely abolished when Met(17) was replaced by Thr, indicating that leaky ribosomal scanning occurs for normal sheep PrP and that translation from Met(17) is the cause of the aberrant cytoplasmic localization observed for a fraction of the protein. In contrast, the same phenomenon was not detected upon expression of similar constructs for mouse PrP. Analysis of samples from sheep brain allowed immunological detection of N-terminal PrP fragments, indicating that sheep PrP is subject to similar processing mechanisms in vivo.
Highlights
PrPC 2 is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases [1, 2]
Cytoplasmic prion protein (PrP) could be a result of retro-translocation from the endoplasmic reticulum (ER), as part of an unfolded protein response [21,22,23] or from attenuated ER import of PrP under conditions of lumenal stress in the ER [24, 25]
No signs of stress or pathology could be detected in neurons of wild-type mice expressing cytoplasmic PrP [14], which led to the suggestion that the cytoplasmic appearance of PrP could constitute a physiologically relevant, but minor, pathway for the protein
Summary
PrPC 2 is a cell surface glycoprotein with an essential role in the pathogenesis of transmissible neurodegenerative prion diseases [1, 2]. Cytoplasmic localization of the prion protein (PrP) has been observed in different species and cell types.
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