Alternative translation initiation generates cytoplasmic sheep prion protein.
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.
Lund
Christoffer
C
Institute of Basic Sciences and Aquatic Medicine, Department of Biochemistry and Physiology, Norwegian School of Veterinary Science, 0033 Oslo, Norway.
Olsen
Christel M
CM
Skogtvedt
Susan
S
Tveit
Heidi
H
Prydz
Kristian
K
Tranulis
Michael A
MA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D045744:Cell Line, Tumor; D018387:Codon, Initiator / Q000235:genetics*; D003593:Cytoplasm / Q000378:metabolism*; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D008715:Methionine / Q000235:genetics; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D011134:Polysaccharides / Q000378:metabolism; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D018387:Codon, Initiator; D011134:Polysaccharides; D011328:Prions; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008715:Methionine