NR ATOK
AU Knetsch,H.; Nikles,D.; Gauczynski,S.; Weiss,S.
TI The cellular prion protein lacking the N-terminal signal peptide is located in the nucleus
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Human prions, risk of blood products, and therapy HUMAN-34
PT Konferenz-Poster
AB
Recently, we identified the 37 kDa/67 kDa-laminin receptor (LRP/LR) as a prion receptor (1-3) and demonstrated the receptor-mediated endocytosis of PrPc. In order to investigate the subcellular localization of cellular PrP in more detail, we used fluorescent-tagged versions of moPrP as a tool to visualize protein trafficking by confocal microscopy.
Murine PrP was generated as EGFP-, EYFP- and DsRed-fusion protein, respectively. The corresponding plasmids pEGFP-moPrP and pEYFP-moPrP include the signal peptide (SP) of moPrP. In addition, we generated pDsRed-moPrP(-SP) lacking the N-terminal SP sequence. As expected, different glycosylated forms of EGFP- and EYFP-PrP were detected in BHK and HeLa cells after transfection. In contrast, DsRed-PrP(-SP) showed only the non-glycosylated form with a molecular weight of approx. 50 kDa.
Confocal microscopy on transfected HeLa and BHK cells showed that EGFP-PrP and EYFP-PrP were localized at the cell surface and concentrated within a perinuclear compartment. A similar distribution of the EGFP-fused PrP was shown in living SN56 cells (4). In contrast, we observed a nuclear localization of DsRed-moPrP(-SP) in both cell types. This phenomenon confirmed previous studies suggesting the existence of two nulear localization signals within the N-terminal domain of PrP responsible for the nuclear localization (5).
1. Rieger, R. et al. (1997) Nat Med, 3, 1383-8.
2. Gauczynski, S. et al. (2001) EMBO J, 20, 5863-75.
3. Hundt, C. et al. (2001) EMBO J, 20, 5876-86.
4. Lee, K. S. et al. (2001) J Neurochem, 79, 79-87
5. Gu, Y. et al. (2003) Neurobiol Dis, 12, 133-149.
AD H.Knetsch, D.Nikles, S.Gauczynski, S.Weiss, Laboratorium für Molekulare Biologie - Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen-Str. 25, 81377 München, Germany
SP englisch
PO Deutschland