Abstract
Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.
Highlights
The abbreviations used arePrion protein (PrP), prion protein; TM, transmembrane; GPI, glycosylphosphatidylinositol; ER, endoplasmic reticulum; SS, signal sequence; RM, rough microsomes; PAGE, polyacrylamide gel electrophoresis; Endo H, endoglycosidase H; SRP, signal recognition particle
At steady state in normal brain the 35-kDa glycoprotein Prion protein (PrP) is anchored in the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) moiety exposing the polypeptide to the extracellular face of the plasma membrane [4, 5]
These findings suggest that early stages in the biogenesis of PrP may be important for understanding the generation of some variant forms of PrP that lead to neurodegeneration
Summary
PrP, prion protein; TM, transmembrane; GPI, glycosylphosphatidylinositol; ER, endoplasmic reticulum; SS, signal sequence; RM, rough microsomes; PAGE, polyacrylamide gel electrophoresis; Endo H, endoglycosidase H; SRP, signal recognition particle. Membrane, PrP exists in two transmembrane forms These span the membrane with the same hydrophobic region, TM1, but in opposite orientations. Disease-associated mutants within PrP affected the proportion of the three topological forms in vitro and in transgenic animals expressing these mutant forms of PrP [5] These findings suggest that early stages in the biogenesis of PrP may be important for understanding the generation of some variant forms of PrP that lead to neurodegeneration. For generating CtmPrP the SS may not be engaged, and the TM1 or TM2 may function as internal topogenic sequences To test this hypothesis, we analyzed whether in the absence of SS other hydrophobic regions in PrP can target the protein to the ER membrane and mediate membrane insertion. Targeting by TM2 occurs post-translationally and leads to the preferential formation of the CtmPrP form
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