Specific Features of the Prion Protein Transmembrane Domain Regulate Nascent Chain Orientation

Abstract

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.

Cytosolic prion protein is the predominant anti-Bax prion protein form: Exclusion of transmembrane and secreted prion protein forms in the anti-Bax function

“…Together, these experiments confirm the expression and topology of PrP mutants in MCF-7 cells, but show that in vivo, a relatively large amount of lumenal Sec-PrP is generated from both the Ctm PrP and Ntm PrP-encoding constructs. This has also been reported for Ctm PrP in transgenic mouse brain microsomes [37,50].…”

“…Furthermore, while the Triton X-100 detergent eliminates protection against proteinase K of the full length PrP, it does not eliminate the protection against the Ntm PrP isoform. This indicates that this fragment becomes resistant to detergents in a manner similar to that of transmissible PrP and CHO-transfected A120L and L129 PrP mutants [50]. These experiments confirm that the N4A120L generates some transmembrane Ntm PrP but it also makes significant Sec PrP.…”

“…Indeed, whereas the PrP mutant constructs have been well characterized to principally generate Ctm PrP, Ntm PrP or Sec PrP from in vitro translations in the presence of dog pancreatic microsomes [35,37,55,56], the expression of PrP from these same constructs in MCF-7 cells generated mostly Sec PrP and only a small amount of Ctm PrP or Ntm PrP. Others have observed Sec PrP generated from transmembranegenerating PrP constructs [37,50,57]. Furthermore, the A120L construct generates different PrP isoforms in N2a and MCF-7 cells and the function varies in MCF-7 and human neurons indicating that the A120L can give rise to different topologies in different cellular environments.…”

New insights into cellular prion protein (PrPc) functions: The “ying and yang” of a relevant protein

“…Furthermore, the transmembrane part of PrP c holds specific residues that regulate chain orientation when the protein is anchored to the cell membrane (Ott et al, 2007) promote neurotoxicity in neuronal cell cultures (Forloni et al, 1993) because of their self-aggregative properties. However, these studies are subject to debate since the requirement of PrP c expression for synthetic peptide toxicity is controversial (see for example (Brown, 2000;Fioriti et al, 2005;Gavin et al, 2005;Kunz et al, 1999;Singh et al, 2002)).…”

Pathogenic mutations in the glycosylphosphatidylinositol signal peptide of PrP modulate its topology in neuroblastoma cells

“…Likely scenarios include modulation of events prior to translocation of precursor PrP into the endoplasmic reticulum (ER), or interference with GPI-SP cleavage and anchor addition following ER translocation. In the former case mutations in this region would alter ER translocation or membrane topology of translocated PrP (Kim and Hegde, 2002;Ott et al, 2007), and in the latter cause accumulation of GPI-SP containing mutant PrP in the ER (Moran et al, 1991;Udenfriend and Kodukula, 1995;Jin et al, 2000) or secretion of anchorless PrP into the extracellular milieu (Chesebro et al, 2005). Support for the first hypothesis comes from a study demonstrating GPI-SP mediated posttranslational ER translocation of PrP C in an in vitro transcription-translation reaction.…”