One hallmark of prion diseases is the accumulation of the abnormal isoform PrP Sc of a normal cellular glycoprotein, PrP c , which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP c molecule, should play a role in promoting the conversion into PrP Sc . By using persistently scrapie-infected mouse neuroblastoma (Sc+-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP c that are capable of blocking the conversion of endogenous, wild-type PrP c into PrP Sc . We constructed a deletion mutant (PrP c Δ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP c or PrP c Δ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP c versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc+-MNB cells revealed that PrP c Δ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP Sc derived from endogenous PrP c . Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP c plays an important role in the conversion process of PrP c into PrP Sc and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP Sc accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.

contributed equally to this work Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the ribosome-associated membrane protein 4 (RAMP4). RAMP4 is a small, tail-anchored protein of 66 amino acid residues that is homologous to the yeast YSY6 protein. YSY6 suppresses a secretion defect of a secY mutant in Escherichia coli. The interaction of RAMP4 with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with RAMP4 contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with RAMP4 and inefficient N-glycosylation. We propose that RAMP4 controls modification of Ii and possibly also of other secretory and membrane proteins containing specific RAMP4-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.

The poliovirus polyprotein is cotranslationally linked to myristic acid at its amino-terminal glycine residue. We investigated the role of myristoylation in the viral replication cycle by site-directed mutagenesis of this glycine codon. Synthetic full-length RNA transcripts carrying a Gly-to-Ala mutation (G4002A) gave no infectious virus on transfection into permissive cells (HeLa). However, mutant viral RNA was replicated in the transfected cells, albeit at a reduced level. The virus-specific polypeptide P1, the precursor for the capsid proteins, was found in HeLa cells transfected with wild-type or mutant RNA, but only the wild-type P1 was myristoylated; the G4002A mutant P1 was not myristoylated. We also introduced the G4002A mutation into an in vitro transcription-translation vector encoding poliovirus P1 precursor. Processing of the mutant precursor by poliovirus-infected cell lysate (providing 3CPrO and 3CDPrF activities) was severely inhibited, whereas the normally inefficient cleavage by purified 3CPrT was not affected. These results suggest that the myristic acid moiety of the P1 precursor may be required for efficient processing by 3CDPr .

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