Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

Maria E Juanes ,Gema Elvira,Aranzazu Garcia-Grande,Miguel Calero,Maria Gasset

doi:10.1074/jbc.m804051200

Abstract

The cellular prion protein PrP(C) is synthesized as a family of four distinct forms. Of these, (Cyt)PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that (Cyt)PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G(0)/G(1) phase. The identification of this PrP isoform and its properties provides novel insight into PrP(C) physiological and pathological functions.

Highlights

The cellular prion protein (PrPC)2 underlies a group of fatal tained in its N-terminal signal sequence [15] prompted us to neurodegenerative diseases through its conversion into self- decipher this code and use it as a tool to isolate its synthesis perpetuating and neurotoxic forms [1,2,3,4]
The presence of a second Met residue at the h-region boundary of the signal sequence determines the alternative translation initiation event. This process permits the synthesis of an isoform translated either from Met-15 in HaPrP or from Met-8 in HuPrP
This isoform represents a novel chain differing from the conventional mature form in the retention of the c-region of the N-terminal signal sequence and the full C-terminal hydrophobic region

Summary

RESULTS

N-terminal Signal Peptides of PrP Contain a Dual Methionine Motif—N-terminal signal peptides display a tripartite organization into n-, h-, and c-regions, with the hydrophobic central region (h-region) essential for co-translation membrane integration and translocation process. We identified two in-frame triplets (CUG and GUG coding HaPrP L9 and V13) that could sustain translation initiation by means of a single base difference These non-AUG codons are conserved in all species. The MM Motif Allows a Dual Translation Start and the Existence of PrP Isoforms—To test whether the downstream AUG codons found in the signal sequence regions of PrP mRNA in Group I and Group II could sustain translation initiation, we generated a series of point mutations in both the HaPrP and the HuPrP open reading frames HaPrP and HuPrP constructs The HaPrP open reading frame, cloned into pcDNA4.1 under BglII/EcoRI targets, and the HuPrP open reading frame, cloned into pcDNA3.1 under BamHI/EcoRI targets, were used as templates for the generation of point, reading shift, and deletion mutants using standard molecular biology protocols

Forward oligonucleotide

These results support the idea that

Analysis of bromodeoxyuridine incorporation showed that

DISCUSSION