DIFFERENTIAL RESISTANCE TO PROTEINASE-K DIGESTION OF THE YEAST PRION-LIKE (URE2P) PROTEIN SYNTHESIZED IN-VITRO IN WHEAT-GERM EXTRACT AND RABBIT RETICULOCYTE LYSATE CELL-FREE TRANSLATION SYSTEMS

The Ure2p yeast prion-like protein was translated in vitro in the pres ence of labeled [S-35]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-Gee systems, When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was prote olysed much more slowly compared to that synthesized in RRL; this disp lays fragments of about 31-34 kDa, persisting over 8 min, Thus, the di gestion rate and pattern of the protein synthesized in WGE, unlike tha t synthesized in RRL, revealed characteristic features of the [URE3] p rion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B . (1995) Science 270, 93-95]. Chloramphenicol acetyltransferase, synth esized under the same conditions, differed fundamentally in its proteo lytic sensitivity toward proteinase K (PK); in the RRL system it was m ore slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems, Posttranslational addition of the WGE to the R RL-synthesized Ure2p does not protect Ure2p from efficient PK degradat ion either, The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesi zed in WGE; obviously, they yield a protein that mimics the behavior o f the Ure2p in [URE3] yeast strains, The present data suggest that par ticular conditions of the Ure2p protein translation and/or certain cel lular components (accessory proteins and extrinsic factors), as wen as the nature of the translation process itself, could affect the intrac ellular folding pathway of Ure2p leading to the de novo formation of t he prion [URE3] isoform. (C) 1997 Federation of European Biochemical S ocieties.