Fig. 2. Design and mechanistic analysis of a constitutive pQC variant of PrP.
(A) Schematic diagram of cytosolic quality control (cQC), pre-emptive quality control (pQC), and ER-associated degradation (ERAD). Of these, only the pQC pathway has been demonstrated to be utilized by PrP during ER stress (Kang et al., 2006; Orsi et al., 2006).
(B) The signal sequences and cleavage site (arrowhead) for constructs used in this study. Lysine residues used for crosslinking analyses are in bold.
(C) Crosslinking to cytosolic proteins of ribosome-associated nascent chains (RNCs) synthesized up to PrP residue 150. PrP (P), Ifn-PrP (I), Opn-PrP (O), and ΔSS-PrP (D) are analyzed. The arrowhead indicates crosslinks to SRP54, confirmed by immunoprecipitation (right panel). Asterisk indicates the position of uncrosslinked nascent chains.
(D) Crosslinking to ER proteins of RNCs synthesized to PrP residue 150. After crosslinking, the products were fractionated into membrane-associated and lumenal proteins, shown in the left and middle panels, respectively. Open arrow indicates crosslinks to the translocon component Sec61α (verified by immunoprecipitation; not shown), and closed arrow indicates crosslinks to the lumenal chaperone PDI, identified by immunoprecipitation in the right panel.
(E) PrP was synthesized in the absence or presence of ER-derived rough microsomes (RM) in a lysate supplemented with His-tagged ubiquitin. Ubiquitin-conjugated products were captured on immoblized Co+2. The positions of PrP species representing precursor (pre), signal-cleaved (s.c.), glycosylated (glyc) and ubiquitinated (Ub) products are indicated. Also shown are the Ubiquitin-conjugated products for ΔSS-PrP, illustrating its relatively poor ubiquitination.
(F) Ubiquitination analysis (as in panel E) of PrP, Ifn-PrP, and Opn-PrP in the absence and presence of RMs. The lower panel shows the total products and the upper panel the Ubiquitin-conjugated species captured via the His-tagged ubiquitin.