Figure 4. Zn-induced folding blocks translocation at a post-ER targeting step.

a) Cartoon of pPL^45-Zn 87-mer, showing approximate location of the zinc finger domain within the exit tunnel of translocon-bound ribosome. b) Protocol Schematic: pPL 86-mer and pPL^45-Zn 87-mer were translated in RRL + CRMs in the absence of Zn⁺². Cycloheximide was added ± Zn⁺², and nascent chains were treated with puromycin or denatured in SDS followed by RNase digestion. Panels show phosphorimages of translation products treated as indicated. Migration of signal cleaved (translocated) and uncleaved polypeptide are designated by empty and filled circles, respectively. c) Schematic of pPL^45-Zn showing location of cysteine residues. d, e) Puromycin-treated samples of pPL^45-Zn prepared as in panel b in absence of Zn⁺² (panel d) or presence of Zn⁺² (panel e) were incubated with PEG-mal alone or after addition of 1% digitonin (dig) or 0.5M NaCl as indicated. Signal uncleaved and cleaved polypeptides are indicated by filled and empty circles, respectively. Arrows indicate ladder of peptides containing 1, 2, 3 or 4 PEG moieties. (Uncropped gel images for panels b, d and e are shown in Supplementary Fig. 2h, i, and j.)