Figure 4.

Analysis of cargo recognition and Pex14p interaction of N-terminally ubiquitinated Ub_(1–6Δ)Pex5p. Purified GST and N-terminal GST fusion constructs of Pex5p and Ub_(1–6Δ)Pex5p were combined with the PTS1 cargo protein Pcs60p (A) and the peroxisomal membrane docking protein _HisPex14p (B) and loaded onto GSH–agarose. Bound proteins were eluted with buffer containing 50 mm reduced GSH. Equal volumes of load, flow through (Fl), and 3× concentrated eluate fractions were subjected to SDS–PAGE and Coomassie staining. The asterisk marks a degradation product of _GSTUb_(1–6Δ)Pex5p, which represents _GSTUb. The N-terminally monoubiquitinated Pex5p binds its PTS1 cargo (Pcs60p) and the docking protein (Pex14p) with a similar efficiency as WT Pex5p. The amounts of recovered Pcs60p and _HisPex14p in the eluates were 8–10 and 30%, respectively.