Figure 2.

In vitro pulldown assay of ubiquitin and Pex1p–Pex6p. Recombinant GST and C-terminal GST fusion constructs of WT and mutant (I44A) ubiquitin were combined with purified recombinant _HisPex1p (A), _HisPex6p (B), or the assembled _HisPex1p–_HisPex6p complex (C) and loaded onto GSH–agarose. Bound proteins were eluted with buffer containing 50 mm reduced GSH. Equal volumes of load, flow through (Fl), and 10× concentrated eluate fractions were subjected to immunoblot analysis using antibodies against Pex1p, Pex6p, and GST. A comparison of all eluate fractions is shown in D. Pex6p alone and in combination with Pex1p, but not Pex1p alone, did bind ubiquitin. A maximum of 1–2% of loaded AAA proteins could be recovered in the eluate. The I44A mutation reduced binding to Pex6p.