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. 2015 Sep 14;11(8):1326–1340. doi: 10.1080/15548627.2015.1061846

Figure 6.

Figure 6.

Monoubiquitinated PEX5-EGFP trapped at the DTM in the presence of ATP is only partially protease protected. Radiolabeled PEX5(L), PEX5(L)-EGFP and PEX5(L)C11A-EGFP were subjected to in vitro import assays in the presence of Ub aldehyde and either ATP or AMP-PNP, as indicated. After incubation at 37°C, organelle suspensions were treated with proteinase K. NEM-treated organelles were then isolated, and subjected to SDS-PAGE under reducing (+ DTT) and non-reducing (− DTT) conditions. The autoradiographs (upper panels) and a section of the corresponding Ponceau S-stained membranes (lower panels) are shown. a and b represent DTM-inserted PEX5(L) exposing 2 kDa of its N terminus to the cytosol and DTM-embedded monoubiquitinated PEX5(L), respectively. 21 The asterisks mark a set of PEX5(L)-EGFP-derived fragments that are protease resistant. Lanes I1, I2, I3, 5% of the radiolabeled protein used in the assays.