FIGURE 3.

Human and P. falciparum SUMO E1 and E2 enzyme interactions are distinct. a, P. falciparum enzymes modify RanGAP1 at the consensus site lysine. In vitro transcribed and translated wild type RanGAP1 (WT) or consensus sumoylation site mutants (E528A and K526A), were incubated with recombinant human (Hs) or P. falciparum (Pf)-conjugating enzymes. Conjugation was detected by autoradiography. b, human and P. falciparum E1-conjugating enzymes activate both human and P. falciparum SUMOs. Increasing concentrations of E1-activating enzymes were incubated with human or P. falciparum GFP-SUMO in the presence of ATP and reaction products were separated by non-reducing SDS-PAGE and analyzed by immunoblot analysis. The asterisk indicates a nonspecific GFP-SUMO-2 band. c, P. falciparum and human SUMOs are interchangeable. SUMO conjugation assays were performed in the presence of the indicated recombinant human or P. falciparum proteins. Reaction products were analyzed by immunoblot analysis. d, human and P. falciparum SUMO E2 enzymes are not interchangeable. SUMO conjugation assays were performed in the presence of the indicated recombinant human or P. falciparum proteins. Reaction products were analyzed by immunoblot analysis. e, E2∼SUMO thioesters are not formed in reactions containing heterologous E1 enzymes. The indicated recombinant proteins were incubated in the presence of ATP and reaction products were separated by non-reducing SDS-PAGE and analyzed by immunoblot analysis.