Fig 2: PfDdi1 hydrolyzes both peptide substrates and proteins. (a) Coomassie-stained SDS-PAGE and Western blot analysis using a-His antibodies to detect the purified recombinant PfDdi1 (~44 kDa) and its processed fragment (~34 kDa). The soluble, recombinant protein was purified using the Ni-NTA resin (see also Figure S1a–c). (b) Mice or rabbit ant-Ddi1 antibodies detected a band of ~49 kDa from a parasite lysate. (c) Kinetics of substrate hydrolysis. Triplicate enzyme assays were carried out for 4 h at 37 °C. Both the DABCYL-Gaba-SQNYPIVQ-EDANS and Suc-LLVY-AMC cleavage signals were measured at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. On the other hand, an excitation and emission wavelength of 340 and 425 nm, respectively, was used to monitor the hydrolysis of Bz-RGFFP- 4MßNA. The fluorescence signals were captured at 15-min intervals with the VICTOR Multilabel plate reader (VICTOR X3). The purified enzyme (PfDdi1) was more active on the retropepsin (DABCYL-Gaba-SQNYPIVQ-EDANS) substrate compared to the proteasome (Suc-LLVY-AMC) substrate. On the other hand, PfDdi1 did not hydrolyze the pepsin/cathepsin D (Bz-RGFFP-4MßNA; Figure S1d). (d) Western blot analysis showing the cleavage of polyubiquitin substrate (K48-linked substrate) by the PfDdi1 enzyme. The test assay (substrate/PfDdi1) or the control (substrate alone) were incubated at 37 °C for 2 h, and then resolved in a 12% SDS-PAGE. Cleavage of the polyubiquitin substrate was probed using rabbit anti-ubiquitin antibodies (U5379, Sigma Aldrich). (e) Coomassie stained SDS-PAGE showing degradation of BSA by the PfDdi1 enzyme. The test assay (BSA/PfDdi1) or the control (BSA alone) were incubated at 37 °C for 2 h, at pH 5.0, and then resolved in a 12% SDS-PAGE. (f) Quantification of the control and the degraded BSA band intensities (~66 kDa). There was no degradation of BSA at pH 7.0 (Supplementary Figure S3a). The units are arbitrary (AU) and the bars show the mean ± standard error for three independent reactions.

Fig 3: Polyubiquitination and proteasome degradation are involved in the turnover of CycB2/cyc6 during mitosis.Cells arrested in late S-phase by hydroxyurea treatment were released for synchronous growth for 8 hours in the absence (A) or the presence (B) of 20 µM MG132. Hourly cell samples were lysed, subjected to immunoprecipitation with anti-HA mAb and fractionated with SDS-PAGE. Western blotting was used to analyze the time course of the level of CycB2/cyc6-3HA with anti-HA mAb (upper panels) or polyubiquitination staining with anti-Ubiquitin (Sigma, U5379) antibody (middle panels). Anti-Bip was used as loading controls. A quantitative analysis of CycB2/cyc6-3HA relative abundance and ubiquitin staining intensity for each experiment were plotted versus time in the bottom panels. Error bars represent the SD from two independent experiments.