Text S1 - An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Provides supplemental materials and methods used for the Bioinformatic analysis. We also provides additional information for the T. gondii plasmids, cell line and cell culture used; the apicoplast protein import assay; the antibody based assay; the cloning and purification of recombinant P. falciparum proteins and finally the Phylogenetic analysis and homology modelling of T. gondii TgE2Ap. Figure S1 presents the 3×HA tagging strategy used for PfE3cAp. Figure S2 shows the gene disruption strategies used for PfE1LAp, PfE3cAp (PFC0740c) and PfE3wAp. Figure S3 shows the multiple sequence alignment for E2 enzymes using MUSCLE. Figure S4 shows additional cyro-electonmicroscopic images of TgE2Ap. Table S1 presents the apicoplast candidates for E1, E2, E3 and Dub enzymes in both P. falciparum and T. gondii and lastly table S2 present a list of primers used for the cloning of recombinant proteins. Figure S1. (A) 3×HA tagging of PfE3cAp (PFC0740c) strategy is shown in the left panel. V-H indicates primer pairs that amplify only if there is targeted integration of the 3×HA plasmid to the PfE3cAp gene. After recovery, there was a diminishment of V-H PCR products over time (right panel), indicating that transfected strains exhibited a delayed-death effect of properly integrated vectors, leaving only recovered strains with non-integrated plasmids. The number of weeks (W1, W6, W12) starts from the time we observed recovered strains. (B) 3D7 parasite strains were transfected with plasmids that had a GFP fused to the c-terminus of the PfE3wAp (PFC0510w) for targeted integration by homologous recombination. Transfected strains (PFC0510w-GFP) were screened by PCR, where primers pairs (Ver) only amplified a product if proper integration had taken place. (C) RT-PCR reveals that GFP fused to the c-terminal of PFC0510w is being transcribed in the PFC0510w-GFP strains. Figure S2. (A) Gene disruption strategy of PfE1LAp (PF13_0182), PfE3cAp (PFC0740c) and PfE3wAp (PFC0510w). PCR of transfected strains with disruption vectors show that only PFC0510w was successfully disrupted. (B) RT-PCR of sub-cloned PFC0510w disrupted strains show no transcription of the RING domain of PFC0510w. (C) CGH microarray analysis show that the PFC0510w gene disruption vector was able to integrate into the genome of the transfected strain. Figure S3. Multiple sequence alignment for E2 enzymes using MUSCLE. Figure S4. Additional cyro-electonmicroscopic images of TgE2Ap. Sections were incubated with anti-HA antibody and gold conjugated anti-immunoglobulin as detailed in Figure 2. Table S1. Apicoplast candidates for E1, E2, E3 and Dub enzymes in both P. falciparum and T. gondii. Table S2. List of primers used for the cloning of all recombinant proteins. (DOC)