Fig 1.
PfPanK1 and PfPanK2 are part of similar-sized protein complexes that possess PanK activity.
(A) Confocal micrographs showing the subcellular location of PfPanK2-GFP within trophozoite/schizont-stage P. falciparum-infected erythrocytes. The nuclei of the parasites are stained with DAPI. From left to right: Brightfield, GFP-fluorescence, DAPI-fluorescence, and merged images. Arrows indicate the plasma membranes of the erythrocyte (red) or the parasite (white). Scale bars represent 5 μm. (B) Denaturing and native western blot analyses of the GFP-tagged proteins in the PfPanK1-GFP and PfPanK2-GFP parasite lines. The expected sizes of the proteins are ~87 kDa for PfPanK1-GFP and ~118 kDa for PfPanK2-GFP. For reference, the molecular mass of the GFP tag is ~27 kDa. Western blots were performed with anti-GFP antibodies and each of the blots shown is representative of three independent experiments, each performed with a different batch of parasites. (C) The phosphorylation of [14C]pantothenate (initial concentration of 2 μM, ~10,000 counts per minute) over time by the immunopurified complex from lysates of parasites expressing PfPanK1-GFP (black circles), PfPanK2-GFP (white circles) and untagged GFP (grey circles). Data shown are representative of two independent experiments, each performed with a different batch of parasites and carried out in duplicate. Error bars represent range/2 and are not shown if smaller than the symbols.
Fig 2.
PfPanK1 and PfPanK2 are part of a single PanK complex that includes Pf14-3-3I.
(A) The four most abundant proteins identified in the MS analysis of proteins immunoprecipitated with GFP-Trap from the PfPanK1-GFP and PfPanK2-GFP lines. Data shown are representative of two independent analyses (1st and 2nd rep), each performed with a different batch of parasites. Proteins detected in the untagged GFP line or wild-type 3D7 parasite immunoprecipitations (negative controls) were removed. Only proteins with three or more peptides detected in both replicate co-immunoprecipitation experiments are shown. Proteins identified but which did not meet these criteria are shown in S1 Table. Proteins are listed in descending order according to the total number of peptides detected across all replicates (total peptides in all four columns). (B) The phosphorylation of [14C]pantothenate (initial concentration of 2 μM) over time by (i) lysates generated from Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasite lines (reproduced from [27]) and (ii) proteins immunoprecipitated with GFP-Trap from Parent+PfPanK2-GFP (white circles), PanOH-A+PfPanK2-GFP (black triangles), PanOH-B+PfPanK2-GFP (black squares) and CJ-A+PfPanK2-GFP (black diamonds) parasite lysates. Values in (ii) are averaged from three independent experiments, each performed with a different batch of parasites and carried out in duplicate. Error bars represent SEM and are not shown if smaller than the symbols. (C) Native western blot analysis of the lysates and denaturing western blot analyses of the different GFP-Trap immunoprecipitation fractions generated from PfPanK1-GFP and PfPanK2-GFP parasite lines, with the untagged GFP line as a control. Protein samples used in the denaturing western blot were derived from the same immunoprecipitation depicted in S2 Fig. Western blots were performed with pan-specific anti-14-3-3 antibodies (previously shown to detect Plasmodium 14-3-3 [32]). Arrows indicate the position of 14-3-3-containing complexes of comparable masses in all three lines (solid arrow) and the complexes found only in the PfPanK1-GFP and PfPanK2-GFP lines (dashed arrow). The native blot shown is a representative of three independent experiments, while the denaturing blot is a representative of two independent experiments, each performed with a different batch of parasites.
Fig 3.
TgPanK1 and TgPanK2 are part of a single protein complex with PanK activity.
(A) Denaturing and native western blot analyses of the HA-tagged proteins in TgPanK1-mAIDHA and TgPanK2-mAIDHA parasite lines. The expected sizes of TgPanK1-mAIDHA and TgPanK2-mAIDHA are ~143 kDa and ~189 kDa, respectively. Western blots were performed with an anti-HA antibody and each blot shown is a representative of three independent experiments, each performed with different batches of parasites. Denaturing western blots were also probed with anti-TgTOM40, which served as a loading control. (B) Western blot analysis of proteins from TgPanK1-GFP/TgPanK2-mAIDHA parasite lysates immunoprecipitated with GFP-Trap and anti-HA beads (TgPanK1-GFP is 160 kDa). Protein samples were collected before immunoprecipitation (Total), from the fraction not bound to the GFP-Trap/anti-HA beads (Unbound), and from the fraction bound to the GFP-Trap/anti-HA beads (Bound). Membranes were probed with anti-GFP and anti-HA antibodies, and the blot shown is representative of three independent experiments, each performed with different batches of parasites. TgTOM40 served as a control protein that is part of an unrelated protein complex. Bound fractions contain protein from 4 × as many cells as the total and unbound lanes. (C) The phosphorylation of [14C]pantothenate (initial concentration 2 μM) over time by protein samples immunoprecipitated with GFP-Trap from TgPanK1-GFP/TgPanK2-mAIDHA (black circles),TgPanK1-HA/TgPanK2-GFP (white circles) and untagged GFP (grey circles) lines. Data shown are representative of two independent experiments, each performed with a different batch of parasites and carried out in duplicate. Error bars represent the range/2 and are not shown if smaller than the symbols.
Fig 4.
Expression of both TgPanK1 and TgPanK2 is necessary for PanK activity and for T. gondii tachyzoite proliferation.
(A) IAA-induced knockdown of TgPanK1-mAIDHA or TgPanK2-mAIDHA protein over time. Western blot analysis of TgPanK1-mAIDHA and TgPanK2-mAIDHA lines incubated with either 100 μM IAA (+IAA) for 1, 2 and 4 h or an ethanol vehicle control (0 h). Membranes were probed with anti-HA antibody to detect the TgPanK1-mAIDHA and TgPanK2-mAIDHA proteins, and with anti-TgTom40 as a loading control. Western blots shown are representative of three independent experiments, each performed with a different batch of parasites. (B) The effect of TgPanK1-mAIDHA or TgPanK2-mAIDHA knockdown on T. gondii tachyzoite proliferation. Parent, TgPanK1-mAIDHA and TgPanK2-mAIDHA lines (all expressing tdTomato RFP) were cultured over 7 days in the presence (red circles) or absence (black circles) of 100 μM IAA. Parasite proliferation was measured over time by assessing the RFP expression using a fluorescence reader. Graphs shown are representative of three independent experiments carried out in triplicate, each performed with a different batch of parasites. Error bars represent SD and are not shown if smaller than the symbols. (C) Complementation of TgPanK1 and TgPanK2 knockdown with SaPanK. SaPanK was constitutively expressed (+) in TgPanK1-mAIDHA+SaPanK-Ty1 (green bars) and TgPanK2-mAIDHA+SaPanK-Ty1 (blue bars) parasites. These lines were cultured alongside the non-complemented (-) TgPanK1-mAIDHA (green bars), TgPanK2-mAIDHA (blue bars) and parent lines (grey bars). All parasite lines were cultured either in the presence (+) or absence (-) of 100 μM IAA. Parasite proliferation was monitored 1–2 times daily for 7 days. Proliferation was compared when the Parent strain cultured in the absence of IAA was at the mid-log phase of parasite proliferation. Values are averaged from three independent experiments, each performed with a different batch of parasites and carried out in triplicate. Error bars represent SEM.
Fig 5.
TgPanK2 is required for PanK activity in T. gondii parasites.
(A) Denaturing western blot analysis of the GFP- and HA-tagged proteins in the TgPanK1-GFP/TgPanK2-mAIDHA parasite line cultured in the absence or presence of IAA. TgPanK1-GFP/TgPanK2-mAIDHA parasites were incubated with either 100 μM IAA (+IAA) for 1, 2, 4 and 6 h, or an ethanol vehicle control (0 h). TgTOM40 served as a loading control. (B) Denaturing western blot analysis of the GFP- and HA-tagged proteins in the TgPanK1-GFP/TgPanK2-mAIDHA parasite line before and after immunopurification. TgPanK1-GFP/TgPanK2-mAIDHA parasites were incubated with either 100 μM IAA (+IAA), or an ethanol vehicle control (-IAA) for 2–3 h. The cells were lysed and a sample of total lysate (Total) was incubated with GFP-Trap. The GFP-Trap-immunoprecipitated protein (Bound) samples were then separated from the supernatant (Unbound) and all three fractions analysed by western blot. TgTOM40 served as a control protein that is not expected to be in the TgPanK complex. The western blot shown is a representative of three independent experiments, each performed with a different batch of parasites. (C) The phosphorylation of [14C]pantothenate (initial concentration of 2 μM) over time by GFP-Trap-immunoprecipitated protein samples from TgPanK1-GFP/TgPanK2-mAIDHA parasite lysates. The lysates were generated from parasites that were incubated in either the presence (+IAA) or absence (-IAA) of 100 μM IAA for 2–3 h. Data shown are averaged from three independent experiments, each performed with a different batch of parasites and carried out in duplicate. Error bars represent SEM and are not shown if smaller than the symbols.