PMC

TgTom22 is essential for parasite growth. A, Western blotting of rTom22/Δtom22 parasites grown for 0–2 days on ATc and probed with anti-HA antibodies to detect HA-Tom22 protein or anti-GRA8 antibodies as a loading control. B and C, fluorescence growth assays of TgTom22 knockdown (rTom22/Δtom22) (B) and TgTom22-complemented (rTom22/Δtom22/Tom22WT) (C) strains. Parasites were grown in the absence (black) or presence (red) of ATc for 0–8 days. Error bars represent the standard deviation of three technical replicates.

T. gondii harbors a TOM complex of ∼400 kDa that contains TgTom40 and TgTom22. A, Western blotting probing TgTom40 proteins separated by BN-PAGE extracted in 0.5% (w/v) digitonin, 1% (w/v) digitonin, 1% (w/v) dodecyl maltoside (DDM), 1% (w/v) octyl β-d-glucopyranoside (OGP), and 1% (w/v) Triton X-100 (TX-100). B, Western blotting probing TgTom40, HA-Tom22, and Sam50-cMyc proteins separated by BN-PAGE in a first dimension and SDS-PAGE in a second dimension. C, Western blottings of immunoprecipitated parasite proteins extracted in 0.5% digitonin and pulled down with anti-Tom40 antibodies (left) or anti-HA antibodies (right). Western blottings were probed for TgTom40 (top), HA-Tom22 (middle), and Sam50-cMyc (bottom). Total proteins, unbound proteins, and bound proteins were loaded for each experiment, and each lane contains proteins extracted from an equivalent number of parasites. The heavy chain of anti-Tom40 antibodies was detectable in the anti-Tom40 Western blotting.

TgTom7 is important for parasite growth, mitochondrial protein import, and TOM complex assembly. A, Western blotting of proteins extracted from TATi/Δku80/rTom7 grown for 0–3 days in ATc. Blots were probed with antibodies against HA₃-Tom7, TgGRA8 (loading control), TgTom40, and TgHsp60. Precursor and mature forms of TgHsp60 are indicated. This experiment was performed in three biological replicates, and a representative experiment is shown. B–D, fluorescence growth assays of TATi/Δku80 parental control (B). C, TgTom7 knockdown (TATi/Δku80/rTom7) and D, TgTom7 complemented (TATi/Δku80/rTom7/Tom7wt) strains. Parasites were grown in the absence (black) or presence (red) of ATc for 0 to 6 days. Error bars represent the standard deviation of three technical replicates. E, anti-Tom40 Western blotting of proteins extracted from TATi/Δku80/rTom7 parasites grown for 0–3 days in ATc and separated by BN-PAGE. F, Western blotting of proteins extracted from TATi/Δku80/rTom7 parasites grown in the absence (top) or presence of ATc for 2 days (bottom). Proteins were separated by BN-PAGE in a first dimension and SDS-PAGE in a second dimension, and probed with anti-Tom40 antibodies.

Localization of putative mitochondrial import proteins in T. gondii. A, B, D, F, H, J, L, and N, target proteins are labeled in green, and in all cases they co-localize with a mitochondrial marker (either RFP fused to the mitochondrial matrix-targeting leader sequence of TgHsp60 or anti-Tom40 antibodies; red). Scale bars, 2 μm. C, E, G, I, K, and M, Western blottings of each protein are shown to the right of each panel. For the TgTom40 Western blotting, protein extracts from wild-type (WT) and the HA-tagged TgTom40 strain were probed with anti-TgTom40 antibodies. Our transfection experiments did not produce tagged TgPam18 in sufficient quantity to detect by Western blotting. O, schematic depicting the putative composition of the TOM, SAM, TIM23, PAM, and TIM22 complexes in T. gondii. Proteins localized in this study are depicted in light green, and proteins identified bioinformatically are depicted in dark green, and notable proteins without homologues in T. gondii are depicted in gray. OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane.

TgTom22 is a mitochondrial membrane protein with homology to Tom22 proteins from other eukaryotes. A, multiple protein sequence alignment of TgTom22 with homologues from P. falciparum (PfTom22), humans (HsTom22), A. thaliana (AtTom22), and S. cerevisiae (ScTom22). Transmembrane domains of each protein (predicted using TMHMM) are highlighted by a box. B, immunofluorescence assay demonstrating co-localization of HA-Tom22 protein (green) with the mitochondrion marker Tom40 (red). Scale bar, 2 μm. A Western blotting analysis of HA-Tom22, probed with anti-HA antibodies, reveals a protein with a mass of 10 kDa. C, Western blotting analysis of HA-Tom22-expressing parasites, where proteins were extracted in 1% Triton X-100 (TX-100) or in sodium carbonate (Na₂CO₃) at pH 11.5. HA-Tom22 partitions into the pellet phase of the Na₂CO₃ extraction, much like the integral mitochondrial membrane protein Tom40 and unlike the soluble mitochondrial matrix protein ATP synthase β subunit, which partitions predominantly into the supernatant (S/N) phase.

TgTom7 is a mitochondrial protein and a constituent of the TOM complex. A, multiple protein sequence alignment of the long and short isoforms of TgTom7 with homologues from N. caninum (NcTom7), P. falciparum (PfTom7), S. cerevisiae (ScTom7), A. thaliana (AtTom7-1), and humans (HsTom7). Transmembrane domains of each protein (predicted using TMHMM) are highlight by a box. B, immunofluorescence assay demonstrating co-localization of HA-tagged version of the short isoform of TgTom7 (HA₃-Tom7; green) with the mitochondrial marker Tom40 (red). Scale bar, 2 μm. C, Western blotting analysis of HA₃-Tom7, probed with anti-HA antibodies, reveals a protein of 11 kDa. D, Western blotting of proteins extracted from the TATi/Δku80/rTom7 parasite strain and separated by BN-PAGE in a first dimension and SDS-PAGE in a second dimension. Blots were probed with anti-Tom40 antibodies (top) and anti-HA antibodies to detect HA₃-Tom7 protein (bottom). E, Western blottings of parasite proteins extracted from TATi/Δku80/rTom7 strain parasites in 0.5% digitonin, immunoprecipitated with anti-Tom40 antibodies, and separated by SDS-PAGE. Blots were probed with anti-Tom40 antibodies (top) and anti-HA antibodies (bottom). Total proteins, unbound proteins, and bound proteins were loaded for each experiment, and each lane contains proteins extracted from an equivalent number of parasites. The heavy chain of anti-Tom40 antibodies was detectable in the anti-Tom40 Western blotting.

Generation of mutant parasite strains. A, schematic describing knock-out of the native TgTom22 gene. The native TgTom22 locus (orange) was replaced with a chloramphenicol acetyltransferase (CAT)-selectable marker (blue), in a background strain expressing an ATc-regulatable copy of TgTom22 (data not shown). B, PCR screen of 12 parasite clones transfected with the TgTom22 knock-out construct with primers 21 and 22. The primer pair will produce a band of 0.8 kb if the native gene is present and a band of 1.6 kb when the regulatable copy of TgTom22 is present. Clones 3 and 5–11 lack the native gene, indicating successful knock-out of the TgTom22 native locus in these parasites. C, schematic describing replacement of the native TgTom7 promoter with an ATc-regulated promoter (t7s4) and 5′ HA tag. The TATi/Δku80 parasite strain was transfected with a vector containing 5′ and 3′ flanks of the TgTom7 gene, the t7s4 ATc-regulated promoter, a 5′ HA tag, and a pyrimethamine-resistant T. gondii DHFR-selectable marker. D, PCR screen of six parasite clones transfected with the promoter replacement vector and screened with primer pairs 31 and 32 to detect the native TgTom7 locus (top) or primer pairs 32 and 33 to detect the modified locus (bottom). DNA from wild-type (WT) TATi/Δku80 parasites was used as a control. Clones 1, 3, and 4 lack the band corresponding to the presence of the native gene but harbor the band corresponding to the modified locus, indicating successful promoter replacement at the TgTom7 locus in these parasites.

TgTom22 is essential for mitochondrial protein import and functions in assembly of the TOM complex. A, Western blottings of HA-Tom22 parasites expressing c-Myc-tagged mouse DHFR fused to the mitochondrion-targeting leader sequence of TgHsp60 (Hsp60_L-DHFR-cMyc). Parasites were grown for 0–3 days in ATc, and protein extracts were probed with anti-HA, anti-Hsp60, anti-cMyc, anti-TgTom40, and anti-GRA8 antibodies as a loading control. Precursor and mature forms of the Hsp60 and Hsp60_L-DHFR-cMyc protein are indicated. This experiment was performed in three biological replicates, and a representative experiment is shown. B, import of radiolabeled Hsp60_L-DHFR-cMyc protein into the mitochondrion of rTom22/Δtom22 parasites grown for 0 (no ATc) or 2 (d2 +ATc) days on ATc. Intracellular parasites were incubated for 10, 30, and 60 min in growth medium containing [³⁵S]cysteine and [³⁵S]methionine. Proteins were extracted in detergent, and Hsp60_L-DHFR-cMyc protein was purified by immunoprecipitation before separation by SDS-PAGE. Precursor (pHsp60_L-DHFR-cMyc) and mature (mHsp60_L-DHFR-cMyc) isoforms of the Hsp60_L-DHFR-cMyc protein were detected. This experiment was performed in two biological replicates, and a representative experiment is shown. C, anti-Tom40 Western blotting of proteins extracted from rTom22/Δtom22 parasites grown for 0–3 days in ATc and separated by BN-PAGE. D, Western blotting of proteins extracted from rTom22/Δtom22 parasites grown in the absence (top) or presence of ATc for 2 days (bottom). Proteins were separated by BN-PAGE in a first dimension and SDS-PAGE in a second dimension and then probed with anti-Tom40 antibodies. E and F, fluorescence growth assays of rTom22/Δtom22 parasites (E) and rTom22/Δtom22 (F) complemented with DD-tagged wild-type TgTom22 (rTom22/Δtom22/DD-Tom22WT) and grown in the absence (black) or presence (red) of ATc or in the presence of ATc and Shld1 (blue). Note that the red +ATc line in E is not visible behind the blue +ATc/+Shld1 line. Error bars represent the standard deviation of three technical replicates. G, anti-Tom40 Western blotting of proteins extracted from rTom22/Δtom22/DD-Tom22WT parasites separated by BN-PAGE. Parasites were grown in the absence of ATc or in the presence of ATc for 2 days. Parasites grown in ATc were also grown for 0–48 h in Shld1.