EMBO/SourceData huggingface dataset:
Figure 1A. ATPase activity of ABCC3 wild-type (WT) and E1451Q in nanodiscs. The data points for WT and E1451Q are fitted with a Michaelis-Menten equation. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.B. Substrate-stimulated ATPase activities of ABCC3 WT and E1451Q upon addition of substrate E217βG or DHEAS. All data points are the average of three independent experiments in nanodiscs, and error bars represent standard deviation.
Figure 2C. Relative ATPase activities of ABCC3 WT and mutants in the presence of 2 mM ATP upon addition of E217βG. The relative activity represents the substrate-stimulated activity of ABCC3 WT or its mutants harboring mutation of residues at the substrate-binding pocket. For (C) the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.D. Transport [3H]-E217βG activity assays of ABCC3 WT and mutants. For (D), the activities of ABCC3 WT are set as 100%. One-way analysis of variance (ANOVA) is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.
Figure 3C. Relative ATPase activities of ABCC3 WT and mutants in nanodiscs upon addition of DHEAS and 2 mM ATP. The relative activity represents the substrate-stimulated activity of ABCC3 WT or mutants harboring mutation of residues at the substrate-binding pocket.E. Relative ATPase activities of ABCC3 WT and mutants K318A, T535A upon addition of E217βG. For (C) and (E), the activities of ABCC3 WT are set as 100%. One-way ANOVA is used for the comparison of statistical significance of mutants and WT. The P values of <0.05, 0.01, and 0.001 are indicated with *, ** and ***, respectively.
Figure 1(E) Western blot analysis with an anti-HA antibody or in-house-generated anti-SMC3 antibody (indicated to the left of each blot) of nuclear extracts from synchronous SMC1-3HA-dd (D) or STAG-3HA-dd (E) late-stage parasites before (input, "In") and after (flow-through, "FT") immunoprecipitation with an antibody against HA ("𝛼-HA IP"). An antibody against histone H3 is used as a control. Immunoprecipitation with IgG ("IgG IP") was used as a control in the right panel of (E). Molecular weights are shown to the right. SMC1-3HA-dd has a predicted molecular weight of approximately 230 kDa and STAG-3HA-dd has a predicted molecular weight of approximately 298 kDa (of which 3.3 and 11.9 kDa correspond to the 3HA tag and the ddFKBP, respectively).
Figure 2Western blot analysis of nuclear extracts of ring (R), trophozoite (T), and schizont (S) stages from a synchronous population of SMC3-3HA-glmS parasites. SMC3-3HA is detected with an anti-HA antibody. An antibody against histone H3 is used as a control. Molecular weights are shown to the right. SMC3-3HA has a predicted molecular weight of 147.3 kDa (of which 3.3 kDa correspond to the 3HA tag).(C) Immunofluorescence assays of fixed RBCs infected with trophozoite or schizont stage SMC3-3HA-glmS parasites. DNA was stained with DAPI (blue) and SMC3-3HA was detected with anti-HA (green in B and magenta in C) antibody. HP1 was detected with anti-HP1 antibody (green in C). DIC, differential interference contrast. Scale bars equal 10 µm (B) and 5 µm (C).
Figure 3Zoomed-in view of SMC3 ChIP-seq data from clone A corresponding to chromosome 4 (604,345 - 709,167 bp), including the centromere (represented with dark red line below the x-axis). For 12 (blue), 24 (coral), and 36 (green) hpi, the y-axis is enrichment (ChIP/Input), with vertical lines below representing significant peaks obtained from peak calling algorithm macs2 (present in both clones, q-value < 0.05). The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction.
Figure 4Western blot analysis of nuclear extracts at 12, 24, and 36 hpi from a clonal population of SMC3-3HA-glmS parasites in the absence (-) or presence (+) of glucosamine (GlcN). SMC3-3HA is detected with an anti-HA antibody. An antibody against histone H3 is used as a control. Molecular weights are shown to the right.Growth curve showing parasite growth rate (parasitemia at Day X/parasitemia at Day 1) over five days for WT and two clones of SMC3-3HA-glmS parasites in the absence or presence of glucosamine (GlcN). Glucosamine treatment was started 96 h (two IDC cycles) before Day 1 to ensure SMC3 knockdown during the days sampled Uninfected red blood cells (Blood) served as reference of background. Error bars indicate standard deviation of three technical replicates (n = 3). A two-way ANOVA with Tukey post hoc test was used for statistical analysis. No significant differences were found.
Figure 5RNA-seq of an SMC3-3HA-glmS clone shows smc3 transcript levels (FPKM) at 12 (blue, q = 8.5 x 10-3), 24 (coral, q = 1.3 x 10-39), and 36 (green, q = 4.1 x 10-54) hpi in the absence (filled circles) or presence (empty circles) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 q = Bonferroni corrected P-value. Asterisks indicate q values < 0.5.
Figure 6ChIP-seq data showing enrichment of SMC3 (y-axis = ChIP/Input) at 12 (blue), 24 (coral), and 36 (green) hpi in clonal SMC3-3HA-glmS parasites at the rhoptry-associated protein 2 (rap2, PF3D7_0501600) and the glideosome-associated protein 45 (gap45, PF3D7_1222700) gene loci. The x-axis is DNA sequence, with the gene represented by a black box with white arrowheads to indicate transcription direction. One replicate (clone A) was used for ChIP-seq. ATAC-seq data from closely corresponding time points (15, 25, and 35 hpi) are shown in grey, with the y-axis representing ATAC-seq (RPM)/gDNA (RPM).RNA-seq of an SMC3-3HA-glmS clone shows transcript levels (FPKM) for rap2 (PF3D7_0501600) at 12 (q = 3 x 10-2) and 24 (q = 3.3 x 10-2) hpi and gap45 (PF3D7_1222700) at 12 (q = 8 x 10-1) and 24 (q = 1.6 x 10-2) hpi in the absence (black) or presence (grey) of glucosamine (GlcN). Error bars represent standard deviation of three technical replicates except for the untreated 12 hpi parasites, for which there were two replicates. P-values are calculated with a Wald test for significance of coefficients in a negative binomial generalized linear model as implemented in DESeq2 (Love et al, 2014). q = Bonferroni corrected P-value.Expression values of rap2 (PF3D7_0501600) and gap45 (PF3D7_1222700) genes across the IDC (indicated on the x-axis by hpi) from the transcriptomics time course Data corresponding to 12 (blue), 24 (coral), and 36 (green) hpi time points are highlighted.
Figure 1(A-B) Mitochondria isolated from (A) hTim8aKO HEK cells with and without hTim8aFLAG re-expression, or (B) hTim8bKO HEK cells with and without hTim8bFLAG re-expression were compared to mitochondria isolated from control HEK293 cells by SDS-PAGE and immunoblot. Protein expression of the hTim8 protein was induced with tetracycline (Tet) for the indicated time. Representative of n=2 biological replicates.Affinity enrichment mass spectrometry. Mitochondria isolated from (C) hTim8aKO HEK cells expressing hTim8aFLAG, (D) hTim8bKO HEK cells expressing hTim8bFLAG, (E) hTim8aMUT SH cells expressing hTim8aFLAG, (F) hTim8bKO SH cells expressing hTim8bFLAG were treated with DSP crosslinker prior to immunoprecipitation with anti-FLAG resin. The log2 fold change in mean LFQ intensity is plotted against unpaired Student's t-test p-value (n=3 biological replicates). Curve indicates significantly enriched proteins. FDR<0.01; (C) s0=5.5, (D) s0=4.5, (E) s0=2, (F) s0=5, Functional annotations manually curated and coloured: TIMM family proteins (purple), Complex I subunits and assembly factors (orange), Complex IV subunits and assembly factors (green), MIA import substrates (blue), significantly enriched (black).
Figure 2Changes in protein abundance of hTim8a and hTim8b interactors in mitochondria isolated from (B) hTim8aKO HEK and hTim8bKO HEK, or (C) hTim8aMUT SH and hTim8bKO SH cells; determined by quantitative mass spectrometry as published in Quadrants are labelled with 8a/8b ↑ or ↓ to indicate the increase or decrease of protein abundances in each knock-out cell line. The log2 fold change in mean LFQ intensity between knock-out and control cells is plotted on each axis. Lines indicate fold change in abundance of ±1.5. n=3 biological replicates for each cell line. Functional annotations manually curated and coloured: TIMM family proteins (purple), Complex I subunits and assembly factors (orange), Complex IV subunits and assembly factors (green), MIA import substrates (blue), significantly altered (black).
Figure 3(B-C) Mitochondria isolated from (B) COX17MUT HEK and (C) COX17KO SH cells were analysed by quantitative mitochondrial proteomics relative to control cells. The log2 fold change in mean LFQ intensity is plotted against unpaired Student's t-test p-value (n=3 biological replicates). Curve indicates significantly altered proteins; FDR<0.05; s0=0.5. Functional annotations manually curated and coloured: Complex I subunits and assembly factors (orange), Complex II subunits and assembly factors (yellow), Complex III subunits and assembly factors (red) Complex IV subunits and assembly factors (green), Complex V subunits and assembly factors (pink), TIMM family proteins (purple), significantly altered (black).(D) Mitochondria isolated from control cells, COX17MUT HEK and COX17KO SH cells were analysed by BN-PAGE and immunoblotting with the indicated antibodies. COX4I1-containing complexes indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Representative of n=2 biological replicates.(F) Intact cells or mitochondria isolated from hTim8bKO HEK and control HEK293 cells were subject to ICP-MS analysis to measure copper (Cu) and iron (Fe) content. Abundance is shown as mean ±S.D for metal content per gram of protein. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.(G) Recombinant COA6, hTim8a and hTim8b were purified and incubated, in increasing concentrations, with Cu(I) and bathocuproine disulfonic acid (BCS). Protein-Cu(I) binding outcompetes BCS2-Cu(I) binding and the decrease in BCS2-Cu(I) is shown by absorbance, particularly at 482 nm. n=1 biological replicates.
Figure 4(A) hTim8aKO HEK, hTim8bKO HEK and control HEK293 cells were pulsed with [35S]-Met/Cys for 2 h in the presence of anisomycin and chased for the indicated times. Isolated mitochondria were analysed by SDS-PAGE, auto-radiography and immunoblot. n=1 biological replicates.[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (B) hTim8aMUT SH control SH-SY5Y Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (C) hTim8bKO HEK and control HEK23 cells. Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.(F) Mitochondria isolated from control, hTim8aMUT SH and hTim8bKO HEK cells were analysed by BN-PAGE and immunoblotting. COX4I1-containing complexes indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Levels of COX4I1 present in subcomplex (*) was quantified as mean ±S.D percentage of COX4I1 present in mature Complex IV (CIV) for each cell line. Significance determined by unpaired Student's t-test (hTim8bKO HEK n=6 biological replicates; hTim8aMUT SH n=4 biological replicates). **, p<0.01.
Figure 5(B-C) Profile plots of iBAQ intensity for Complex IV subunits across the complexome profile of mitochondria isolated from (B) hTim8aMUT SH and control SH-SY5Y cells; and (C) hTim8bKO HEK and control HEK293 cells. Subunits are plotted by Complex IV module and iBAQ intensity relative to the maximum intensity of plotted subunits. Solid line indicates subunits from control cells, dashed line from knock-out cells. Complex IV assemblies are indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Representative of n=2 biological replicates. Data presented as iBAQ intensity, normalised as in (A) and expressed as a ratio of the maximum iBAQ intensity.
Figure 6(A) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria from control SH-SY5Y cells were analysed by SDS-PAGE and immunoblotting. Amount of protein after 12 h induction (lanes 4 and 10) was quantified as mean ±S.D percentage of control SH-SY5Y mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). *, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001; 'ns' indicates not significant, p>0.05.(C) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria isolated from control SH-SY5Y cells analysed by BN-PAGE and immunoblotting. COX4I1-containing complexes are indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Amount of COX4I1 present in mature monomeric Complex IV (CIV) was quantified as mean ±S.D percentage of control SH-SY5Y mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.001; ***, p<0.001; 'ns' indicates not significant, p>0.05.(D) Mitochondria isolated from hTim8bKO HEK cells with and without hTim8aFLAG expression compared to mitochondria isolated from control HEK293 cells by BN-PAGE. hTim8aFLAG expression induced by tetracycline for 8 h. Native protein complexes detected by immunoblot. COX4I1-containing complexes by immunoblot indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Amount of COX4I1 present in mature monomeric Complex IV (CIV) was quantified as mean ±S.D percentage of control HEK293 mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.
Figure 1C: Movie stills (first 4 panels in each row) and maximum intensity projections (5th panel in each row) of a 90-second video of P. berghei sporozoites undergoing normal circular gliding motility (top row) and patch gliding (bottom row). Scale bars: 5 µm.
Figure 2A: Numbers of oocysts in infected midguts of the different lines. Values (n) above dot plots indicate numbers of observed midguts. Shown are data from three biological replicates. Numbers above horizontal bars indicate p-values. Data were tested for significance using the Kruskal-Wallis-test. Images show cFluo oocysts in an infected midgut without (top) and with (bottom) green fluorescence. Scale bar: 200 µm.B: Western blots using an anti-TRAP antibody of the control lines cFluo and S210C in comparison to S210C/Q216C (open) and S210C/F224C (closed). Anti-CSP antibody mAb 3D11 was used as loading control, revealing the two expected CSP-bandsC: Surface expression of TRAP mutants on fixed but not permeabilized sporozoites. Differential interference contrast (DIC) and immunofluorescence images of representative examples of hemolymph derived sporozoites of the indicated parasite lines stained with anti-TRAP antibodies shown in red Hoechst was used to reveal the sporozoite nuclear DNA shown in blue. Cytoplasmic GFP or staining with anti-CSP antibodies was used to validate the integrity of sporozoites. Scale bar: 5 µm.
Figure 3A: Numbers of hemolymph (HL) derived sporozoites reveal no difference between the parasite lines. B: Numbers of salivary gland (SG) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines. C: Ratios of hemolymph (HL) derived versus oocyst-derived sporozoites reveal no difference between the parasite lines. D: Ratios of salivary gland (SG) derived versus hemolymph (HL) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines.
Figure 4A: Blood stage parasitemia after injection of 10,000 hemolymph (HL) derived sporozoites of the different lines into C57Bl/6 mice from day 3 to day 9 post infection. Shown is the mean +/- SEM. Only data of infected mice have been plotted. In shown are pooled data from 3 (S210C), 2 (S210C/F224C), 3 (S210C/Q216C) and 1 (wild type) biological replicates using 3 or 4 mice, respectively.B: Kaplan Meier plot showing the percentage of parasite free mice until day 9 of infection. , shown are pooled data from 3 (S210C), 2 (S210C/F224C), 3 (S210C/Q216C) and 1 (wild type) biological replicates using 3 or 4 mice, respectively.D: Blood stage parasitemia after C57Bl/6 mice were bitten by mosquitoes (ten mosquitoes/mouse) infected with the indicated parasite lines at day 0. Shown is the mean +/- SEM where appropriate. Only data of infected mice have been plotted for all but the S210C/F224C (closed I domain) infections, where all mice stayed negative. each condition represents pooled data from 3 biological replicates using 2-4 mice, respectively.E: Kaplan Meier plot showing the percentage of parasite free mice after mosquito bites until day 9 of infection. each condition represents pooled data from 3 biological replicates using 2-4 mice, respectively.G: Blood stage parasitemia after injection of 10.000 salivary gland (SG) derived sporozoites of the indicated parasite lines into C57Bl/6 mice at day 0. Shown is the mean +/- SEM. Only data of infected mice have been plotted. shown are pooled data from 3 (S210C), 2 (S210C/Q216C) and 3 (wild type) biological replicates using 2-4 mice, respectively.H: Kaplan Meier plot showing the percentage of parasite free mice after salivary gland sporozoite injection until day 9 of infection. shown are pooled data from 3 (S210C), 2 (S210C/Q216C) and 3 (wild type) biological replicates using 2-4 mice, respectively.
Figure 5A: Movement type analysis of hemolymph derived sporozoites (HLS) of the indicated parasite lines. Top: Classification of attached sporozoites by motility. Bottom: Classification by motility type. n indicates the number of observed sporozoites. Floaters have been omitted in the analysis presented in the top graph and are not included in n. Shown are pooled data from 3 (S210C), 2 (S210C/F224C), 2 (S210C/Q216C) and 2 (wild type) biological replicates. Shown is the mean +/- SEM.D: Percentage of gliding and non-gliding control hemolymph sporozoites (HLS) at increasing concentrations of DTT. n indicates the number of observed sporozoites. Floaters are included in the non-motile fraction. Each condition represents pooled data from 2 biological replicates. Shown is the mean +/- SEM. E: Percentage of gliding hemolymph sporozoites (HLS) expressing the open TRAP I domain at increasing concentrations of DTT. n indicates the total number of observed sporozoites. Floaters are included in the non-motile fraction. Shown are pooled data from 3 (0mM DTT), 2 (50mM DTT), and 3 biological replicates for concentrations of 100mM DTT in the S210C/Q216C line. For concentrations of 200mM and 330mM DTT in the S210C/Q216C line, data shown represent pooled data from 1 biological replicate. Several comparable screening experiments previously performed showed no productive motility at these concentrations. Shown is the mean +/- SEM.F: Maximum projections (1 min time lapse, 3 s per frame) of single S210C/Q216C sporozoites in presence or absence of 50 mM DTT. A projection from a motile sporozoite of the cFluo line treated with 50 mM DTT is shown as control. Scale bar: 5 µm.
Figure 1C The binding of His-SOS2 to different PA species. The amount of each PA spot is 5 nmol. PA species: natural PA from soy, dipalmitoyl PA (di16:0 PA), distearoyl PA (di18:0 PA), dioleoyl PA (di18:1 PA), dilinoleoyl PA (di18:2 PA), palmitoyl-oleoyl PA (16:0-18:1 PA), palmitoyl-linoleoyl PA (16:0-18:2 PA), stearoyl-oleoyl PA (18:0-18:1 PA), stearoyl-linoleoyl PA (18:0-18:2 PA). For Mock control, no PA was incubated with the proteins.D The His-SOS2 binding to the liposome containing PA. Liposomes were made up of 16:0-18:2 PC only or the mixtures of 16:0-18:2 PC+16:0-18:2 PA. After incubation with the recombinant proteins, liposomes were pelleted by centrifugation. The liposome-conjugated proteins in pellet and proteins remaining in supernatant were detected by immunoblot using anti-His antibody. For the Mock control, no liposomes were incubated with the proteins.E Microscale thermophoresis (MST) assay between His-SOS2 and PA. His-SOS2 was labeled and kept at a constant concentration (1.0 μM). His-RbohD1-100, as the negative control, was labeled and kept at a constant concentration (4.5 μM). PA was hydrated in TBST buffer (0.005% tween 20), and diluted 16 times in a 1:1 ratio.
Figure 2A The PA synthesis defective mutants pldα1 pldδ-1 and pldα1 pldδ-2 are salt-sensitive. Six-d-old seedlings were grown in half-strength Murashige and Skoog (MS), and then transferred to half-strength MS medium with or without 100 mM NaCl for 7 d.G, H Analysis of the average values of the net Na+ (G) and K+ (H) fluxes of Col-0, sos2, C-1 and C-2. Six-d-old seedlings were treated with or without 50 mM NaCl for 24 h. Error bars represent SD (n ≥ 3).I-N The contents of shoot Na+ (I), shoot K+ (J), root Na+ (K) and root K+ (L) as well as the relative ratios of shoot Na+/K+ (M) and root Na+/K+ (N) of Col-0 and pldα1 pldδ mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 100 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. O-T The contents of shoot Na+ (O), shoot K+ (P), root Na+ (Q) and root K+ (R) as well as the relative ratios of shoot Na+/K+ (S) and root Na+/K+ (T) of Col-0, sos2, C-1 and C-2 mutants. Six-d-old seedlings were transferred to half-strength MS medium with or without 50 mM NaCl treatment for 7 d. Error bars represent SEM of three independent biological repeats. D