Functional characterisation of components in two Plasmodium falciparum Cullin-RING-Ligase complexes

Abstract

Ubiquitination is the key eukaryotic post-translational modification that governs protein degradation, localisation, and activity which is mediated by a concerted enzyme cascade. The largest superfamily of these enzymes include the Cullin-RING-Ligase (CRL) complexes. Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, encodes the critical proteins required for ubiquitination, but we do not yet understand the function of this pathway. Here the P. falciparum CRL complexes were characterised to reveal an essential but minimal repertoire controlled by two Cullin scaffolds. A PfCullin1-linked CRL complex, recruiting a single substrate receptor, was identified as being required for parasite inner-membrane biogenesis and DNA replication. A second CRL complex functioning through a PfCullin4 scaffold was identified that utilised a previously unidentified adaptor protein and receptors to support DNA replication. These results show that the P. falciparum CRL complexes are essential in both nuclear maintenance and membrane integrity.

Keywords: P. falciparum; Cullin-ring-ligase; E3 ligase; Malaria; Ubiquitination.

Fig. 1

Identification of core PfCRL complex components. (a) Immunoblots showing C-terminal tagging of DiCre-PfRBX1-HA and DiCre-PfSKP1-HA transgenic strain parasites by anti-HA antibodies. (b) Immunofluorescence imaging of ring, trophozoite and schizont stages of DiCre-PfSKP1-HA parasites. PfSKP1, parasite ER membrane and DNA levels were visualised using anti-HA, anti-ERC antibodies and DAPI staining respectively. (c) Immunofluorescence imaging of ring, trophozoite and schizont stages of DiCre-PfRBX1-HA parasites. RBX1, parasite ER membrane and DNA levels were visualised using anti-HA, anti-ERC antibodies and DAPI staining respectively.

Fig. 2

PfRBX1 and PfSKP1 are essential for intra-erythrocytic P. falciparum survival. (a) Schematic of protein size of wild type, HA-tagged and truncated PfSKP1 in DiCre-PfSKP1-HA transgenic parasite strain. FBOX-binding domain in blue, HA tag in red and Neon Green domain in green colours respectively. (b) Schematic of protein size of wild type, HA-tagged and truncated PfRBX1 in DiCre-PfRBX1-HA transgenic parasite strain. RING domain in yellow, HA tag in red and Neon Green domain in green colours respectively. (c) Immunoblots of schizont stage parental DiCre, DICre-PfSKP1-HA and DiCre-PfRBX1-HA parasites treated with 0 or 10 nM rapamycin for 48 h. DiCre-Rapamycin mediated excision, protein loading, and total ubiquitination of parasites assessed by anti-HA, anti-Aldolase, and anti-Ubiquitin antibodies respectively. Full-length and truncated PfSKP1-HA and PfRBX1-HA is denoted by one and two blue circles respectively. (d) Analysis of ring stage DiCre, DiCre-PfSKP1-HA and DiCre-PfRBX1-HA parasites grown on DMSO or 10 nM Rapamycin for 3 intraerythrocytic cycles. Parasitemia is assessed as a percentage compared to growth of control parasite grown on DMSO. Data are mean ± s.e.m for n = 3 biological replicates with 2 technical duplicates and compared by two-way Anova (Dunnett’s multiple comparison test. (e) Growth of ring stage DiCre, DiCre-PfSKP1-HA and DiCre-PfRBX1-HA parasites on DMSO or 10nM Rapamycin for 1.5 intraerythrocytic cycles. Parasitemia was assessed every 24 h following treatment and assessed as a percentage compared to growth of control parasite grown on DMSO. Data are mean ± s.e.m for n = 3 biological replicates and compared by two-way Anova (Dunnett’s multiple comparison test. (f) Immunofluorescence imaging of DMSO (top panels) and Rapamycin (bottom panels) treated DiCre-PfRBX1-HA schizonts. Parasite membrane integrity and DNA levels visualised using anti-PfGAP45 antibodies and SPY505 DNA dye respectively. (g) Immunofluorescence imaging of DMSO (top panels) and Rapamycin (bottom panels) treated DiCre-PfSKP1-HA schizonts. Parasite membrane integrity and DNA levels visualised using anti-PfGAP45 antibodies and SPY505 DNA dye respectively. (h) Transmission electron microscopy of Rapamycin treated parental DiCre, DiCre-PfRBX1-HA and DiCre-PfSKP1-HA schizont stage parasites. The nuclei and rhoptries have been manually annotated and coloured blue and yellow respectively using a trace feature from Adobe Illustrator that identifies these organelles.

Fig. 3

Identification of PfSKP1 and PfRBX1 interactome. (a) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from parasite lysate comparing DiCre (green) with DiCre-PfSKP1-HA (red). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Proteins of interest to this study are further labelled with PlasmoDB accession number. (b) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from parasite lysate comparing DiCre (green) with DiCre-PfRBX1-HA (red). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Proteins of interest to this study are further labelled with PlasmoDB accession number. (c) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from schizont stage parasite lysate comparing DMSO (green) with Rapamycin (red). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Up and down regulated proteins of specific interest to this study are highlighted in green and red respectively. (d) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from schizont stage parasite lysate comparing DMSO (green) with Rapamycin (red). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Up and down regulated proteins of specific interest to this study are highlighted in green and red respectively.

Fig. 4

Identification of PfFBOX01 substrate receptor. (a) Immunoblots showing C-terminal tagging of DiCre-PfFBOX01 transgenic strain parasites by anti-HA antibodies. (b) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from parasite lysate comparing DiCre (red) and DiCre-PfFBOX01-HA (blue). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Proteins of interest to this study are further labelled with PlasmoDB accession number. (c) Immunoblots of schizont stage DiCre-PfFBOX01-HA parasites treated with 0 or 10nM rapamycin for 48 h. DiCre-Rapamycin mediated excision and protein loading assessed by anti-HA and anti-Aldolase antibodies respectively. Full-length and truncated PfRBX1-HA is denoted by one and two cyan circles respectively. (d) Growth curve of ring stage DiCre and DiCre-PfFBOX01-HA parasites grown on DMSO or 10nM Rapamycin for 1–3 intraerythrocytic cycles. Parasitemia is assessed as a percentage compared to growth of control parasite grown on DMSO. Data are mean ± s.e.m for n = 3 biological replicates with 2 technical duplicates and compared by two-way Anova (Dunnett’s multiple comparison test. (e) Immunofluorescence analysis of DMSO and Rapamycin treated DiCre-PfFBOX01-HA schizont stage parasites. FBOX01 levels, parasite membrane integrity and DNA levels were visualised using anti-HA, anti-PfGAP45 antibodies and DAPI staining respectively. Inset shows magnification of merozoite within the schizont stage parasite. (f) Transmission electron microscopy of Rapamycin treated DiCre-PfFBOX01-HA schizont stage parasites. The nuclei and rhoptries have been manually annotated and coloured blue and yellow respectively using a trace feature from Adobe Illustrator that identifies these organelles. (g) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from schizont stage parasite lysate comparing DMSO and Rapamycin treated DiCre-PfFBOX01-HA. Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Up regulated proteins of specific interest to this study are highlighted in green.

Fig. 5

Identification of MCM complex in P. falciparum. (a) Immunoblots showing C-terminal tagging of PfMCM4 in DiCre transgenic strain parasites by anti-Flag antibodies. (b) Volcano plot illustrating the log₂ protein ratios in anti-FLAG immunoprecipitations from parasite lysate comparing DiCre (red) and PfMCM4-Flag (blue). Proteins were deemed differentially regulated if the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. Proteins of interest to this study are further labelled with protein names. (c) Immunoblot of MCM4-Flag containing DiCre, PfSKP1-HA and PfRBX1-HA schizont stage parasites treated with DMSO or rapamycin. Anti-Flag, Aldolase and HA antibodies were used to identify levels of PfMCM4, loading and PfSKP1/PfRBX1 protein levels respectively. Densitometry of n = 2 western blots with mean ± s.d are plotted next to immunoblot. (d) Immunoblot of anti-Flag immunoprecipitation of MCM4-Flag from DiCre-PfSKP1-HA + MCM4-Flag transgenic schizont stage parasites pre-treated with DMSO or 10 nM Rapamycin. Input (I), Unbound (U) and Eluate (E) fractions were analysed using antibodies against MCM4 (anti-Flag), Ubiquitin (anti-ubiquitin) and PfSKP1 (anti-HA). Full-length and truncated PfSKP1-HA is denoted by one and two blue circles respectively. Note that the anti-HA panel has been overexposed so that the SKP1 HA-Neon Green fusion can be observed. (e) Immunoblot of anti-Flag immunoprecipitation of MCM4-Flag from DiCre-PfRBX1-HA + MCM4-Flag transgenic schizont stage parasites pre-treated with DMSO or 10nM Rapamycin. Input (I), Unbound (U) and Eluate (E) fractions were analysed using antibodies against MCM4 (anti-Flag), Ubiquitin (anti-ubiquitin) and PfRBX1 (anti-HA). Full-length and truncated PfRBX1-HA is denoted by one and two red circles respectively. (f) Immunofluorescence analysis of MCM4-Flag tagged DiCre-PfSKP1-HA strain schizont stage parasites. Parasites were treated with DMSO or Rapamycin to induce PfSKP1 deletion and PfMCM4, parasite membrane integrity and DNA levels were visualized with anti-Flag, anti-PfGAP45 antibodies and DAPI staining respectively. (g) Immunofluorescence analysis of MCM4-Flag tagged DiCre-PfRBX1-HA strain schizont stage parasites. Parasites were treated with DMSO or Rapamycin to induce PfRBX1 deletion and PfMCM4, parasite membrane integrity and DNA levels were visualized with anti-Flag, anti-PfGAP45 and DAPI respectively.

Fig. 6

Characterisation of PfCPSFA-HA and role in PfMCM4 levels. (a) Immunofluorescence analysis of DMSO treated DiCre-PfCPSFA-HA schizont stage parasites. PfCPSFA levels, parasite membrane integrity and DNA levels were visualised using anti-HA, anti-PfGAP45 antibodies and DAPI staining respectively. Inset shows magnification of merozoite within the schizont stage parasite. (b) Schematic of protein size of wild type, HA-tagged and truncated PfCPSFA in DiCre-PfCPSFA-HA transgenic parasite strain. CPSFA domain in blue, HA tag in red and Neon Green domain in green colours respectively. (c) Immunoblot of MCM4-Flag containing DiCre and PfCPSFA-HA schizont stage parasites treated with DMSO or rapamycin. Anti-Flag, Aldolase and HA antibodies were used to identify levels of PfMCM4, loading and PfSKP1/PfRBX1 protein levels respectively. Full-length and truncated PfCPSFA-HA is denoted by one and two green circles respectively. (d) Growth curve of ring stage DiCre and DiCre-PfCPSFA-HA parasites grown on DMSO or 10nM Rapamycin for 1–3 intraerythrocytic cycles. Data are mean ± s.e.m for n = 3 biological replicates with 2 technical duplicates and compared by two-way Anova (Dunnett’s multiple comparison test. Parasitemia is assessed as a percentage compared to growth of control parasite grown on DMSO. (e) Volcano plot illustrating the log₂ protein ratios in anti-HA immunoprecipitations from schizont stage parasite lysate comparing DMSO (red) and Rapamycin treated DiCre-PfCPSFA-HA (green). Proteins were deemed differentially regulated if they exhibited an adjusted p ≤ 0.05. Up and down regulated proteins of specific interest to this study are highlighted in green and red respectively.

Fig. 7

Model of PfCRL complex function in P. falciparum schizonts. PfSCF and PfCRL complexes identified to date function during P. falciparum schizogony and are essential for DNA replication, rhoptry biogenesis and normal merozoite development.