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. 2017 Sep 12;6:e27480. doi: 10.7554/eLife.27480

Figure 4. ASP3 is involved in the processing of rhoptry proteins.

(A), (B) Immunoblots evaluating the processing of the rhoptry bulb proteins ROP1, ROP2-4, ROP7, ROP13, and ROP18 upon ASP3 knockdown. ROP18 was Ty-tagged at endogenous locus in ASP3-iKD. Catalase was used as a loading control. Arrowheads represent pro and mature forms of the proteins. (C), (D) Immunoblots evaluating the processing of the rhoptry neck proteins RON2, RON4, RON5 and RON9 upon ASP3 knockdown. RON2 and RON5 were Ty-tagged at the endogenous locus in ASP3-iKD. (E) ASP3ty but not asp3ty-D299A can rescue the processing of ROP2-4 in absence of ASP3myc. (F) IFAs evaluating localization of ROP1, ROP18, RON4, and RON9 upon ASP3 knockdown. No alteration was observed in absence of ASP3. (G) ASP3 depletion did not impact on the formation of the RON4-RON2 complex as demonstrated by co-immunoprecipitation of RON2 with RON4 antibodies. (H) Schematic for the processing events and products of TLN1. (I) Immunoblot evaluating TLN1 showed that ASP3 depletion abolished the pro-domain processing of TLN1.

Figure 4.

Figure 4—figure supplement 1. Effect of ASP3 depletion on rhoptry proteins.

Figure 4—figure supplement 1.

(A) and B) ASP3 knockdown did not impact on the localization of rhoptry proteins.
Figure 4—figure supplement 2. SφXE motifs in ROPs.

Figure 4—figure supplement 2.

Schematic of some ROPs and positions of the putative cleavage site motif SφXE (φ is hydrophobic, X is any amino acid). Green arrowheads represent sites that have been previously validated.
Figure 4—figure supplement 3. SφXE motifs in RONs.

Figure 4—figure supplement 3.

Schematic of some RONs and positions of the putative cleavage site motif SφXE (φ is hydrophobic, X is any amino acid).
Figure 4—figure supplement 4. SUB2 has no impact on the processing of ROPs/RONs/MICs.

Figure 4—figure supplement 4.

(A) Endogenously epitope tagged SUB2 in ASP3-iKD showed no alteration of processing upon condition knockdown of ASP3 compared to the parental RHΔku80 tagged strain. Catalase was used as loading control. (B) Schematic representation of the 2-gRNA CRISPR/Cas9 mediated knockout of SUB2 (in the SUB2ty strain) by replacing a region of the endogenous SUB2 with CAT cassette. (C) PCR analyses on the SUB2 knockout strains to check for integration of the cassette at the targeted locus. (D) Deletion of the SUB2 in the two knockout clones was assessed by western blot using Ty antibodies. Catalase was used as loading control. (E) SUB2 knockout did not impact on the processing of ROP1, ROP2-4, RON4, RON2, MIC3, M2AP, and MIC6. Catalase was used as loading control. Arrowheads indicate the pro and mature forms of the protein as described before.