RhopH2 and RhopH3 export enables assembly of the RhopH complex on P. falciparum-infected erythrocyte membranes

RhopH complexes consists of Clag3, RhopH2 and RhopH3 and are essential for growth of Plasmodium falciparum inside infected erythrocytes. Proteins are released from rhoptry organelles during merozoite invasion and trafficked to the surface of infected erythrocytes and enable uptake of nutrients. RhopH3, unlike other RhopH proteins, is required for parasite invasion, suggesting some cellular processes RhopH proteins function as single players rather than a complex. We show the RhopH complex has not formed during merozoite invasion. Clag3 is directly released into the host cell cytoplasm, whilst RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Export of RhopH2 and RhopH3 from the parasitophorous vacuole into the infected erythrocyte cytoplasm enables assembly of Clag3/RhopH2/RhopH3 complexes and incorporation into the host cell membrane concomitant with activation of nutrient uptake. This suggests compartmentalisation prevents premature channel assembly before intact complex is assembled at the host cell membrane.

Recombination of the homology regions with the homologous sequences in the genomic DNA enables construct integration and the tagging of the endogenous protein. We didn't manage to obtain fluorescently-tagged Clag3.1 but the same approach was utilised to tag it with HA or

Supplementary Figure 2. Processing of RhopH3 using double-tagged line
Parasites with RhopH3 tagged with mScarlet at the N-terminus and mNeonGreen at the Cterminus were imaged using live super-resolution microscopy revealing the presence of both termini in the rhoptries. This indicates that either full-length RhopH3 or processed RhopH3 with both termini associated are trafficked to rhoptries. On the other hand, membrane associated signal contains no mScarlet and only mNeonGreen suggesting it comes from the cleaved C-terminus. Details in the main body of the manuscript. Scale bar 2 μm.
Supplementary Figure 3. RhopH2 and RhopH3 colocalization using live super-resolution microscopy. Parasites expressing both RhopH2-mNeonGreen and RhopH3-mScarlet were imaged live using super-resolution microscopy at trophozoite and early to late schizont stages.
Scale bar 2 µm. Intensity plots along the drawn line are displayed on the right side and arrows point to peaks of intensity. The peaks show colocalization of RhopH2 and RhopH3 in the rhoptries. The colocalization is weak at early stages as evident from peaks of one colour (late trophozoite and schizont, arrows). The colocalization of both proteins increases in late schizonts shortly before the egress as evident from the intensity peaks displaying strong signals of both mScarlet and mNeonGreen (a) and the percentage of colocalising loci in early vs late schizonts is quantified in (b).

Supplementary Figure 4. The localization of RhopH3-HA-mNeonGeen during invasion
Merozoites of the RhopH3-HA-mNeonGreen were fixed during invasion on red blood cells at 1 min 30 sec or 10 min time points. RON4 (in magenta) was used as a marker of the tight junction to assess the stage of invasion (from early invasion to a complete entry). Scale bar 2 µm. Despite the processing of the C-terminal tag (Supplementary Figure 2), the localization of RhopH3-HA-mNeonGreen is similar to the one in Figure 2 of the main manuscript body, where N-terminally Flag-tagged RhopH2 was used. These data suggest that upon successful invasion, RhopH3 is localised in the newly-formed parasitophorous vacuole. Protein ladder shows molecular weights in kDa.

Supplementary Table 1. List of proteins detected by Mass Spectrometry following RhopH-
HA pulldown from various developmental stages: free merozoites, rings and trophozoites.
Wild type 3D7 parasites were used as a control. The table shows the log2 fold change compared to the control sample and the values have been colour-coded with the highest values in blue and the lowest in red. Statistical significance (p-value) is shown on the right and results defined as real changes have been highlighted on the right-hand side as TRUE (light green) and FALSE (red) for not-statistically significant differences. Each sample is an average of 3 biological replicates.

Supplementary Table 2. List of proteins detected by Mass Spectrometry in the purified
RhopH complex following size-exclusion chromatography ( Figure 5). This complex was used in the membrane incorporation studies (Figure 5b and 5c).
POPE was purchased from Anatrace, PA from sigma and the rest of the lipids from Avanti Polar lipids Inc.

FRET
Samples were imaged on a Zeiss 980 confocal microscope with a 63x 1.4 NA oil immersion objective. Both the donor (mNeonGreen or Alexa-488) and acceptor (mScarlet or Alexa-594) channels were imaged for either 20 or 40 frames before the acceptor was photobleached. Both channels were imaged for 20 or 40 frames after acceptor photobleaching. The mean intensity of the photobleached region in both channels were calculated for the entire time series.