Merozoite of a schizont bounded by parasite plasma membrane (PPM) within the PVM and erythrocyte membrane. PfSUB1 and PMX are stored in exonemes. C1 blocks exoneme and microneme discharge, while PMX inhibitor WM4 and dual PMX/PMIX inhibitor WM382 block parasitophorous vacuole membrane (PVM) and erythrocyte membrane degradation as SUB1 is inactive. b Following exoneme fusion and discharge, SUB1 and PMX are discharged into the parasitophorous vacuole (PV). Here, SUB1 processes several PV-resident proteins (such as SERA5) culminating in a ruptured PVM and a permeabilised erythrocyte membrane. A subclass of microneme is postulated to contain EBAs, PfRhs, AMA1, PMX and PCRCR. In this study, ~30% of PfRh5 was PMX-processed to the Rh5proc form, while 70% remains unprocessed, as depicted in the microneme contents. Rh5proc: PMX-processed PfRh5; Rh5unproc: Unprocessed PfRh5; Rh5PD; PfRh5 prodomain. c Following microneme fusion and discharge, the EBAs, PfRhs and PCRCR complex are translocated to the parasite membrane at the apical tip, while AMA1 initially inserts into the merozoite membrane, then spreads over the merozoite surface. Further membrane lytic events result in a degraded PVM and perforated erythrocyte membrane. d, e Merozoites egress from the erythrocyte ready to engage fresh erythrocyte. PfRh and EBAs are involved in early invasion steps, followed by binding of PCRCR to basigin (BSG), which culminates in release of RON complex proteins into the RBC membrane. Processed AMA1 on the merozoite surface engages with inserted RON complex, to form the tight junction for merozoite entry. We propose that SUB2 ‘sheddase’ is contained in a second microneme subset also containing PMX, that fuses at the rhoptry neck and merozoite membrane, releasing activated SUB2 to shed proteins from the merozoite surface. f Proteins from the merozoite fraction of a 3D7 processing inhibition assay treated (+/−) C1, were probed with an anti-Rh5 mAb and the PfRh5 unprocessed and processed band intensities calculated. Mean values are shown for the level of PfRh5 processing, shown in the histogram below. Band intensities for PfRh5 unprocessed/processed were calculated from Control and C1-treated merozoites in three independent experiments. Error bars represent standard deviation. g, h Data comparing PfRh5 processing levels in C1-treated merozoites (Fig. 6f) suggests two possible models for microneme heterogeneity. The first model (g) posits there are two early microneme subsets (type 1 and 2), only one of which contains PMX. Under control conditions, this subset fuses at the rhoptry neck before PfRh5 has been completely PMX-processed, giving a mix of 28% processed and 34% unprocessed. The other subset has no PMX, so upon rhoptry neck fusion, contributes a further 38% unprocessed PfRh5. Under C1 conditions where there is no rhoptry fusion, PfRh5 processing in the microneme subset goes to completion at 62% and leaving 38% unprocessed. Triglia T, Scally SW, Seager BA, Pasternak M, Dagley LF, Cowman AF. Plasmepsin X activates the PCRCR complex of Plasmodium falciparum by processing PfRh5 for erythrocyte invasion. Nat Commun. 2023 14(1):2219.