The first 69 residues of KAHRP or 99 residues of GBP130, containing a native or mutated PEXEL,were fused to GFP or YFP. All GBP130 chimaeras and the KAHRP_WT chimaera were expressed from the HSP86 promoter (19). The remaining KAHRP chimaeras were expressed from the PfCRT promoter as in-frame fusions with GFPmut2.

Immunoblot(A) and coomassie gel (B) of the GBP130_WT chimaera after affinity purification from the saponin supernatant. Immunoblot (C) and coomassie gel (D) of the KAHRP_WT chimaera after affinity purification from the saponin supernatant. The bands indicated by an arrow in (B) and (D) were excised and subjected to MS. The lowest band (∼26 kDa) is degraded protein (YFP only). E) Mass spectra of the most N-terminal peptide from the band in (B) showing that exported GBP130 was processed in the PEXEL after leucine and acetylated at the new N-terminus.

Immunoblot(A) and coomassie gel (B) of KAHRP_R>A chimeric proteins after immunoaffinity purification from the saponin pellet. The bands indicated by arrows in (B) were excised and subjected to MS. C) Mass spectra of the most N-terminal peptide from the second upper band (▪) in (B) showing that the chimaera is processed at the site predicted by SignalP (³²LKC-SNN³⁶) and N acetylated. D) Mass spectra of the most N-terminal peptide from the uppermost band (♦) in (B) showing that the chimaera contains residues present in the signal sequence (i.e. N-terminal to the SignalP processing site; ³¹VLKC³⁴) and has retained the signal sequence.

Images captured by immunofluorescence microscopy show substantial colocalisation of GBP130 chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild-type PEXEL chimaera is efficiently exported to the erythrocyte cytosol (uppermost panels). All mutants show some accumulation within the parasitophorous vacuole and low-level fluorescence in the erythrocyte cytosol (lower panels), and this is evident, upon careful examination, in previously published images of these chimaeras (19). The presence of GBP130_RILE>A in the saponin supernatant in Figure 4 suggests localisation predominantly in the parasitophorous vacuole (signal sequence processed). This is confirmed by immunofluorescence microscopy (compare GBP130_RILE>A in this figure to KAHRP_RLQ>A in Figure 6, the latter of which retains the signal sequence and localises more in the ER as a result). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.

Two proposedmodels of PEXEL-mediated export are shown (A, 1 and 2). A1) After cotranslational insertion through Sec61 at the rough endoplasmic reticulum (rER) using the signal sequence, proteins to be exported are either processed by signal peptidase (red pac-man) or sequestered and/or processed by the PEXEL protease (yellow pac-man) and sorted at the transitional endoplasmic reticulum (tER) for transport through the Golgi to the parasitophorous vacuole by the default secretory pathway. There, proteins to be exported (xE/Q/D after PEXEL processing; green protein), are recognised and trafficked across the parasitophorous vacuole membrane by a translocon. Secreted or mutated PEXEL proteins are depicted as red proteins. A2) After entry at the rER and sequestration and/or processing by either signal peptidase (red pac-man) or the PEXEL protease (yellow pac-man), proteins to be exported are differentially sorted either at the tER or at the Golgi into vesicles. This may occur through a specific transmembrane PEXEL cargo receptor that enriches functionally distinct vesicles for exported proteins (green proteins), which are targeted to subcompartments (the ‘necklace of beads’; depicted as white compartments in the parasitophorous vacuole) of the parasitophorous vacuole that houses the translocon. Exported transmembrane proteins then presumably diffuse laterally from the translocon and traffic with forming Maurer's clefts (white structures in the erythrocyte). Secreted proteins (red) traffic through the default secretory pathway to alternative compartments of the parasitophorous vacuole that do not contain the translocon. The default pathway may involve bulk flow, depicted as free red proteins in the ER that ‘sample’ the budding membrane. Uncharacterised vesicles are depicted by ‘?’. Close up (box) of the possible sorting mechanism is shown in (B) and (C). B) After cotranslational insertion into the rER PEXEL proteins are sequestered/processed by the PEXEL protease and sorted into vesicles through a transmembrane cargo receptor that interacts with the COPII machinery. C) Secreted proteins are not recognised by the PEXEL receptor but bind either a different receptor or traffic through bulk flow. The role of the Golgi is unclear but similar sorting may occur there.

Immunoblots with α-GFP antibodies against KAHRP chimaeras from tetanolysin pellets (A) and supernatant (B) or GBP130 chimaeras from tetanolysin pellets (C) and supernatant (D) are shown. Antibodies to aldolase (48) were used as a permeabilisation control, as described previously (49) and reflects the quantity of protein loaded in each lane. While equal proportions of pellet and supernatant were loaded for each chimaera, equal loadings could not be achieved between different chimaeras because of differences in episomal expression. The densitometric analyses below (see G and H) were thus limited to comparing only between pellet and supernatant fractions of the same chimaera. Upper bands in (D, lanes 2 and 3) represent slight vacuolar leakage. E) Predicted protein sizes of KAHRP chimaeras after differential N-terminal processing; ♦ represents full-length chimaeras with signal sequence; ▪ represents processing at/near the site predicted by SignalP; H represents chimaeras processed downstream of prediction by SignalP (i.e. within the PEXEL); ▴ represents degradation to GFP/YFP only (confirmed by MS; Figure S2). The linker upstream of YFP in KAHRP_WT (Figure 1) is not depicted here. While the N-termini of each KAHRP chimaera are the same, except for the mutations shown, the C-terminal YFP linker in KAHRP_WT explains the minor size shift in (A) and (B) between WT and other chimaeras processed at the PEXEL (i.e. those depicted with *). F) Predicted protein sizes of GBP130 chimaeras after differential N-terminal processing. Varying exposures within the linear range of the blots represented in (A–D) were scanned at high resolution and densitometry was undertaken to approximately quantify the differential N-terminal processing and cellular localisation of KAHRP (G) and GBP130 (H) chimaeras. For each chimaera, percentages were calculated by dividing the intensity of each band in the supernatant (exported to host) or pellet (ER or parasitophorous vacuole) by the sum of the band intensities for that chimaera (total tagged chimaera) and multiplying by 100. Percentages between chimaerae are directly comparable. WT, wild type; SS, signal sequence; PV, parasitophorous vacuole.

Immunoblot with α-GFP antibodies against GBP130 chimaeras fractionated with saponin indicates that processing occurred in the parasite fraction (pellet) before export. Antibodies to aldolase were used to validate parasite membrane integrity following saponin treatment (note the presence of aldolase in the pellet fractions but absence from the supernatant fractions) and reflects the quantity of protein loaded in each lane. More of the GBP130_R>A and GBP130_L>A chimaera samples were loaded because of rapid loss of fluorescence over time in parasites overexpressing these mutant chimaeras. The GBP130_RILE>A chimaera traffics predominantly to the parasitophorous vacuole (see supernatant fraction). That unprocessed bands of GBP130_R>A, GBP130_L>A and GBP130_RILE>A are present in the supernatant fraction confirms breakdown of the parasitophorous vacuole membrane by saponin.

Images captured by immunofluorescence microscopy show substantial colocalisation of KAHRP chimaeras with the endoplasmic reticulum protein ERC (left panels). Intraparasitic fluorescence is also evident when chimaeras were colocalised with the parasitophorous vacuole membrane protein EXP-2 (right panels). Only the wild type (WT) PEXEL chimaera (uppermost panels) is efficiently exported to the erythrocyte cytosol, but low-level fluorescence in the erythrocyte cytosol was observed for the KAHRP_Q>A chimaera. All mutants show some accumulation within the parasitophorous vacuole but while the KAHRP_Q>A chimaera colocalises with ERC less than the other KAHRP mutants (left panel), it accumulates more in the parasitophorous vacuole in slightly older parasites (right panel). Accumulation of the KAHRP_Q>A chimaera in the parasitophorous vacuole appeared inverse to the ‘necklace of beads’ observation described previously (7; see yellow arrows in right panel). The white bar in the last panel corresponds to 2 μm for each of the panels within the figure.

Immunoblot (A) and coomassie gel (B) of the GBP130_E>A chimaera after affinity purification from the saponin pellet. Immunoblot (C) and coomassie gel (D) of the GBP130_E>A chimaera after affinity purification from the saponin supernatant. The bands indicated by an arrow in (B) and (D) were excised and subjected to MS. The lowest band (∼26 kDa) is degraded protein (YFP only). E) Mass spectra of the most N-terminal peptide from the band in (B) showing that GBP130_E>A was processed in the parasite (pellet) within the PEXEL after leucine and N acetylated. F) Mass spectra of the most N-terminal peptide from the band in (D) showing that GBP130_E>A in the parasitophorous vacuole (supernatant) was processed in the PEXEL after leucine and N acetylated.