Fusion with the redox sensitive BPTI reveals a second translocation step for TM proteins. ( A-D  and  F-L ) Representative images of live  P .  falciparum  parasites expressing the constructs shown schematically above each panel. Hydrophobic regions (SP, signal peptide; TM, transmembrane domain) are in black, the PEXEL motif in yellow. Numbers refer to amino acids (aa). Red boxes labelled C, additional REX2 C-termini. Interrupted yellow box, mutated BPTI (BPTImut). DIC, differential interference contrast. Size bars: 5 μm. ( E ) Schematic for the protease K (PK) protection assay. Left, intact infected RBC with 3 possibilities (I, II, III) for the location of the fusion construct: I, protein is integral to PVM; II, protein is freely accessible in the PV; III, protein is integral to PPM. Middle, after permeabilisation of the erythrocyte plasma membrane (EPM) with tetanolysin the N-terminus of the construct will be digested if it is in the PVM (I), but remains intact in situation II and III. Right, after permeabilisation of the PVM with saponin, the constructs will be digested if it is in the PVM (I) or the PV (II) but if in the PPM (III), an N-terminally truncated fragment will be generated. Red, exported protein; white box, TM; yellow, BPTI with double cysteine bonds; green, GFP. ( F ) Western analysis of a protease protection assay according to ( E ). Digestion is visible after saponin treatment only. As no protected fragment is detectable, the protein is freely accessible in the PV (situation II). The faint bands (asterisk) represent protein degraded down to GFP. REX2-BPTI-GFP was detected using anti-GFP antibodies. Control for PVM integrity was SERA5 (PV resident), for the PPM aldolase (resident in parasite cytoplasm). Release of REX3 (resident of host cell cytosol) demonstrated efficient permeabilisation of the EPM. The marker is indicated in kDa. ( G ) Schematic of the location of REX2 based on the protease protection assay shown in ( F ). Translocation machines are indicated as two black ellipses. Other features are as in ( E ).