ARTICLES https://doi.org/10.1038/s41564-018-0219-2 1 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia. 2 Department of Medical Biology, University of Melbourne, Parkville, Victoria, Australia. 3 Burnet Institute, Melbourne, Victoria, Australia. *e-mail: cowman@wehi.edu.au P lasmodium falciparum, the major human malaria parasite, causes over 450,000 deaths each year 1 . Disease symptoms occur during the asexual stage of the life cycle, in which the para- site invades human erythrocytes. Following invasion, the parasite induces major changes to its biochemical and biophysical properties, a process known as remodelling (reviewed in ref. 2 ). Here, hundreds of parasite proteins are trafficked via the parasite export pathway to their final destinations in the erythrocyte cytoplasm or specific membranes of the host cell 3 . At this stage, the parasite resides in a membrane-bound parasitophorous vacuole that separates it from the erythrocyte cytosol. The presence of the parasitophorous vacu- ole membrane prevents classical secretory trafficking beyond its confines; thus, additional parasite-derived elements are required for the export of effector proteins into the host cell. For export, ~300 exported parasite proteins contain a signal sequence and a downstream pentameric amino-terminal motif known as the Plasmodium export element (PEXEL) or the vacu- olar transport signal motif, RxLxE/Q/D, at the N terminus 4,5 . It is thought that these PEXEL proteins gain access to the endoplasmic reticulum (ER) lumen through their hydrophobic signal sequence 6 . Contrary to classical eukaryotic signal sequences, many PEXEL proteins generally have a hydrophobic sequence indented from the N terminus, known as recessed signal sequences. It is hypothesized that recessed signal sequences are removed by signal peptidase, fol- lowed by PEXEL cleavage 7,8 by the ER-localized aspartyl protease plasmepsin V (PMV) 9,10 . PMV cleaves at the conserved P 1 Leu resi- due and the resulting mature protein is acetylated and exported into the host cell 9–11 . PMV is essential for the maturation of effector proteins and can be inhibited either by the expression of a dominant-nega- tive mutant of the protease 10 , conditional gene knockdown 12 or inhibition with peptidomimetic compounds 12,13 . PMV inhibition causes an accumulation of uncleaved PEXEL proteins both with and without their signal sequences intact, suggesting that these signal sequences of PEXEL proteins are inefficiently cleaved 8,9,12 . Thus far, the molecular basis for the recruitment of PEXEL proteins for export by PMV is unknown. The current hypothesis suggests that proteins destined for export enter the ER via the Sec61 translocon where the signal sequence is co-transla- tionally cleaved by signal peptidase (PfSPC21), then PEXEL rec- ognition by PMV, followed by cleavage and subsequent export to the host cell 14 . In this study, we identified unique PMV-interacting proteins and the mechanism by which a large subset of PEXEL effector proteins are recognized for export. We show that the P. falciparum ortho- logue of signal peptidase complex 25 (PfSPC25), a non-catalytic component of the signal peptidase complex (SPC) 15 , binds to PMV and is required for entry of effector cargo into the ER. PfSPC25 and PMV also bind to the Sec61 ER translocon as well as an ancillary protein PfSec62, which identifies a critical subset of effector pro- teins for export. This interactome is functionally distinct to the established Sec61–SPC that is required for classical secretion. These results imply that these PEXEL proteins are recognized at transla- tion and are sorted to a distinct location at the ER membrane to initiate export. Results PMV interacts with PfSPC25 in the P. falciparum ER. We used stable isotope labelling of amino acids in cell culture (SILAC) and liquid chro- matography–tandem mass spectrometry analysis (LC–MS/MS) to identify specific PMV-interacting proteins. Transgenic P. falciparum strains expressing carboxy-terminal haemagglutinin (HA) epitope- tagged PMV (PMV-HA) 12 and, as a control, signal peptide pepti- dase (PfSPP-HA) (PF14_0543) were used. SPP is a protease of the Plasmepsin V cleaves malaria effector proteins in a distinct endoplasmic reticulum translocation interactome for export to the erythrocyte Danushka S. Marapana 1,2 , Laura F. Dagley 1,2 , Jarrod J. Sandow 1,2 , Thomas Nebl 1,2 , Tony Triglia 1 , Michał Pasternak  1,2 , Benjamin K. Dickerman 3 , Brendan S. Crabb 3 , Paul R. Gilson 3 , Andrew I. Webb 1,2 , Justin A. Boddey  1,2 and Alan F. Cowman  1,2 * Plasmodium falciparum exports hundreds of virulence proteins within infected erythrocytes, a process that requires cleavage of a pentameric motif called Plasmodium export element or vacuolar transport signal by the endoplasmic reticulum (ER)-resident protease plasmepsin V. We identified plasmepsin V-binding proteins that form a unique interactome required for the transloca- tion of effector cargo into the parasite ER. These interactions are functionally distinct from the Sec61–signal peptidase complex required for the translocation of proteins destined for the classical secretory pathway. This interactome does not involve the signal peptidase (SPC21) and consists of PfSec61, PfSPC25, plasmepsin V and PfSec62, which is an essential component of the post-translational ER translocon. Together, they form a distinct portal for the recognition and translocation of a large subset of Plasmodium export element effector proteins into the ER, thereby remodelling the infected erythrocyte that is required for parasite survival and pathogenesis. NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology