Surface iodination experiments followed by immunoprecipitation with a pool of human immune sera showed that both FCR3CD36 and D10 displayed a protein of apparent high molecular weight of approximately 300 kDa present in the TX100ins-SDSsol fraction. Whereas the positive control FCR3CD36 was readily digested by 10 mg/ml of trypsin (Fig. 2B and C, lanes 1–3), the PfEMP1 expressed by D10 was quite insensitive to digestion with even 100 mg/ml of trypsin (Fig. 2B and C, lanes 4–6) or 100 mg/ml of chymotrypsin (data not shown). A ,75 kDa fragment correspond- ing to the intracellular domain of PfEMP1 was detected by Western blotting exclusively in the FCR3CD36 trypsin-treated extracts reacted with anti-ATS antibodies (Fig. 2D lane 1–3). In contrast, these antibodies reacted with the full-length PfEMP1 of D10 treated extracts (Fig. 2D lane 4–6). PfEMP1 localization was also confirmed by surface IFA of live PRBC following trypsin treatment. As predicted surface labelling was sensitive to trypsin in FCR3 and resistant in D10 parasites (Fig. 2E). Furthermore, cell integrity was verified by propidium iodide stanning (data not shown). These findings indicated that a trypsin-resistant PfEMP1 is expressed on the surface of D10 that has lost its adhesive properties. Protein trafficking by P. falciparum D10 To further investigate the non-binding phenotype we analyzed by immunofluorescence whether intracellular protein trafficking was affected in D10 parasites. Several P. falciparum polypeptides are Adhesion Is Not Linked to clag9 Deletion exported through the red blood cell cytoplasm in association with the Maurer’s clefts, and these may be essential for the correct assembly of the cytoadherence complex at the PRBC surface [48]. PRBC double-labelled with the Maurer’s clefts marker Pf332 and PfEMP1 antibodies (Fig. 3A), showed that PfEMP1 was associated with the Maurer’s clefts in transit through the RBC cytoplasm. Comparable results were obtained with the FCR3CD36 PRBC (Fig. 3A), however, the D10 Maurer’s clefts labelling detected with anti-Pf332 appeared more diffuse than in FCR3CD36 suggesting a different morphology. A similar altered Maurer’s clefts architec- ture was observed in REX1 mutant parasites [49], one of the 25 genes lost in the chromosome 9 deletion of D10. Furthermore, mAb89 reacted with knobs at the RBC membrane of D10-infected erythrocytes indicating that KAHRP, which interacts with the PfEMP1 ATS region, is correctly addressed to the RBC membrane (Fig. 3B). These data suggest that trafficking and export of the studied marker proteins and PfEMP1 are apparently not affected in D10 parasites. Because PfEMP1 is anchored to the cytoskeleton of the infected erythrocyte through interactions with the knob component KAHRP, we verified if the knobs from D10 had a structure similar to parasites expressing functional PfEMP1 on their surface. The visualisation by AFM of the PRBC revealed only subtle changes in the knob morphology between D10 and FCR3CD36 (Fig. 4A). No difference was observed in the amount of knobs and their distribution on the erythrocyte surface for these strains. The Figure 3. Localization of parasite exported proteins. A. Double-labelling of synchronous air-dried D10 (top row) and FCR3CD36 (bottom row) with guinea pig anti-PfEMP1 antibodies (1:500) and purified rabbit anti-Pf332 IgG (1:200) revealed with a mixture of goat anti-guinea pig Alexa Fluor 488 (1:500) and Alexa Fluor 594-conjugated anti-rabbit IgG (1:1000). B. Labelling of PfEMP1 (1:500, green) and knobs on the PRBC surface with monoclonal anti-KAHRP (mAb89, 1:800, red), followed by anti-guinea pig Alexa Fluor 488 and anti-mouse Alexa Fluor 594. Scale bars: 5 mm. doi:10.1371/journal.pone.0029039.g003 PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29039