Figures
Figure 1. P2’ Ala and Lys mutations reduce with the proteolytic processing of the PEXEL motif. (A)5 µM fluorogenic peptides were incubated with 2 nM recombinantPv PMV and assayed at 20oC. Fluorescence data was normalised to the WT substrates (n=3). PEXEL motifs are indicated below the x-axis with mutated resides indicated in red. Statistical significance was determined using ordinary one-way ANOVA. ****, p-value<0.0001, ***, p-value<0.001). (B)Representative IFA images (n=3 independent replicates) of HSP101-HAglmS parasites expressing WT and P2’ K of Hyp1, STEVOR, & KAHRP-Nluc-mDH-FL reporter proteins. Parasite cells were probed with anti-EXP2 and anti-HA antibodies to visualise the PTEX components within the cell. The Nluc-mDH-FL proteins were localised using anti-Nluc antibody. The red bar in the schematic picture of the construct indicates the location of the PEXEL motif. Scale bars, 5μm. DIC, Differential Interference Contrast. DAPI (4’,6-diamidino-2-Phenylindole; Blue) was used to stain parasite nuclei. (C) Quantification of the fluorescence signal of STEVOR and KAHRP-Nluc-mDH-FL constructs within the parasite cells from A . Quantification was performed using at least 20 cells expressing each construct. Box and whisker plot represents 25th-75th and 5th-95th percentiles, respectively. Statistical significance was determined using ordinary one-way ANOVA. ****, p-value<0.0001). (D) Representative Western blot (n=3) of lysates made from mid-stage trophozoites expressing Nluc-mDH-FL constructs probed with anti-FLAG and anti-Nluc IgGs. The identity of various protein species was based on their observed sizes. ***, Full length proteins. , Mis-cleaved P2’ K Hyp1-Nluc-mDH-FL (58.1 ± 1.2 kDa, n=10). **, Mis-cleaved P2’ K KAHRP (51.4 ± 1.2 kDa, n=3) and STEVOR (53.8 ± 1.7 kDa, n=3)-Nluc-mDH-FL. *, correctly PEXEL-cleaved species of the Nluc-mDH-FL proteins. < is a cross-reactive protein. Hyp1-Nluc-mDH-FL blots are shown separate to KAHRP and STEVOR blots as they were run on different gels. The Neg lane contains protein from HSP101-HAglmS parasites not transfected with the NL-mDH-FL reporter and the blots­ were probed with anti-PTEX150 antibody as a loading control.
Figure 2. Solubility profile of Nluc-mDH-FL proteins.(A) Predicted full-length and correctly processed sizes of Hyp1, KAHRP and STEVOR-Nluc-mDH-FL reporter proteins. (B)Western blot analysis of infected RBCs (mid-stage trophozoites) sequentially extracted with 5 mM Tris-Cl pH 8.0 (Tris Sn), 0.1M Na2CO3 pH 11.3 (Carb Sn), and 1% Triton X-100 buffer (TX-100 Sn) to partition proteins based on their association with cellular membranes. Insoluble fraction represents the final pellet obtained after Triton X-100 extraction. GAPDH, HSP101, and EXP2 were used as a control for the release of soluble, peripheral, and integral protein, respectively. *, PEXEL-cleaved species. **, mis-cleaved species. ***, full-length protein. # WT Hyp1-NL-mDH-FL blot (lanes 1-4) has been published previously42, and has been included as a comparator to the KAHRP and STEVOR blots.
Figure 3. Sequential truncation of the spacer region caused trapping of the Nluc-mDH-FL constructs in the ER and PV of the parasite. (A)Representative IFA images (n=3 independent replicates) of HSP101-HAglmS parasites expressing Hyp1/STEVOR/KAHRP-Nluc-mDH-FL proteins with various spacer lengths (see cartoon representations). Parasite cells were probed with anti-EXP2 and anti-HA antibodies to visualise the PTEX components within the cell. Anti-Pf ERC antibody was used to visualise parasite’s ER. The Nluc-mDH-FL proteins were either probed with anti-Nluc or anti-FLAG antibodies. Scale bars, 5 μm. DIC, Differential Interference Contrast. DAPI (4’,6-diamidino-2-Phenylindole; Blue) was used to stain parasite nuclei. (B) Quantification of export of the spacer truncation Nluc-mDH-FL constructs presented in A . Export signal of >20 cells were recorded for each construct. Box and whisker plot represents 25th-75th and 10th-90th percentiles, respectively. Statistical significance was determined using ordinary one-way ANOVA. ****, p-value<0.0001; **, p-value<0.01; *, p-value = 0.0159).
Figure 4. Truncation of the spacer region of Hyp1-Nluc-mDH-FL reduces its interaction with HSP101. (A ) Western blot of anti-HA immunoprecipitation (IP) of HSP101-HAglmS expressing various truncations of the Hyp1 spacer region (n=3) as indicated above each lane. HSP101-HAglmS parasites not transfected with a Hyp1-Nluc-mDH-FL reporter are represented by ‘-‘. Immunoblots were performed to detect other PTEX components (EXP2 and PTEX150) as a positive control and GAPDH as a negative control. Hyp1 spacer mutants were visualised using anti-FLAG antibody. (B) Western blot of the reciprocal anti-Nluc IP of the truncated spacer reporters (n=4). Immunoblots were performed using anti-HA to detect HSP101 and GAPDH as a negative control. The length (C) and (D) , Densitometry of the IP performed on A (left) & B (right). For anti-HA IP, the intensity of co-immunoprecipitated 13aa and 3aa spacer Hyp1-Nluc-mDH-FL were normalised to the value of the 51aa spacer. For anti-Nluc IP, the intensity of co-immunoprecipitated HSP101 bands in the 13aa and 3aa spacers were normalised to the 51aa spacer. Error bars, ±SD. Statistical significance was determined using an Ordinary one-way ANOVA. ****, p value<0.0001.
Figure 5. The sequence requirements of the minimal 13aa spacer for export are relatively unconstrained. (A) Alignment of the sequences of Hyp1 spacers inclusive of the PEXEL motif who trafficking was investigated. (B) Fluorescence microscopy of Hyp1 spacers detected with rabbit Nluc and mouse EXP2 mAb as a PVM marker that 51aa, ∆NT13aa, 13aa and 13aa.Ser reporter were exported efficiently whereas the Hyp1 13aa.Glu and Hyp1 3aa appeared to be more strongly retained in the parasite. (C) Quantification of the % of exported protein signal beyond the EXP2 boundary into the infected RBC compartment indicated that the degree of export was progressively reduced in the Hyp1 13aa.Ser, Hyp1 13aa.Glu and Hyp1 3aa parasites relative to the Hyp1 13aa parasites. The Hyp1 ∆NT13aa parasites exported more efficiently Hyp1 13aa parasites. Export signal of >60 cells were recorded for each construct. Box and whisker plot represents 25th-75th and 10th-90th percentiles, respectively. Statistical significance was determined using ordinary one-way ANOVA. P-value, * <0.05. (D) Nanoluciferase export assay with the same parasite lines produced similar trends as the microscopic imaging but specifically indicated that most of the reduction in export in the mutant Hyp1 13aa and Hyp1 3aa spacer was due to retention in the PV rather than the parasite. Statistical significance was determined using ordinary Dunnett’s T3 multiple comparisons test in Graphpad Prism. P-values, *<0.05, *<0.01.
Figure 6. Hypothetical model for the HSP101-cargo recognition. (A) Side and top views of scale model of PTEX based on cryoelectron microscopy structure showing how Hyp1spacer with attached Nluc (red) could extend into PTEX’s central cavity 19. (B) Enlarged view of central HSP101 cavity showing the spacer length required to engage the helical protein binding regions of HSP101-nucleotide binding domain 2 (NBD2) which could apply an unfolding force to the cargo proteins for translocation.