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.