2.2 P2’ Lysine mutations reduces export
efficiency in multiple PEXEL proteins through the inhibition of
plasmepsin V cleavage
To clarify the effects of P2’ mutations of different
PEXEL proteins expressed under the same conditions we synthesised
several fluorogenic peptides containing wildtype PEXEL motifs and
mutations thereof of three different PEXEL proteins and determined how
well they were cleaved by recombinantly-expressed P. vivaxplasmepsin V (Pv PMV) (Table S2) 41.Pv PMV was employed as this protease was more experimentally
amenable than the P. falciparum equivalent41,42. Our first peptide from KAHRP sequence (RTLAQ)
containing P3 R to A and P1 L to A
mutations (ATAAQ) served as a control and was barely cleaved at all byPv PMV compared to the wildtype sequence (Fig 1A). The next Hyp1
(PF3D7_0113300) and STEVOR (subtelomeric variable open reading frame,
PF3D7_0200400) fluorogenic peptides (RLLTE and RLLAQ, respectively) had
their P2’ resides exchanged from E to K for Hyp1 and
from Q to K for STEVOR. In both cases, the P2’ K
fluorogenic peptides were barely cleaved indicating that
P2’ K strongly inhibits PEXEL processing (Fig 1A).
Previously, a GFP-tagged STEVOR with P2’ A mutation was
shown to be trapped at the parasite periphery by microscopy33. However, the PEXEL processing status of this
mutant was not investigated. We therefore included P2’ A
mutants of both Hyp1 and STEVOR in our cleavage assay. Compared to the
wildtype peptides, cleavage of the P2’ A mutants of Hyp1
and STEVOR were moderately inhibited, but this was only statistically
significant for Hyp1 (58%). The P2’ A mutation thus
likely represents an intermediate between the WT and the charge reversal
P2’ K mutation (Fig 1A).
We next sought to determine how the P2’ K mutations
would affect the trafficking and proteolytic processing of these
proteins in parasite infected RBCs. To generate reporters specific for
these proteins we fused the first 113aa of Hyp1 containing the PEXEL
motif RLLTE and a P2’ K mutant version, to a reporter
cassette comprising nanoluciferase (Nluc), murine dihydrofolate
reductase (mDH) and three FLAG epitopes (FL) 42. For
STEVOR, we fused the first 99 amino acids of the protein that includes
the PEXEL motif RLLAQ, and the corresponding P2’ K
mutant version to Nluc-mDH-FL. For KAHRP we fused the first 105aa
including PEXEL motif RTLAQ and the P2’ K mutant version
to the same reporter. All six constructs were transfected into the
HSP101-HAglmS parasite background line to enable direct
comparison between the wildtype and P2’ K mutants. We
note that microscopy and western blot data of wildtype (WT) and
P2’ K Hyp1-Nluc-mDH-FL have been previously published
but these are included them here as a comparator to KAHRP and STEVOR42. All P2’ wildtype
(P2’ WT) reporters were exported with most of the signal
in the RBC compartment (Fig 1B-D). For the P2’ K
mutants, Hyp1 differed from STEVOR and KAHRP with Hyp1 being largely
trapped around the nucleus in the ER and P2’ K STEVOR
and P2’ K KAHRP being trapped in the ER and around the
parasite circumference in the PV (Fig 1B). Quantification of the
reporter signals in KAHRP and STEVOR infected RBCs parasites indicated
there was more reporter within the parasite with the P2’
K reporters than the P2’ WT reporters (Fig 1C).
We have previously shown that the Hyp1 P2’ K reporter
was not efficiently cleaved at the PEXEL motif by Pv PMV and that
this could be why this reporter was not exported and remained trapped in
the ER 42. To determine if incorrect cleavage of the
KAHRP and STEVOR reporters could also be responsible for the increased
trapping of the reporters in the parasite we performed western blot
analysis on parasite lysates expressing WT and P2’ K
Hyp1-, -STEVOR- and KAHRP-Nluc-mDH-FL constructs. In the western blot
analysis of Hyp1 parasite lysates, the predominant correctly cleaved
species of WT Hyp1 migrates at 50 kDa and the full-length pre-processed
species migrates at about 70 kDa (Fig 1D, lane 1, single and triple
asterisks, respectively). In contrast, cleavage of the Hyp1
P2’ K reporter appears to be upstream of the PEXEL
probably near the transmembrane domain (Fig 1D, lane 2, arrow)42. Incorrect cleavage correlates with the high ER
retention observed by IFA although the mechanism for this is unknown
(Fig 1B and D) 42. In contrast, P2’
Lys STEVOR and KAHRP constructs migrated predominantly at the same size
as their WT counterpart on a western blot (Fig 1D, lanes 4-7),
indicating that the P2’ K STEVOR and KAHRP are mostly
processed within their PEXEL which would explain why they visually
appear more efficiently trafficked to the PV relative to Hyp1
P2’ K (Fig 1B). However, we noted an additional low
abundance mis-cleaved band for the KAHRP and STEVOR P2’
K reporters that are approximately 3-4 kDa bigger than the PEXEL cleaved
species (Fig 1D, lanes 5 and 7, double asterisks). We were able to
detect these mis-cleaved species using anti-FLAG antibody, suggesting
they are not C-terminally truncated form of the full-length protein (Fig
1D, Table S3). We therefore conclude that the mis-cleaved species likely
represent aberrant N-terminally processed forms of P2’ K
KAHRP and STEVOR reporters that arise from less efficient PEXEL
processing, that may account for the small reduction in export.
Collectively, both western blot and biochemical analyses suggest that a
P2’ mutation, particularly to a positively charged
residue, can reduce efficient cleavage for Hyp1 and cause ER retention
but not for KAHRP and STEVOR. For the latter two proteins, cleavage of
the P2’ K PEXEL is much more efficient and the proteins
traffic to the PV but are translocated less effectively into the RBC
than WT reporters.
2.3
ER trapped Hyp1 P2’ K is retained in a more insoluble
form than PV trapped P2’ K STEVOR and KAHRP
We have previously observed that the ER-trapped mis-cleaved Hyp1
P2’ K reporter was poorly soluble which may partly
account for its ER retention 42. To determine if PV
trapping of STEVOR and KAHRP P2’ K reporter proteins was
due to reduced solubility, we performed protein solubility assays on
parasite lines expressing the WT and P2’ K Nluc-mDH-FL
reporters (Fig 2A). We reported previously that the higher MW
mis-cleaved species of P2’ K Hyp1-Nluc-mDH-FL was mostly
concentrated in the TX-100 fraction (Fig 2B lane 7, double asterisk),
suggesting that it could be membrane-associated 42. In
contrast, correctly processed P2’ K Hyp1-Nluc-mDH-FL
(Fig 2B lane 5, single asterisk) was readily extracted by hypotonic
lysis (Fig 2B, Tris Sn). The cleaved forms of P2’ K
KAHRP- (Fig 2B, lanes 13-16, single asterisk) and STEVOR-Nluc-mDH-FL
(Fig 2B, lanes 21-24, single asterisk) were largely found in the soluble
fraction, although some was present in the other fractions as well (Fig
2B). In contrast to the Hyp1 P2’ K reporter, the
mis-cleaved forms of KAHRP and STEVOR were evenly distributed in all
fractions (Fig 2B, double asterisks). For all KAHRP and STEVOR
reporters, the larger uncleaved forms were also found in the soluble
fraction which was surprising considering these proteins would still
retain their signal sequence transmembrane domains (Fig 2B, lanes 9, 13,
17 and 21, triple asterisks). We speculate that mis-cleaved
P2’ K reporters, particularly Hyp1, may remain trapped
in the ER because they are less soluble although the mechanism behind
this is not obvious since both size of the mis-cleaved
P2’ K proteins and previous mass spectrometric analysis
suggests they lack their hydrophobic signal peptides42. When correctly processed however, the
P2’ K reporters are more soluble and traffic beyond the
ER to at least the PV.
2.4.
The length of the spacer region is essential for protein translocation
across the parasitophorous vacuole membrane.
Thus far our data have indicated that the amino acid occupying the
P2’ residue is important for the correct cleavage of
Hyp1 but less so for KAHRP and STEVOR indicating that other residues
within and/or bordering the PEXEL motif may also be important for
accurate cleavage. Earlier work has shown that truncation of the amino
acid sequence (termed spacer region) that separates the PEXEL motif from
a downstream folded protein such as GFP, influences export22,24. Interestingly, the N-terminal regions of PNEPs
are functionally exchangeable with this spacer region of PEXEL proteins38 and replacement of the spacer region with the
N-terminal sequence of a PV-resident protein inhibits export4, suggesting that this region may comprise abona fide export signal. To investigate whether the spacer region
has a role in binding to PTEX, the spacer region of the Hyp1, STEVOR,
and KAHRP-Nluc-mDH-FL constructs were C-terminally truncated, from their
original lengths of ~50aa, down to 13aa and 3aa
preceding the folded domain of Nluc (Fig 3A and 3B). IFAs of
trophozoite-infected RBCs expressing the truncation constructs showed
reduced export with reducing spacer length in all three constructs.
Quantification of the exported signal across the cell population further
revealed that truncation from ~50aa to 13aa reduces
export by ~10-20%, while export was strongly reduced in
3aa spacer constructs, showing a marked ~ 80% reduction
in fluorescence signal relative to the control (Figs 3A-C). This
observation contrasts with the mutations of the P2’
PEXEL motif alone, performed in the previous section (Fig 1) and other
studies, which displayed variable export-blocking phenotypes with
different PEXEL protein sequences 1,14,33,36.
Co-labelling of microscopy images with EXP2 (PV marker) and Pf ERC
(ER marker) further indicated that the Hyp1-Nluc-mDH-FL with a 3aa
spacer accumulated mainly in the PV with some signal in the ER
overlapping with HSP101-HA (Fig 3A panels 3-7).
IFAs using anti-Nluc with STEVOR-Nluc-mDH-FL and KAHRP- Nluc-mDH-FL
parasites showed that these reporters behaved similarly to the
Hyp1-reporter (Fig 3B, panels 2, 3 and 5, 6) and also displayed the
highest co-localisation with the HA-tagged translocon component HSP101,
which we have shown resides within the ER in addition to the PV42,43.
Truncation of the spacer did not appear to reduce processing of the
PEXEL motif in this context as western blots of the Hyp1-Nluc-mDH-FL
truncation constructs showed that each reporter protein migrated
according to a predicted mass consistent with PMV-processed versions of
the proteins (Fig 4A and B, lanes 2-4, Table S4). Taken together, these
results show that the length of the spacer mutant is important for
export, post-PEXEL processing.
2.5.
Truncation of the spacer region reduces cargo binding with HSP101.
The observed co-localisation of all spacer constructs with the
translocon components EXP2 and HSP101 in the PV was perplexing as the
truncated proteins appeared to have processed PEXEL N-termini suggesting
they were unable to engage with PTEX to be exported. We therefore sought
to determine if the truncated PEXEL proteins could bind HSP101 by
co-immunoprecipitation. To do this, the Hyp1 truncation constructs
(51aa, 13aa, and 3aa Hyp1-Nluc-mDH-FL) were transfected into the
HSP101-HAglmS parasite line 42. These parasites
were grown to the ring stage and treated with WR99210 for 24 hours to
stabilise the folding of the murine DHFR, that had previously been
demonstrated to stall the cargo unfolding process within PTEX, thereby
trapping and stabilising the cargo’s interaction with PTEX16,43-45. The whole trophozoite infected RBCs were
lysed and incubated with anti-HA-IgG agarose to immunoprecipitate the
HA-tagged HSP101 from the sample. Western blot analysis of the eluates
revealed a significantly reduced amount of Hyp1-Nluc-mDH-FL co-eluted
with HSP101 with decreasing length of the spacer, with 60% and 90%
reduction (n=3) observed in the 13aa and 3aa spacer, respectively,
relative to the 51aa spacer Hyp1-Nluc-mDH-FL (Figs 4A, lanes 6-8 and
4C). Importantly, the experiment was performed in the presence of a
stabilising ligand WR99210, suggesting that the 13aa and 3aa spacer
truncation mutants did not proceed to the unfolding step within PTEX.
The same samples were also subjected to a reciprocal
co-immunoprecipitation using anti-Nluc antibodies to pull-down the
Hyp1-Nluc-mDH-FL truncation proteins and we consistently saw a gradual
reduction in the amount of HSP101 co-eluted as the length of the spacer
was shortened (Figs 4B lanes 6-8 and Fig 4D). We plotted the normalised
% exported as observed by IFA and the amount of cargo
(Hyp1-Nluc-mDH-FL) bound with HSP101 and we saw a remarkable correlation
between these two variables, suggesting that the level of cargo binding
with HSP101 determines its exportability (compare Fig 3C with Figs 4B
and 4D). Taken together, these results demonstrated that the spacer
region regulates cargo engagement with PTEX, particularly with HSP101
which is possibly the first point of contact cargo has with PTEX19,46.