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\begin{document}

\title{Sequence elements within the PEXEL motif and its downstream region
modulate PTEX dependent protein export in Plasmodium falciparum .}



\author[1]{Paul R. Gilson}%
\author[1]{Mikha Gabriela}%
\author[1]{Claudia B. G. Barnes}%
\author[1]{Dickson Leong}%
\author[2]{Brad E. Sleebs}%
\author[1]{Molly P. Schneider}%
\author[3]{Dene R. Littler}%
\author[1]{Brendan S. Crabb}%
\author[4]{Tania F. de Koning-Ward}%
\affil[1]{Burnet Institute}%
\affil[2]{Walter and Eliza Hall Institute of Medical Research}%
\affil[3]{Monash University Monash Biomedicine Discovery Institute}%
\affil[4]{Deakin University School of Medicine}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
The parasite \emph{Plasmodium falciparum} causes the most severe form of
malaria and to invade and replicate in red blood cells (RBCs), it
exports hundreds of proteins across the encasing parasitophorous vacuole
membrane (PVM) into this host cell. The exported proteins help modify
the RBC to support rapid parasite growth and avoidance of the human
immune system. Most exported proteins possess a conserved
\emph{Plasmodium} Export Element (PEXEL) motif with the consensus
RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage
recognition site within the parasite's endoplasmic reticulum (ER).
Cleavage occurs after the P \textsubscript{1} L residue and is thought
to help release the protein from the ER so it can be putatively escorted
by the HSP101 chaperone to the parasitophorous vacuole space surrounding
the intraerythrocytic parasite. HSP101 and its cargo are then thought to
assemble with the rest of a \emph{Plasmodium} Translocon for Exported
proteins (PTEX) complex, that then recognises the xE/D/Q capped N
terminus of the exported protein and translocates it across the vacuole
membrane into the RBC compartment. Here, we present evidence that
supports a dual role for the PEXEL's conserved P \textsubscript{2}'
position E/Q/D residue, firstly, for plasmepsin V cleavage in the ER,
and secondly, for efficient PTEX mediated export across the PVM into the
RBC. We also present evidence that the downstream `spacer' region
separating the PEXEL motif from the folded functional region of the
exported protein controls cargo interaction with PTEX as well. The
spacer must be of a sufficient length and permissive amino acid
composition to engage the HSP101 unfoldase component of PTEX to be
efficiently translocated into the RBC compartment.%
\end{abstract}%



\sloppy


\textbf{Title.}

\textbf{Sequence elements within the PEXEL motif and its downstream
region modulate PTEX dependent protein export in \emph{Plasmodium
falciparum}.}

\textbf{Running Title.}

Protein export in \emph{Plasmodium falciparum} .

\textbf{Authors.}

Mikha Gabriela\textsuperscript{\#1,2}, Claudia B. G.
Barnes\textsuperscript{\#1}, Dickson Leong\textsuperscript{1}, Brad E.
Sleebs\textsuperscript{3,4}, Molly P. Schneider\textsuperscript{1}, Dene
R. Littler\textsuperscript{5}, Brendan S.
Crabb\textsuperscript{1,4,6,7}, Tania F. de
Koning-Ward\textsuperscript{2,8}, and Paul R. Gilson\textsuperscript{1,6
*}

\textsuperscript{1} Malaria Virulence and Drug Discovery Group, Burnet
Institute, Melbourne, Australia

\textsuperscript{2} School of Medicine, Deakin University, Geelong,
Victoria, Australia

\textsuperscript{3} The Walter and Eliza Hall Institute of Medical
Research, Parkville, Australia

\textsuperscript{4} Department of Medical Biology, The University of
Melbourne, Parkville, Australia

\textsuperscript{5}Infection and Immunity Program and Department of
Biochemistry and Molecular Biology, Biomedicine Discovery Institute,
Monash University, Clayton, Victoria, Australia,

\textsuperscript{6} Department of Immunology and Microbiology,
University of Melbourne, Parkville, Australia

\textsuperscript{7} Department of Immunology and Pathology, Monash
University, Melbourne, Australia

\textsuperscript{8}Institute for Mental and Physical Health and Clinical
Translation (IMPACT), Deakin University, Geelong, Australia

* Corresponding Author: paul.gilson@burnet.edu.au

\# These authors contributed equally

Format for \emph{Traffic} .

\textbf{Synopsis.}

The malaria parasite \emph{Plasmodium falciparum} , exports hundreds of
proteins into the red blood cells (RBC) it infects to help it grow and
replicate. Most exported proteins possess a five amino acid barcode that
acts as a proteolytic cleavage site and licenses these proteins for
export into the RBC compartment by protein translocating gateways
surrounding the parasite. We report here that the conserved fifth amino
acid position of the barcode plays an important role in the proteolytic
maturation of exported protein and, together with the downstream
flexible spacer region preceding the folded region of the exported
protein, are important for efficient transport into the RBC
via\emph{Plasmodium} Translocon of Exported Proteins (PTEX).

\textbf{Abstract}

The parasite \emph{Plasmodium falciparum} causes the most severe form of
malaria and to invade and replicate in red blood cells (RBCs), it
exports hundreds of proteins across the encasing parasitophorous vacuole
membrane (PVM) into this host cell. The exported proteins help modify
the RBC to support rapid parasite growth and avoidance of the human
immune system. Most exported proteins possess a
conserved\emph{Plasmodium} Export Element (PEXEL) motif with the
consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic
cleavage recognition site within the parasite's endoplasmic reticulum
(ER). Cleavage occurs after the P\textsubscript{1} L residue and is
thought to help release the protein from the ER so it can be putatively
escorted by the HSP101 chaperone to the parasitophorous vacuole space
surrounding the intraerythrocytic parasite. HSP101 and its cargo are
then thought to assemble with the rest of a \emph{Plasmodium} Translocon
for Exported proteins (PTEX) complex, that then recognises the xE/D/Q
capped N terminus of the exported protein and translocates it across the
vacuole membrane into the RBC compartment. Here, we present evidence
that supports a dual role for the PEXEL's conserved P\textsubscript{2}'
position E/Q/D residue, firstly, for plasmepsin V cleavage in the ER,
and secondly, for efficient PTEX mediated export across the PVM into the
RBC. We also present evidence that the downstream `spacer' region
separating the PEXEL motif from the folded functional region of the
exported protein controls cargo interaction with PTEX as well. The
spacer must be of a sufficient length and permissive amino acid
composition to engage the HSP101 unfoldase component of PTEX to be
efficiently translocated into the RBC compartment.

\textbf{Keywords.}

\emph{Plasmodium falciparum} , malaria, protein export, PTEX, virulence,
PEXEL, chaperone.

\textbf{Introduction}

The pathogenesis of malaria lies upon the ability of
\emph{Plasmodium}parasites, the causative agent of the disease, to
propagate asexually within red blood cells (RBCs) while avoiding the
immune mechanisms of its host. To accomplish this, the parasite exports
hundreds of its proteins into its host cell to transform it into a
hospitable niche and to avoid host immunity \textsuperscript{1-5}. The
intraerythrocytic stage of the parasite resides within a membranous
compartment called the parasitophorous vacuole (PV)
\textsuperscript{6,7}. Consequently, parasite proteins destined for
export must traverse two membranes, the parasite plasma membrane and the
encasing parasitophorous vacuole membrane (PVM), to reach the host RBC.
The \emph{Plasmodium t} ranslocon of \emph{ex} ported proteins (PTEX)
mediates the translocation of proteins across the PVM
\textsuperscript{8} and is the only protein channel known to reside at
the PVM. PTEX is comprised of three core components, HSP101, PTEX150,
and EXP2, that are all essential for parasite survival, making it an
attractive drug target candidate\textsuperscript{8-10}.

Most parasite proteins destined for export into the RBC begin their
journey with entry into the endoplasmic reticulum (ER) via the Sec61
translocon, followed by cleavage of the protein within the ER by the
aspartyl protease, plasmepsin V (PMV) \textsuperscript{11-13}. The
cleavage step occurs within a pentameric amino acid motif near the
N-terminus of the exported protein destined for export, termed
the\emph{Plasmodium} export element (PEXEL) \textsuperscript{1,2,14}.
The resulting mature proteins then travel via the vesicular transport
pathway to the parasite plasma membrane where they are secreted into the
parasitophorous vacuole \textsuperscript{15}. Here the exported proteins
are unfolded and translocated across the PVM and into the host-cell
compartment in an ATP-dependent manner by PTEX \textsuperscript{16}. It
is thought the AAA+ ATPase HSP101 first engages and unfolds cargo
proteins in an ATP-dependent manner and threads them through a
tetradecamer membrane-spanning channel consisting of the scaffold
protein PTEX150 and the pore-forming protein
EXP2\textsuperscript{17-19}. However, the information that is contained
within proteins destined for export that allows HSP101 to specifically
recognise these proteins prior to the unfolding step is still
unknown\textsuperscript{20,21}.

Historically, the N-terminal region of exported proteins (and the
protein's transmembrane domain in some cases) have been found to be
sufficient to mediate the trafficking of proteins into the RBC
compartment \textsuperscript{22-26}. This N-terminal region usually
contains a recessed signal peptide to promote entry of the proteins into
the parasite's ER \textsuperscript{27} or a hydrophobic stretch of amino
acids that serve the same function \textsuperscript{28}. The discovery
of the five-amino acid PEXEL motif in the N-terminal region of many
exported proteins was the first export-specific
signal\textsuperscript{1,2} that was proven to be predictive for
exported proteins \textsuperscript{29} and allowed the identification of
\textgreater{}450 putative exported proteins in \emph{P.
falciparum}\textsuperscript{4}. Interestingly, however, the presence of
the PEXEL motif alone does not guarantee export, as its location within
a protein's primary structure \textsuperscript{30} and the presence of
\textasciitilde{}12 amino acids downstream of the motif have also been
shown to be required to achieve efficient export \textsuperscript{24}.
Furthermore, PEXEL-negative exported proteins (PNEPs) are also present
in the \emph{Plasmodium} exportome \textsuperscript{5,31,32}, suggesting
that PEXEL is not strictly required for passage through PTEX.

The function of the PEXEL motif has been relatively well studied and its
consensus sequence is RxLxE/D/Q where x can be almost any amino acid.
The position P\textsubscript{3} R and P\textsubscript{1} L residues are
the most conserved of the PEXEL motif and in \emph{P. falciparum} have
been shown to be necessary for efficient cleavage by
PMV\textsuperscript{12-14}. It is assumed therefore that
P\textsubscript{1}and P\textsubscript{3} facilitate PEXEL cleavage and
thus help to release the exported proteins from the ER membrane where
they are originally anchored following ER import \textsuperscript{11}.
In comparison, the purpose of the last conserved P\textsubscript{2}'
(E/Q/D) residue of PEXEL is less certain. Some evidence suggests that
following cleavage, the P\textsubscript{1}' and P\textsubscript{2}'
resides which now cap the mature PEXEL protein, play a role in promoting
export across the PVM \textsuperscript{1,14,33}. After cleavage, the
exposed xE/Q/D motif becomes N-terminally acetylated\textsuperscript{34}
resulting in an Ac- xE/Q/D cap that was thought to serve as a 'barcode'
or a recognition motif for HSP101 engagement in the PV
\textsuperscript{35}. Contrary to this, however, other studies have
found that P\textsubscript{2}' amino acid also influences the efficiency
of PEXEL processing or does not influence export at
all\textsuperscript{36-38}.

Here, we present evidence that the P\textsubscript{2}' residue has a
dual function. For some PEXEL proteins, P\textsubscript{2}' mutations
greatly reduce PMV cleavage increasing protein retention in the ER and
reducing export. For other proteins, P\textsubscript{2'} mutations do
not inhibit PMV cleavage as much and PEXEL proteins can reach the PV but
are translocated less efficiently. We also provide evidence that the
length of the region downstream of the PEXEL motif universally regulates
the degree of cargo interaction with HSP101 and ultimately affects
protein export across the PVM.

\textbf{Results}

2.1 A systematic review of PEXEL motif mutants reveals that a single
point mutation of P2' into a positively charged residue produces the
most consistent export-blocking effect.

To better understand the contribution of the PEXEL P\textsubscript{2}'
position on protein export, we conducted a review of P\textsubscript{2}'
mutagenesis experiments that have been performed on various PEXEL
proteins (Table S1). We sorted the mutants based on the amino acids
incorporated into the P\textsubscript{2}' position and linked it to the
export phenotype, PEXEL processing pattern and the N-terminal
acetylation (Table S1 'PMV cleavage' and 'N-acetylation') reported for
the corresponding mutant. This in general revealed that alanine (A)
mutations in the P\textsubscript{2}' position have a limited or
uncertain effect on export (Table S1). For example when the
P\textsubscript{2}' position of KAHRP (knob-associated histidine rich
protein, PF3D7\_0202000) was exchanged with an A, the protein was not
exported and was retained in the parasite \textsuperscript{39}. This was
contrary to the study by \textsuperscript{14}, where the reporter was
mostly trapped in the PV, despite both groups using very similar
constructs (i.e. the first 69aa of KAHRP leader sequence fused to GFP).
The same discrepancy was also observed with P\textsubscript{2}' A
mutations of STEVOR and GBP130 (PF3D7\_1016300) (Table S1), Rather than
regard these findings as contradictory, the different localisation of
P\textsubscript{2}' A mutants could arise because of the continuum from
the ER to the PV that might be dependent on the parasite age and the
different module(s) appended downstream of the PEXEL
motif\textsuperscript{36,38} . P\textsubscript{2}' A mutagenesis study
on other PEXEL proteins, such as RESA (PF3D7\_0102200), PfEMP3
(PF3D7\_0201900), PF3D7\_0532600, and murine \emph{P. berghei} CP1
(PBANKA\_1246500) did not result in any export defect (Table S1).

Mutation of the P\textsubscript{2}' PEXEL position to positively charged
amino acids appears to produce the most robust export-blocking effect on
exported proteins tested in two different studies (Table
S1)\textsuperscript{36,40}. Mutation to basic amino acids would usually
reverse the charge of the P\textsubscript{2}' position, given that the
two most common P\textsubscript{2}' residues are glutamic and aspartic
acids. Curiously, the P\textsubscript{2}' lysine (K) mutant of REX3
(PF3D7\_0936300) PEXEL also moderately inhibited PMV cleavage of the
motif \textsuperscript{36}, suggesting that P\textsubscript{2}' may also
be involved in PEXEL processing, albeit to a lesser extent than
P\textsubscript{1} and P\textsubscript{3}.

P\textsubscript{2}' mutations can also cause changes to the
N-acetylation profile of the mature protein. However, N-terminal
acetylation is not sufficient to determine the fate of exported
proteins, as suggested previously \textsuperscript{12,36,38}. Whilst it
is unclear whether PEXEL proteins without N-terminal acetylation can be
exported or not, having an N-terminal acetylation by itself does not
guarantee successful export, as can be seen in the case of
P\textsubscript{2}' A mutant of GBP130 and P\textsubscript{2}' K of
STEVOR (PF3D7\_0631900) which fail to be exported despite being shown to
be N-terminally acetylated (Table S1)\textsuperscript{14}. We therefore
conclude that substituting the conserved P\textsubscript{2}' PEXEL
reside with a basic amino acid would likely produce a more pronounced
export-blocking phenotype in the parasite.

2.2 P\textsubscript{2}' Lysine mutations reduces export efficiency in
multiple PEXEL proteins through the inhibition of plasmepsin V cleavage

To clarify the effects of P\textsubscript{2}' 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 \emph{P.
vivax}plasmepsin V (\emph{Pv} PMV) (Table S2)
\textsuperscript{41}.\emph{Pv} PMV was employed as this protease was
more experimentally amenable than the \emph{P. falciparum}
equivalent\textsuperscript{41,42}. Our first peptide from KAHRP sequence
(RTLAQ) containing P\textsubscript{3} R to A and P\textsubscript{1} L to
A mutations (ATAAQ) served as a control and was barely cleaved at all
by\emph{Pv} 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 P\textsubscript{2}' resides exchanged from E to
K for Hyp1 and from Q to K for STEVOR. In both cases, the
P\textsubscript{2}' K fluorogenic peptides were barely cleaved
indicating that P\textsubscript{2}' K strongly inhibits PEXEL processing
(Fig 1A).

Previously, a GFP-tagged STEVOR with P\textsubscript{2}' A mutation was
shown to be trapped at the parasite periphery by
microscopy\textsuperscript{33}. However, the PEXEL processing status of
this mutant was not investigated. We therefore included
P\textsubscript{2}' A mutants of both Hyp1 and STEVOR in our cleavage
assay. Compared to the wildtype peptides, cleavage of the
P\textsubscript{2}' A mutants of Hyp1 and STEVOR were moderately
inhibited, but this was only statistically significant for Hyp1 (58\%).
The P\textsubscript{2}' A mutation thus likely represents an
intermediate between the WT and the charge reversal P\textsubscript{2}'
K mutation (Fig 1A).

We next sought to determine how the P\textsubscript{2}' 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 P\textsubscript{2}' K mutant version, to a reporter
cassette comprising nanoluciferase (Nluc), murine dihydrofolate
reductase (mDH) and three FLAG epitopes (FL) \textsuperscript{42}. For
STEVOR, we fused the first 99 amino acids of the protein that includes
the PEXEL motif RLLAQ, and the corresponding P\textsubscript{2}' K
mutant version to Nluc-mDH-FL. For KAHRP we fused the first 105aa
including PEXEL motif RTLAQ and the P\textsubscript{2}' K mutant version
to the same reporter. All six constructs were transfected into the
HSP101-HA\emph{glmS} parasite background line to enable direct
comparison between the wildtype and P\textsubscript{2}' K mutants. We
note that microscopy and western blot data of wildtype (WT) and
P\textsubscript{2}' K Hyp1-Nluc-mDH-FL have been previously published
but these are included them here as a comparator to KAHRP and
STEVOR\textsuperscript{42}. All P\textsubscript{2}' wildtype
(P\textsubscript{2}' WT) reporters were exported with most of the signal
in the RBC compartment (Fig 1B-D). For the P\textsubscript{2}' K
mutants, Hyp1 differed from STEVOR and KAHRP with Hyp1 being largely
trapped around the nucleus in the ER and P\textsubscript{2}' K STEVOR
and P\textsubscript{2}' 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 P\textsubscript{2}'
K reporters than the P\textsubscript{2}' WT reporters (Fig 1C).

We have previously shown that the Hyp1 P\textsubscript{2}' K reporter
was not efficiently cleaved at the PEXEL motif by \emph{Pv} PMV and that
this could be why this reporter was not exported and remained trapped in
the ER \textsuperscript{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 P\textsubscript{2}' 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
P\textsubscript{2}' K reporter appears to be upstream of the PEXEL
probably near the transmembrane domain (Fig 1D, lane 2,
arrow)\textsuperscript{42}. Incorrect cleavage correlates with the high
ER retention observed by IFA although the mechanism for this is unknown
(Fig 1B and D) \textsuperscript{42}. In contrast, P\textsubscript{2}'
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 P\textsubscript{2}' 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
P\textsubscript{2}' K (Fig 1B). However, we noted an additional low
abundance mis-cleaved band for the KAHRP and STEVOR P\textsubscript{2}'
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 P\textsubscript{2}' 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
P\textsubscript{2}' 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 P\textsubscript{2}' 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 P\textsubscript{2}' K is retained in a more
insoluble form than PV trapped P\textsubscript{2}' K STEVOR and KAHRP

We have previously observed that the ER-trapped mis-cleaved Hyp1
P\textsubscript{2}' K reporter was poorly soluble which may partly
account for its ER retention \textsuperscript{42}. To determine if PV
trapping of STEVOR and KAHRP P\textsubscript{2}' K reporter proteins was
due to reduced solubility, we performed protein solubility assays on
parasite lines expressing the WT and P\textsubscript{2}' K Nluc-mDH-FL
reporters (Fig 2A). We reported previously that the higher MW
mis-cleaved species of P\textsubscript{2}' 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 \textsuperscript{42}. In
contrast, correctly processed P\textsubscript{2}' 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 P\textsubscript{2}' 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 P\textsubscript{2}' 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
P\textsubscript{2}' 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
P\textsubscript{2}' K proteins and previous mass spectrometric analysis
suggests they lack their hydrophobic signal
peptides\textsuperscript{42}. When correctly processed however, the
P\textsubscript{2}' 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
P\textsubscript{2}' 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
export\textsuperscript{22,24}. Interestingly, the N-terminal regions of
PNEPs are functionally exchangeable with this spacer region of PEXEL
proteins\textsuperscript{38} and replacement of the spacer region with
the N-terminal sequence of a PV-resident protein inhibits
export\textsuperscript{4}, suggesting that this region may comprise
a\emph{bona 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 \textasciitilde{}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 \textasciitilde{}50aa to 13aa reduces
export by \textasciitilde{}10-20\%, while export was strongly reduced in
3aa spacer constructs, showing a marked \textasciitilde{} 80\% reduction
in fluorescence signal relative to the control (Figs 3A-C). This
observation contrasts with the mutations of the P\textsubscript{2}'
PEXEL motif alone, performed in the previous section (Fig 1) and other
studies, which displayed variable export-blocking phenotypes with
different PEXEL protein sequences \textsuperscript{1,14,33,36}.
Co-labelling of microscopy images with EXP2 (PV marker) and \emph{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
PV\textsuperscript{42,43}.

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-HA\emph{glmS} parasite line \textsuperscript{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
PTEX\textsuperscript{16,43-45}. 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
PTEX\textsuperscript{19,46}.

2.6 The sequence requirements of the 13aa spacer for export are
relatively unconstrained.

Since the 13aa Hyp1 spacer still permitted a reasonable amount of export
compared to the full size 51aa spacer we decided to use this as basis
for subsequent mutagenesis experiments due its small size. The spacers
of PEXEL proteins appear to possess little obvious sequence information
for putative PTEX recognition apart from appearing relatively
unstructured with no conserved domains. To assess whether the minimal
spacer may contain some cryptic trafficking information, we decided to
remove it from the original 51aa spacer and fuse this new [?]NT13aa spacer
to the Nluc-mDH-FL reporter and express this in parasites (Figs 5A).
Western blots of the Hyp1 [?]NT13aa-Nluc-mDH-FL parasites indicated the
reporter protein was of a size consistent with correct PMV cleavage (Fig
S1). Microscopy of the [?]NT13aa reporter showed it was efficiently
exported and quantification of the fluorescence intensity implies a
higher degree of export of the [?]NT13aa reporter than the 13aa spacer
(Fig 5B and C). This result indicated that the [?]NT13aa spacer was
competent for export and did not rely on targeting information contained
within the 13aa spacer for export essential for export of the
Hyp1-NlucmDH-FL reporter.

Next, we investigated whether certain amino acids within the minimal
spacer were necessary for export. As proteins chaperones tend to
recognise misfolded proteins via their hydrophobic
residues\textsuperscript{47}, the four hydrophobic amino acids Y, L, I
and V in the minimal Hyp1 spacer TE\emph{Y} KDT\emph{L} Q\emph{I}
K\emph{V} EQ were mutated to determine if this reduced export. In the
absence of predictive tools for HSP100 chaperones we used a binding
predictor for the ER HSP70 chaperone BIP \textsuperscript{47} that scans
7aa peptide windows and found that mutation of the hydrophobic amino
acids to the polar amino acid serine, produced lower scores than for
other amino acids (Figs 5A and S2). We therefore replaced the
hydrophobic amino acids in the 13aa spacer with serines and transfected
this Hyp1 13aa.Ser repoter into parasites where, by western blot, the
Hyp1 13aa.Ser reporter was of a size consistent with correct PMV
cleavage (Fig S1). Microscopic analysis of the Hyp1 13aa.Ser
parasite-infected RBCs indicated that this mutant was exported less
efficiently than the WT minimal 13aa spacer (Figs 5A-C and S1).

Next, the hydrophobic amino acids of the minimal 13aa spacer were
mutated to glutamic acids (Hyp1 13aa.Glu) as these mutations were scored
most poorly by the BIP binding predictor (Fig S2). Western blots of Hyp1
13aa.Ser-Nluc-mDH-FL parasites indicated PMV processing was correct and
microscopic analysis of parasite-infected RBCs expressing the reporter
indicated the Hyp1 13aa.Glu protein was exported significantly less well
than the Hyp1 13aa parasites (Fig 5B and C). These results indicated
that although there is probably a great deal of flexibility in the amino
acid sequences of spacers, some attributes are required such as the
presence of hydrophobic amino acids that could be important for general
chaperone binding.

As it can be difficult to visibly discern cargo trapped in the PV versus
that inside the parasite we employed a recently-developed protein export
assay based on the release and detection of Nluc bioluminescence from
differentially lysed cellular compartments \textsuperscript{43,48}. We
saw that the Nluc bioluminescence signal exported into the RBC
compartments closely followed the same trend as the spacer reporter
parasites whose export was measured visibly by microscopy (compare Fig
5D to 5C). However, the signal for the Nluc bioluminescence export assay
was however often 10-20\% greater than the microscopy signal probably
because bioluminescence is highly sensitive can detect lower protein
levels dispersed in the large RBC compartment (Fig 5D). Additionally,
microscopy does not measure RBC signal in front or behind the parasite
which may underestimate the total signal. Interestingly, the
bioluminescence signal of the reporters secreted into the PV compartment
increased as export into the RBC compartment decreased indicating that
as the spacer length shrunk, or its hydrophobic residues were mutated,
the proteins were still able to traverse the parasite plasma membrane
but were less efficiently translocated into the RBC by PTEX, leaving
them to accumulate in the PV. It is also worth noting that the signal
retained in the parasite was relatively constant indicating spacer
length or mutations did not reduce secretion to the PV.

\textbf{Discussion}

Here we sought to understand what element(s) of PEXEL proteins govern
recruitment of PTEX to facilitate the export of proteins into the
erythrocyte compartment. It was previously hypothesised that the last
conserved residue of the PEXEL, which remains on the mature protein, is
responsible for interacting with PTEX \textsuperscript{1,2,14}. Our data
partly support this in that mutation of P\textsubscript{2}' residue to K
can lead to increased trapping of some PEXEL proteins in the PV, most
notably for the KAHRP and STEVOR reporters. The P\textsubscript{2}' K
mutation can also greatly reduce the efficiency of PMV cleavage in the
case of Hyp1 but not so for KAHRP and STEVOR and as such, increase
protein trapping in the ER as shown for the Hyp1 reporter. Apart from
the efficiency of cleavage of the PEXEL motif for governing PTEX's
interaction with the cargo proteins, the spacer region downstream of the
PEXEL that separates the PEXEL from the folded functional Nluc region of
the reporter protein is also important for trafficking. As the length of
spacer increased, so did the reporter's binding to PTEX and the
efficiency with which it was exported into the RBC compartment. A small
spacer length of 13aa was found to still result in efficient export with
hydrophobic residues in the spacer important for export efficiency.
Reporters with short or mutated spacers appeared to be still efficiently
cleaved by PMV but interacted weakly with PTEX leaving them trapped in
the PV compartment and unable to be exported. One caveat of this study
is that our reporter had a folded Nluc domain a defined distance from
the upstream PEXEL cleavage site, and we are unsure if similar rules
will generally apply to native PEXEL proteins many of whose structures
and functions are unknown.

Other studies have shown that the amino acid in the last position of
export motifs is important for export. For example, reporter proteins
containing oomycete effector motif RxLR \textsuperscript{30,38}, which
lack the P\textsubscript{2}' residue, were not cleaved by plasmepsin V
and failed to promote export into the host cell \textsuperscript{30,38}.
Of all possible mutations of P\textsubscript{2}', only charge reversal
P\textsubscript{2}' mutations, either to arginine or lysine, strongly
inhibit plasmepsin V cleavage \textsuperscript{36} whilst a single
alanine mutation was observed to cause a variable export
phenotype\textsuperscript{4,36}. This was evident from the small
reduction of cleavage observed in the \emph{in vitro} cleavage assay
with P\textsubscript{2}' A mutation compared to the K mutation. Despite
this, the strong inhibition \emph{in vitro} cleavage of STEVOR
P\textsubscript{2}' K did not translate to what was observed in
parasites as the reporter protein was efficiently cleaved. It seems
likely that suboptimal amino acid variants at PEXEL P\textsubscript{2}'
are still permissive for PMV cleaving in the cellular context,
presumably because of the fast kinetics of the proteolytic
reaction\emph{in vivo} or due to concentrated substrate in the ER that
favours cleavage even under less cleavable substrate. It is also
possible that\emph{Pf} PMV in parasites is more active against the
P\textsubscript{2}' mutants than the \emph{Pv} PMV used in the peptide
cleavage assay\textsuperscript{49,50}.

Having previously observed that the ER-trapped Hyp1 P\textsubscript{2}'
K mutant, which was mis-cleaved by an unknown protease upstream of the
PEXEL, became quite insoluble and remained in the
ER\textsuperscript{42}, we explored how the solubility of cleaved PEXEL
reporters can influence their export. In contrast, to the mis-cleaved
Hyp1 P\textsubscript{2}' K reporter, the solubility of the mis-cleaved
KAHRP and STEVOR P\textsubscript{2}' K reporter proteins did not appear
to decrease, and it is unclear whether the mis-cleaved species
accumulated in the ER or were trafficked to the PV like the correctly
cleaved P\textsubscript{2}' K species. We have shown previously that the
ER-trapped Hyp1 P\textsubscript{2}' K reporter can still bind to
ER-resident HSP101 despite its insolubility \textsuperscript{42}. The
fact that the KAHRP and STEVOR P\textsubscript{2}' K mutants were
efficiently cleaved and trafficked to the PV but were not strongly
exported suggests the P\textsubscript{2}' residue might not be important
for the initial binding to HSP101 but rather for commitment to stronger
downstream interactions with the whole of PTEX required for cargo
unfolding and export.

Our results also argue that PTEX, more specifically HSP101, recognises a
wider region in the cargo than the PEXEL motif and that this is
important for export. Truncation of the spacer region in at least three
different reporter constructs used in this study consistently blocked
export without apparently affecting PEXEL processing. Microscopic
analysis of the 3aa spacer Nluc-mDH-FL constructs clearly showed that
the protein is trapped within the PV area where PTEX is located,
consistent with previous reports \textsuperscript{4,24}. Despite this
co-localisation, we have shown that spacer mutants that bind less
strongly to HSP101 are exported less efficiently.

While the molecular mechanism of PTEX cargo recognition remains to be
elucidated, our data has shed some light into how this process may
occur. We found that while a 13aa spacer was sufficient to facilitate
modest export, a longer 51aa spacer promoted stronger cargo binding to
HSP101, suggesting that perhaps an increase the length of the
unstructured N-terminal polypeptide increases the likelihood of the
cargo will stably binding to HSP101. Clp/HSP100 chaperones generally
require a recognition signal of at least 10-20 broadly diverse amino
acids to initiate the polypeptide unfolding and
translocation\textsuperscript{51,52}. Our data is consistent with this
model and suggest that HSP101 also requires a large region within the
N-terminal portion of the cargo protein to initiate cargo translocation.
This could explain why single P\textsubscript{2}' K point mutation in
the mature PEXEL motif is not enough to prevent the interaction
altogether and why the N-termini of PNEPs, despite the latter lacking a
mature PEXEL motif, can still act as an export signal
\textsuperscript{38}. Cargo recognition in AAA+ ATPases, particularly
the family of Clp/HSP100 chaperones, begins with the pre-unfolding step
that is initiated by a low-affinity probabilistic binding of the
chaperone to a loosely folded or aggregated region of a protein,
followed by a commitment step where the ATPase binds more stably to the
cargo protein before unfolding and threading the protein through the
chaperone's central cavity\textsuperscript{53}. It has been shown for
the AAA+ ATPase ClpXP, that the length of the cargo polypeptide bound to
the inner cavity of the ClpXP affects the commitment step, such that
longer polypeptides seem to promote more successful commitment and
subsequent unfolding\textsuperscript{54,55}. Clp/HSP100 chaperones,
particularly ClpB and HSP104, are thought to have a similar
pre-unfolding step\textsuperscript{52,53,56}. We therefore propose a
model whereby a longer spacer region may increase accessible areas for
the initial probabilistic binding step, or stabilise association of
exported proteins to HSP101, subsequently leading to less frequent
dissociation from the unfoldase (Fig 6A). Consistently, Hyp1-Nluc-mDH-FL
reporter with a short 3aa spacer region exhibited low-level affinity to
HSP101 that greatly reduced export, suggesting that the cargo may have
initially associated with HSP101 but later dissociated from the
unfoldase because there was insufficient net affinity to proceed to the
commitment step.

We also explored which amino acids of the spacer region could promote or
inhibit HSP101 binding. The subunits of the second nucleotide binding
domain (NBD2) of HSP101 contain conserved tyrosine residues which are
thought to bind the unfolded cargo protein via hydrophobic interactions
to help ratchet the cargo through HSP101 \textsuperscript{19}. As the
HSP101 subunits undergo allosteric changes powered by ATP hydrolysis,
the tyrosines move up and down to help grip and pull on the cargo (Fig
6B). Since it is possible that the tyrosine residues may interact with
the hydrophobic residues in the spacer region, we mutated the four
hydrophobic residues in the 13aa Hyp1 spacer and this was shown to
reduce export. Export reduction was particularly strong for the mutation
of hydrophobics to charged (E) residues compared to polar residues (S).
The deletion of the 13aa spacer region from the 51aa acid spacer still
resulted in strong export as the next 13aa downstream from the first
13aa still contained four hydrophobic residues. One possible reason why
the minimal 13aa spacer preceding the globular Nluc region was exported
much better than the 3aa spacer was that the longer spacer could project
further into HSP101's central cavity, down into NBD2 where it could
engage the cargo binding tyrosine residues (Fig 6B).

In conclusion, our data suggests dual functions for the
P\textsubscript{2}' position of PEXEL proteins. The first is that it
forms part of the PMV recognition sequence for cleavage. In some
proteins such as Hyp1, mutation of P\textsubscript{2}' to K greatly
reduces PMV binding and/or successful proteolytic activity but in other
PEXEL proteins such as STEVOR and KAHRP P\textsubscript{2}' is not as
critical for PMV cleavage. This indicates that other amino acids within
and bordering the PEXEL probably have a bearing on how dependent PMV
activity is on the P\textsubscript{2}' residue. A lack of PMV cleavage
probably results in ER retention as evidenced by the substantial
retention for poorly cleaved Hyp1 versus efficiently cleaved STEVOR and
KAHRP. Successful cleavage of the PEXEL P\textsubscript{2}' K mutant
appears to permit trafficking to the PV as STEVOR and KAHRP more
efficiently reach the PV than poorly cleaved Hyp1. Once in the PV,
however, the STEVOR and KAHRP P\textsubscript{2}' K reporters were not
efficiently exported into the RBC compartment. This could be due to the
reporters not being efficiently recognised by PTEX or because the
reporters were in a PTEX-free sub-compartment of the PV. The latter,
however, is unlikely, because recent split-GFP experiments indicate that
PV-resident protein have full access to PTEX \textsuperscript{57}. It is
possible therefore that the charge reversal P\textsubscript{2}' mutant
somehow binds to PTEX less efficiently leading to less engagement,
unfolding and eventual export into the RBC compartment.

\textbf{Materials and Methods}

\emph{4.1. Culture of} P. falciparum transfectants

Asexual blood-stage \emph{Plasmodium falciparum} (3D7 or CS2 background)
was cultured according to the established protocol \textsuperscript{58}.
Cultures were routinely maintained in complete RPMI media containing
RPMI-1640 base media supplemented with 2.5 mM HEPES, 367 \selectlanguage{greek}µ\selectlanguage{english}M
hypoxanthine, 31.25 \selectlanguage{greek}µ\selectlanguage{english}g/mL Gentamicin, 25 mM NaHCO\textsubscript{3}, and
0.5\% (w/v) Albumax II (Invitrogen). Prior to transfection, 100 \selectlanguage{greek}µ\selectlanguage{english}g of
plasmid DNA was resuspended in TE and cytomix (25 mM HEPES, 120 mM KCl,
0.15 mM CaCl\textsubscript{2}, 2 mM EGTA, 5 mM MgCl\textsubscript{2}, 10
mM
K\textsubscript{2}HPO\textsubscript{4}/KH\textsubscript{2}PO\textsubscript{4}pH
7.6) and mixed with packed RBCs as per \textsuperscript{59}. After
electroporation using Gene Pulser XCell System (BioRad), the uninfected
RBCs were then mixed with 20 \selectlanguage{greek}µ\selectlanguage{english}L of HSP101-HA\emph{glmS}trophozoite-stage
parasites \textsuperscript{42} and allowed to invade the transfected
RBCs for 2 cell cycles before starting a selection with 2.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL
blasticidin S.

\emph{4.2 Generation of PEXEL P\textsubscript{2}' mutants and spacer
truncation constructs}

The expression of Hyp1/ STEVOR/ KAHRP-Nluc-mDH-FL reporters was driven
by a bidirectional \emph{Plasmodium berghei} EF1a promoter that also
controlled expression of the blasticidin deaminase drug resistance
cassette. The plasmid pEF-Hyp1-Nluc-mDH-FL was derived from plasmid
pEF-Hyp1-Nluc-DH-APEX \textsuperscript{43}. The Hyp1 component of this
plasmid contained the first 113aa of Hyp1 (PF3D7\_0113300), including
the RLLTE PEXEL motif \textsuperscript{43}. A synthetic murine
dihydrofolate reductase (mDH) gene fragment with C-terminal 3x FLAG
epitopes (Bioneer Pacific) was ligated into the Nluc-DH-APEX plasmid
using \emph{SpeI} and \emph{MluI} enzymes to remove the previous
mDH-APEX gene cassette.

Generation of the P\textsubscript{2}' lysine (lys/K) and alanine (ala/A)
mutation of the Hyp1-Nluc-mDH-FL was performed as follows: Hyp1 region
was first amplified as two overlapping PCR fragments with the primer
pairs 1 \& 2 and 3 \& 4 (Table S4). The overlap region between these two
PCR products contained the mutations indicated above. PCR fragments were
then sewn together with primer pair 1 \& 4 via overlapping PCR, ligated
into the pJET1.2/blunt plasmid (ThermoFisher Scientific). Mutagenesis
was confirmed via standard Sanger sequencing (service provided by Monash
Micromon Genomics) of the isolated plasmids. The mutant
P\textsubscript{2}' K/A Hyp1 fragments were released from pJET1.2/blunt
using \emph{XhoI} and \emph{NcoI} and ligated into pEF-Hyp1-Nluc-mDH-FL
to replace the wildtype Hyp1 fragment.

Generation of the Hyp1-Nluc-mDH-FL plasmids with a truncated spacer
region between the PEXEL motif and the start of the Nluc gene was
carried out as follows: PCRs were performed with primer 1 paired with
primer 7, 8 or 9 to produce spacers of 3aa, 13aa and 51aa, respectively
(Table S4). Note that primers 7, 8 and 9 produced Hyp1 PCR products that
contained one additional amino acid between the last Hyp1 residue and
the start Met of Nluc. The PCR products were ligated into pJET1.2/blunt
and screened as above.

To express wildtype STEVOR (PF3D7\_0200400) and KARHP (PF3D7\_0202000)
in the Nluc-mDH-FL reporter plasmid, synthetic leader sequences encoding
the first 99aa of STEVOR and 105aa of KAHRP containing \emph{XhoI}
and\emph{NcoI} restriction sites were first obtained as string oligos
from GeneART (ThremoFisher Scientific). P\textsubscript{2}' lysine
mutations for both STEVOR and KAHRP leaders were also synthesised as
above. The synthetic DNA sequences were ligated into pJET1.2/blunt and
validated by sequencing prior to transfer via \emph{XhoI} and
\emph{NcoI} sites into the pEF-Hyp1-Nluc-mDH-FL plasmid replacing the
Hyp1 sequence.

To produce pEF-STEVOR-Nluc-mDH-FL reporters with truncated spacers, PCRs
were performed with primers 10 \& 11 and primers 10 \& 12 to produce
spacer fragments of 3aa, and 13aa respectively, using
pEF-STEVOR-Nluc-mDH-FL as a template (Table S5). PCR products were
ligated into pJET1.2/blunt and ligated into pEF-Nluc-mDH-FL
via\emph{XhoI} and \emph{NcoI} cloning sites to produce gene fusions
with only one amino acid between STEVOR/KAHRP leader sequence and Nluc.
To produce the pEF-KAHRP-Nluc-mDH-FL with 3aa and 13aa spacers, PCRs
were performed with primers 10 \& 13 and primers 10 \& 14 (Table S4).
The PCR products were ligated into the Nluc-mDH-FL reporter as per the
STEVOR reporters.

\emph{4.3 Indirect immunofluorescence analysis}

IFA was performed essentially according to \textsuperscript{60}, where
infected RBCs (iRBCs) were settled onto a poly-L-lysine (Sigma, P8920)
coated coverslip and fixed with 4\% paraformaldehyde/0.0075\%
glutaraldehyde. Following fixation, the cells were permeabilised with
0.1 M glycine/0.1 \% Triton X-100 for 12 minutes at room temperature
(RT). Coverslips were probed with rabbit anti-Nluc IgG (12.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL),
mouse anti-EXP2 (10 \selectlanguage{greek}µ\selectlanguage{english}g/mL), rabbit anti-ERC (1:1000), mouse anti-FLAG M2
(Sigma, 10 \selectlanguage{greek}µ\selectlanguage{english}g/mL), mouse anti-HA (Sigma clone HA-7; 1:500). After
washing, goat anti-rabbit Alexa Fluor 594 nm and goat anti-mouse Alexa
Fluor 488 nm (1:2000) secondary antibodies (Invitrogen) were applied for
1 h at RT. Fixed material was mounted in VECTASHIELD with DAPI and
imaged on Zeiss Cell Axio-Observer (Carl Zeiss). Image acquisition was
performed with Zen Blue imaging software.

\emph{4.4 Quantification of Imaging Data}

Image analysis was carried out according to\textsuperscript{42}.

\emph{4.5 Chemical Cross-Linking of P. falciparum Culture and
Immunoprecipitation}

RBCs infected with the HSP101-HA\emph{glmS} /PEXEL-Nluc-mDH-FL parasites
were enriched through magnetic purification and their proteins were
solubilised in 20x pellet volume of IP lysis buffer (1\% Triton X-100,
0.1\% SDS, 150 mM NaCl, 10 mM Tris-HCl pH 7.4) supplemented with
cOmpleteTM Protease Inhibitor Cocktail (Roche) and subjected to 2 freeze
and thaw cycles. The lysate was clarified by centrifugation and
incubated overnight at 4\textsuperscript{o}C with anti-HA monoclonal
agarose (Sigma-Aldrich). Following incubation, the agarose beads were
washed 5x with 1 mL IP lysis buffer and the proteins were eluted with 50
\selectlanguage{greek}µ\selectlanguage{english}L 2x NRSB (100 mM Tris-HCl pH 6.8, 4 mM EDTA, 4\% SDS, 0.01\%
bromophenol blue, 20\% (v/v) glycerol). For Nluc IgG
immunoprecipitations, the parasite lysates were incubated overnight at
4\textsuperscript{o}C with 10 \selectlanguage{greek}µ\selectlanguage{english}g IgG-purified anti-Nluc antibody.
Following incubation, protein A-Sepharose 4B (Invitrogen) was added to
bind the immune complexes and samples were incubated for an additional 1
h at RT. The beads were washed, and proteins eluted as above. In all
cases, both input and elution fractions were reduced in 200 mM DTT at
70\textsuperscript{o}C for 5-10 mins prior to SDS-PAGE separation,
western blot, and immunoblotting.

\emph{4.6 Western Blotting}

Proteins transferred from the gels to a nitrocellulose membrane using
iBlot\selectlanguage{ngerman}\textsuperscript{®} Blotting System (Invitrogen). The blots were
blocked in 1\% casein in PBS and probed with primary antibody (Table
2.3) diluted in the blocking buffer overnight at 4\textsuperscript{o}C.
The blots were washed and probed with goat anti-mouse or goat
anti-rabbit fluorescence-based (Alexa Fluor 700 and 800 nm, Invitrogen)
antibodies diluted 1/10,000 in blocking buffer for 1 h at room
temperature followed by 3 washes with 1x PBS. The fluorescent secondary
antibodies were visualized with a LI-COR Odyssey FC imaging system.
Densitometry analysis was performed with Image Studio v. 1.0.

\emph{4.7 Biochemical PEXEL Cleavage Assays}

The cleavage assay was performed as described in Hodder et al. (Hodder,
Sleebs et al. 2015). 2 nM of \emph{P. vivax} PMV in buffer (25 mM
Tris-HCl pH 6.4 and 25 mM MES pH 6.4) was incubated with 5 \selectlanguage{greek}μ\selectlanguage{english}M FRET
peptide substrates representing WT and mutant KAHRP, STEVOR and Hyp1
sequences (Supplementary Table 5) in a total volume of 20 \selectlanguage{greek}μ\selectlanguage{english}L. Samples
were incubated at 20\selectlanguage{ngerman}°C for 20 h and measurement was carried out using
Envision plate (PerkinElmer) reader (ex. 340 nm; em. 490 nm).
Biochemical \emph{P. vivax} PMV inhibitory assays (20 \selectlanguage{greek}μ\selectlanguage{english}L total volume)
were performed using 2 nM \emph{P. vivax} PMV in buffer (25 mM Tris-HCl
and 25 mM MES, pH 6.4) with 5 \selectlanguage{greek}μ\selectlanguage{english}M FRET KAHRP\_WT fluorogenic peptide.
Assay reactions were incubated at 37\selectlanguage{ngerman}°C for 2 h in the presence of
peptides (10 points dose-response, 1 in 2 dilution series starting at
100 nM) representing STEVOR and Hyp1 sequences (Table 2.2). Fluorescence
was measured with an Envision plate (PerkinElmer) reader (ex. 340 nm;
em. 490 nm). To determine the level of PMV inhibition, ``nonlinear
regression four-parameter to fit analysis'' using Domatics software
(version 5.3.1612.8630) was performed.

\emph{4.8 Protein Solubility Analysis}

Protein solubility profiling was performed according to Gruring et
al.\textsuperscript{38}. 5 \selectlanguage{greek}µ\selectlanguage{english}L of parasite pellet (obtained through
magnetic separation or saponin lysis) was resuspended in 100 \selectlanguage{greek}µ\selectlanguage{english}L
hypotonic buffer (5 mM Tris-HCl pH 8.0) and subjected to 1 cycle of
freeze and thawing. The soluble fraction was separated from the pellet
by centrifugation at 16,000 x \emph{g} for 5 minutes at
4\textsuperscript{o}C. The pellet was then incubated sequentially with
100 \selectlanguage{greek}µ\selectlanguage{english}L 0.1M Na\textsubscript{2}CO\textsubscript{3} pH 11.5 and 1\%
Triton-X in H\textsubscript{2}O for 30 minutes at 4\textsuperscript{o}C.
Soluble fractions from each incubation were transferred into a new tube.
Soluble fractions were mixed with 20 \selectlanguage{greek}µ\selectlanguage{english}L 6x NRSB (300 mM Tris-HCl pH 6.8,
12 mM EDTA, 12\% SDS, and 0.03\% w/v bromophenol blue, 60\% v/v
glycerol) and the pellet was resuspended in 120 \selectlanguage{greek}µ\selectlanguage{english}L 1xNRSB. All samples
were kept at -20\textsuperscript{o}C until used.

\emph{4.9 Statistical Analysis}

Numerical data was mainly visualised using Microsoft Excel for Mac
(v.15.37) and GraphPad Prism version 9.1.0. Statistical analysis was
performed using GraphPad Prism version 9.1.0 unless otherwise stated.

\emph{4.10 Nanoluciferase protein export assays}

The nanoluciferase reporter export assays were performed as
per\textsuperscript{48}.

\textbf{Author Contributions}

Mikha Gabriela, Claudia B.G. Barnes, Dickson Leong, Brad E. Sleebs,
Molly P. Schneider, Dene R. Littler and Paul R. Gilson performed
experimental work. Mikha Gabriela, Claudia G. B. Barnes and Paul R.
Gilson wrote and edited manuscript. Brendan S. Crabb and Paul R. Gilson
and Tania F. de Koning provided funding and student supervision.

\textbf{Acknowledgments}

We thank the Lifeblood Biological Resources for providing blood and the
WEHI Antibody Facility for the EXP2 and Nluc antibodies. We thank
Matthew Dixon and Leann Tilley for kindly providing \emph{Pf} ERC
antibodies.

\textbf{Funding information}

NHMRC Program Grant and NHMRC Investigator Grant. Grant/ Award numbers:
1092789 and 119780521 and Australian Research Council (ARC) grant
DP160102582. This work was supported by funding from the Victorian
Operational Infrastructure Support Program received by the Burnet
Institute. M.G. was recipient of a Deakin University Postgraduate
Research Scholarship.

\textbf{Conflict of interest}

The authors declare no conflicts of interest.

\textbf{Data availability statement}

All relevant data are within the manuscript and its Supporting
Information files.

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\textbf{Figures}

\textbf{Figure 1\emph{.} P\textsubscript{2}' Ala and Lys mutations
reduce with the proteolytic processing of the PEXEL motif.}
\textbf{(A)}5 \selectlanguage{greek}µ\selectlanguage{english}M fluorogenic peptides were incubated with 2 nM
recombinant\emph{Pv} PMV and assayed at 20\textsuperscript{o}C.
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\textless{}0.0001, ***, p-value\textless{}0.001).
\textbf{(B)}Representative IFA images (n=3 independent replicates) of
HSP101-HAglmS parasites expressing WT and P\textsubscript{2}' 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, 5\selectlanguage{greek}μ\selectlanguage{english}m.
DIC, Differential Interference Contrast. DAPI
(4',6-diamidino-2-Phenylindole; Blue) was used to stain parasite nuclei.
\textbf{(C)} Quantification of the fluorescence signal of STEVOR and
KAHRP-Nluc-mDH-FL constructs within the parasite cells from \textbf{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\textless{}0.0001). \textbf{(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
P\textsubscript{2}' K Hyp1-Nluc-mDH-FL (58.1 \selectlanguage{ngerman}± 1.2 kDa, n=10). **,
Mis-cleaved P\textsubscript{2}' 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. \textless{} 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.

\textbf{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. \textbf{(B)}Western blot analysis
of infected RBCs (mid-stage trophozoites) sequentially extracted with 5
mM Tris-Cl pH 8.0 (Tris Sn), 0.1M Na\textsubscript{2}CO\textsubscript{3}
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
previously\textsuperscript{42}, and has been included as a comparator to
the KAHRP and STEVOR blots.

\par\null

\textbf{Figure 3. Sequential truncation of the spacer region caused
trapping of the Nluc-mDH-FL constructs in the ER and PV of the
parasite.} \textbf{(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-\emph{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 \selectlanguage{greek}μ\selectlanguage{english}m. DIC, Differential Interference Contrast.
DAPI (4',6-diamidino-2-Phenylindole; Blue) was used to stain parasite
nuclei. \textbf{(B)} Quantification of export of the spacer truncation
Nluc-mDH-FL constructs presented in \textbf{A} . Export signal of
\textgreater{}20 cells were recorded for each construct. Box and whisker
plot represents 25\textsuperscript{th}-75\textsuperscript{th} and
10\textsuperscript{th}-90\textsuperscript{th} percentiles, respectively.
Statistical significance was determined using ordinary one-way ANOVA.
****, p-value\textless{}0.0001; **, p-value\textless{}0.01; *, p-value =
0.0159).

\textbf{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-HA\emph{glmS} expressing various
truncations of the Hyp1 spacer region (n=3) as indicated above each
lane. HSP101-HA\emph{glmS} 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. \textbf{(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 (\textbf{C)} and (\textbf{D)} ,
Densitometry of the IP performed on \textbf{A} (left) \& \textbf{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, \selectlanguage{ngerman}±SD. Statistical significance was determined using an
Ordinary one-way ANOVA. ****, p value\textless{}0.0001.

\textbf{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. \textbf{(B)} Fluorescence microscopy of Hyp1 spacers
detected with rabbit Nluc and mouse EXP2 mAb as a PVM marker that 51aa,
\selectlanguage{english}[?]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. \textbf{(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 \selectlanguage{english}[?]NT13aa parasites exported more efficiently
Hyp1 13aa parasites. Export signal of \textgreater{}60 cells were
recorded for each construct. Box and whisker plot represents
25\textsuperscript{th}-75\textsuperscript{th} and
10\textsuperscript{th}-90\textsuperscript{th} percentiles, respectively.
Statistical significance was determined using ordinary one-way ANOVA.
P-value, * \textless{}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, *\textless{}0.05, *\textless{}0.01.

\textbf{Figure 6. Hypothetical model for the HSP101-cargo recognition.}
\textbf{(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 \textsuperscript{19}.
\textbf{(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.

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