A Newly Discovered
Protein Export Machine
in Malaria Parasites
Koning-Ward, Tania F. de, Paul R. Gilson, Justin A. Boddey, Melanie Rug, Brian J.
Smith, Anthony T. Papenfuss, Paul R. Sanders, et al.
Nature, 2009
Carolyn Brager
Plasmodium
Lifecycle
Römisch K, 2012 Diversion at the ER
What is the mechanism of protein export?
Plasmodium falciparum translocon of exported proteins (PTEX)
Parasitophorous Host Erythrocyte
Vacuole
? Exported proteins
• 200-300 proteins
• virulence-associated
roles
• essential for blood
stream survival
Figure 4, modified
Known motifs required for protein export
• Transport across plasma membrane
• Proteins are sent to the secretory pathway mediated by
?
a hydrophobic endoplasmic reticulum signal sequence
• Transport across the PVM
• requires a Plasmodium export element (PEXEL) located
approximately 25–30 amino acids downstream of the
ER signal sequence
from Alan Cowman’s lab at the Walter and Eliza Hall Institute of Medical Research
Criteria for candidate translocon proteins
1. Be restricted to the Plasmodium genus only
because PEXEL motifs appear to be absent from even closely related Apicomplexan
parasites
2. Incorporate a power source
likely an ATPase
3. Have dual apical merozoite and ring-stage PVM localization
proteins are known to be exported upon merozoite invasion and during early ring stages
4. Be essential for blood-stage growth
if exported proteins are essential, then the machinery responsible for transporting them
would be as well
5. Specifically associate with their exported protein cargo.
Proteomic analysis of relevant parasite membranes
• lipid-raft-like detergent resistant membranes from ring-stage
parasites were strongly enriched in proteins that are known (or
suspected) to localize to the PVM
analyzed for potential translocon components
First candidate: HSP101
• belongs to the dual AAA+ ATPase domain containing HSP100/ClpA/B
chaperone family
• encodes an N-terminal ER signal sequence for export into the
parasitophorous vacuole
• forms a hexameric ring-shaped complex that generally translocates
proteins through a central pore of the ring in an ATP-dependent
manner
Structural prediction
Supplementary figure 1
Second candidate: PTEX150
• has a putative ER signal sequence and is found throughout
the Plasmodium genus but not in other genera
• has an identical late schizont/early ring transcriptional profile to
HSP101
• appears with HSP101 in two distinct high molecular mass bands,
suggesting they form a large complex
Supplementary figure 2
Methods Overview
• Antibodies were generated against HSP101 and PTEX150
• Generated transgenic P. falciparum (3D7) parasite strains where
endogenous HSP101 and PTEX150 genes were modified to include an
HA tag, termed 3D7-101HA and 3D7-150HA
Slightly larger due to the
addition of the tag
Main techniques: western blotting, immunofluorescence analysis,
co-immunoprecipitation
Figure 1
Translocon Candidates
• PTEX150 and HSP101 co-localize
and are found in the PVM (outer)
MSP1: plasma membrane marker (inner)
• PTEX150 and HSP101 reside at the
apical end of merozoites*,
presumably in secretory
organelles
*arrows point to apical ends, determined by
additional BF/DAPI staining
Figure 1
Do PTEX150 and HSP101 associate?
• HSP101 and PTEX150 co-
precipitated in a specific and
reciprocal manner
• Three additional proteins were
also specifically affinity purified
with both 150HA and 101HA
species
Figure 1
Isolating a 5 member PTEX complex
• So far: identified a core macromolecular complex comprising HSP101,
PTEX150 and EXP2 with two additional, potentially accessory,
proteins, PTEX88 and TRX2
Figure 2, Supplementary figure 4
EXP2
EXP2 interacts with the PTEX150 complex EXP2 and PTEX150 colocalize
EXP2 may be the membrane-associated component
• EXP2 is the most resistant of the three core PTEX components to
extraction with carbonate, consistent with it being the direct
membrane-associated component of the PTEX complex
• p83 fragment of MSP1 is included as a control for solubilization of a
peripherally associated membrane protein
Figure 4
Interaction between PTEX components and
exported proteins
• used antibodies against 3
exported proteins to pull-down
cross-linked parasite protein
extracts
• Unbound and eluate fractions
were probed with antibodies
against PTEX components
exported proteins pulled down
the PTEX components HSP101,
PTEX150 and EXP2 far more than
did the negative control
Figure 3
Proposed Mechanistic Model
1. PTEX complex is injected directly from
apical merozoite organelles into the
vacuole à associates with the PVM
2. Proteins destined for export are unfolded
by HSP101
3. EXP2 is the direct membrane-associated
component of the PTEX complex EXP2 and
may constitute the membrane pore
4. Host erythrocyte HSP70 transforms from a
soluble to a membrane-bound state in
parasitized cells to assist in the passage
and/or refolding of exported proteins
Figure 4
Main Takeaways and Future Directions
this study reveals the
possible identity of a protein
provides an “Achilles heel”
export machine involved in
for possible anti-malarial
the export of proteins that
drug targets
are necessary for Pf’s
virulence and survival