Keywords.
Plasmodium falciparum , malaria, protein export, PTEX, virulence,
PEXEL, chaperone.
Introduction
The pathogenesis of malaria lies upon the ability of Plasmodiumparasites, 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 1-5. The intraerythrocytic
stage of the parasite resides within a membranous compartment called the
parasitophorous vacuole (PV) 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 Plasmodium
t ranslocon of ex ported proteins (PTEX) mediates the
translocation of proteins across the PVM 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 candidate8-10.
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) 11-13. The
cleavage step occurs within a pentameric amino acid motif near the
N-terminus of the exported protein destined for export, termed thePlasmodium export element (PEXEL) 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 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 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 EXP217-19. 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 unknown20,21.
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 22-26. This N-terminal region usually
contains a recessed signal peptide to promote entry of the proteins into
the parasite’s ER 27 or a hydrophobic stretch of amino
acids that serve the same function 28. The discovery
of the five-amino acid PEXEL motif in the N-terminal region of many
exported proteins was the first export-specific signal1,2 that was proven to be predictive for exported
proteins 29 and allowed the identification of
>450 putative exported proteins in P. falciparum4. Interestingly, however, the presence of the PEXEL
motif alone does not guarantee export, as its location within a
protein’s primary structure 30 and the presence of
~12 amino acids downstream of the motif have also been
shown to be required to achieve efficient export 24.
Furthermore, PEXEL-negative exported proteins (PNEPs) are also present
in the Plasmodium exportome 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 P3 R and P1 L residues are
the most conserved of the PEXEL motif and in P. falciparum have
been shown to be necessary for efficient cleavage by PMV12-14. It is assumed therefore that P1and P3 facilitate PEXEL cleavage and thus help to
release the exported proteins from the ER membrane where they are
originally anchored following ER import 11. In
comparison, the purpose of the last conserved P2’
(E/Q/D) residue of PEXEL is less certain. Some evidence suggests that
following cleavage, the P1’ and P2’
resides which now cap the mature PEXEL protein, play a role in promoting
export across the PVM 1,14,33. After cleavage, the
exposed xE/Q/D motif becomes N-terminally acetylated34 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 35. Contrary to this, however, other studies have
found that P2’ amino acid also influences the efficiency
of PEXEL processing or does not influence export at all36-38.
Here, we present evidence that the P2’ residue has a
dual function. For some PEXEL proteins, P2’ mutations
greatly reduce PMV cleavage increasing protein retention in the ER and
reducing export. For other proteins, P2’ 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.
Results