Endoplasmic Reticulum PI(3)P
Lipid Binding Targets Malaria
Proteins to the Host Cell
Souvik Bhattacharjee,1,2Robert V. Stahelin,3,4Kaye D. Speicher,5David W. Speicher,5and Kasturi Haldar1,2,*
1Center for Rare and Neglected Diseases
2Department of Biological Sciences
3Department of Chemistry and Biochemistry
University of Notre Dame, Notre Dame, IN 46556, USA
4Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, South Bend, IN 46617, USA
5Center for Systems and Computational Biology and Molecular and Cellular Oncogenesis Program, Wistar Proteomics Core Facility,
The Wistar Institute, Philadelphia, PA 19104, USA
Hundreds of effector proteins of the human malaria
parasite Plasmodium falciparum constitute a‘‘secre-
tome’’ carrying a host-targeting (HT) signal, which
predicts their export from the intracellular pathogen
into the surrounding erythrocyte. Cleavage of the
HT signal by a parasite endoplasmic reticulum (ER)
protease, plasmepsin V, is the proposed export
mechanism. Here, we show that the HT signal facili-
tates export by recognition of the lipid phosphatidy-
linositol-3-phosphate (PI(3)P) in the ER, prior to and
independent of protease action. Secretome HT
signals, including those of major virulence determi-
nants, bind PI(3)P with nanomolar affinity and amino
acid specificities displayed by HT-mediated export.
PI(3)P-enriched regionsaredetected within thepara-
site’s ER and colocalize with endogenous HT signal
on ER precursors, which also display high-affinity
binding to PI(3)P. A related pathogenic oomycete’s
HT signal export is dependent on PI(3)P binding,
without cleavage by plasmepsin V. Thus, PI(3)P in
the ER functions in mechanisms of secretion and
Malaria continues to extract a major toll on human health. The
most virulent of human malarias is caused by the protozoan
parasite Plasmodium falciparum. It is responsible for a million
deaths in children each year, largely in sub-Saharan Africa
(WHO, 2010). The symptoms and pathologies of disease are
caused by blood stage parasites that infect erythrocytes (Miller
et al., 2002). The intraerythrocytic parasite resides and prolifer-
ates within a parasitophorous vacuolar membrane (PVM; Fig-
ure 1). Proteins are exported from the parasite, across the
PVM into the cytoplasm and membrane of the erythrocyte to
induce changes in transport and membrane properties of the
host cell (Haldar and Mohandas, 2007; Maier et al., 2009; Ngui-
tragool et al., 2011). Several hundred P. falciparum proteins are
predicted to be released into the erythrocyte (Hiller et al., 2004;
Marti et al., 2004; Sargeant et al., 2006; van Ooij et al., 2008)
including adhesins directly linked to severe and fatal disease
pathologies of cerebral malaria and placental malaria (Kyes
et al., 2007; Duffy and Fried, 2003).
Parasite proteins destined for the erythrocyte are expected to
be first recruited into the endoplasmic reticulum (ER) via an
N-terminal signal sequence or a transmembrane domain
(Lopez-Estran ˜o et al., 2003). The presence of a consensus host
(cell) targeting (HT) or Plasmodium export element (PEXEL)
signal RxLxE/D/Q downstream of the signal peptide or trans-
(Hiller et al., 2004; Marti et al., 2004; Sargeant et al., 2006; van
Ooij et al., 2008). As shown schematically in Figure 1, the HT
signal is cleaved in the parasite’s ER (Chang et al., 2008;
Osborne et al., 2010). In vitro data suggest that cleavage is
due to a resident ER protease plasmepsin V (Boddey et al.,
2010; Russo et al., 2010) that functions in lieu of signal peptidase
to release newly synthesized protein from the ER membrane.
Cleavage by plasmepsin V is also proposed to be the mecha-
nism for host targeting (Boddey et al., 2010; Russo et al.,
2010), but the underlying mechanisms remain unknown.
The malarialHTsignalisrelatedboth insequenceand function
to the host-targeting signal of another eukaryotic pathogen, the
oomycete Phytophthora infestans, which infects plant cells
and caused the Irish potato famine (Whisson et al., 2007). The
P. infestans signal is composed of the sequence RxLR.DEER,
which if expressed in P. falciparum, can target reporter proteins
to the host erythrocyte (Bhattacharjee et al., 2006). Conversely
malarial HT signal when expressed in P. infestans catalyzes
protein export to the host plant cell (Dou et al., 2008). In addition,
the oomycete signal RxLR.DEER has recently been shown to
trating the host (Kale et al., 2010; Figure S1 available online). The
Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc. 201
binding of this HT signal to PI(3)P was a surprising finding. Poly-
phosphoinositide binding and binding to the phosphomonoester
phosphatidic acid have been attributed to small but cationic rich
motifs (McLaughlin et al., 2002; Stace and Ktistakis, 2006).
However, to date identified PI(3)P effectors have a well-defined
PI(3)P-binding pocket in FYVE or PX domains and generally
have at least four points of contact with PI(3)P (Kutateladze,
2010). Although the initial specificity of HT signal binding to
PI(3)P was established, the underlying mechanism of binding
remains undefined. The significance of HT signal binding to lipid
for malarial parasites is unknown, because malarial HT signals
are cleaved in the ER and no cell surface PI(3)P was detected
on host erythrocytes (Kale et al., 2010). Thus, malarial effector
proteins cannot utilize PI(3)P to translocate the PVM into the
erythrocyte (Figure 1).
PI(3)P Binds the Malarial HT with Both Affinity
and Specificity Linked to Export and Is Detected
in the Parasite’s ER
To investigate the phosphoinositide (PI) specificity of the HT
signal (RLLYE) of P. falciparum histidine-rich protein II (PfHRPII),
green and red fluorescent protein (GFP and RFP) fusions were
used in lipid sedimentation assays (Figures 2A, S2, and Table
S2). Clearly, both constructs displayed high selectivity for
PI(3)P containing vesicles, whereas mutation of the consensus
sequence (ALAYA) or the introduction of a cleaved (YE)
sequence significantly reduced PI(3)P binding. To further
demonstrate the specific nature of PI(3)P binding, Ins1,3P2was
added to the lipid sedimentation assay to compete with PI(3)P
vesicles for HT binding. In both instances (HT-GFP and HT-
RFP), Ins1,3P2 greatly reduced the amount of HT bound to
PI(3)P containing vesicles (Figure S2D). Additionally, HT-GFP
was able to compete with the high-affinity PI(3)P binding PX
cles in a concentration-dependent manner (Figure 2B).
A lack of quantitative information on the affinity of PI(3)P
binding by HT-targeting signals of oomycete/malaria, precludes
understanding their relative mechanism of action. We therefore
2C–2E, discovered a high-affinity interaction between the HT
signal of PfHRPII and PI(3)P vesicles (36 nM). This was
completely abolished by mutation ALAYA (Kd> 5 mM). The muta-
tion ALLYE (Kd> 5 mM) also abrogated PI(3)P binding, whereas
RLAYE and RLLYA reduced binding by 23- and 7-fold, respec-
tively. The relative contribution of R, L and E residues in the HT
signal to PI(3)P binding correlated with their relative utilization
in the host targeting logo (Figure 2E), suggesting lipid binding
and protein export may be linked. Additional HT signals from
the major virulence determinant P. falciparum erythrocyte
membrane protein 1 (PfEMP1) and other parasite effectors
known to be targeted into the host erythrocyte, also bound
PI(3)P vesicles with nanomolar affinity ranging from 20 to
110 nM. Mutation of R/K with alanine decreased binding by at
least 20-fold (Figure 2E), and, in most cases, binding to PI(3)P
was not detectable, suggesting that HT signals of multiple,
important malarial effector proteins bind PI(3)P. Together these
data led us to consider that PI(3)P binding may be a generalized
property of a wide range of malaria secretome effectors ex-
ported to the erythrocyte.
However, PI(3)P is known to be a cytoplasmic lipid in cells. To
bind the HT signal on newly synthesized parasite effectors
exported to the erythrocyte, it must be present in the lumen of
parasite’s secretory pathway and act prior to processing by
plasmepsin V in the ER. To the best of our knowledge, there
are no reports of the presence of PI(3)P within the secretory
pathway of cells. Thus, to investigate thispossibility in the malar-
ial secretory pathway, we expressed a secretory fusion of
monomeric Cherry (mCherry) and the FYVE domain of early en-
dosomal antigen 1 (EEA1), a protein with nanomolar affinity for
PI(3)P (and is widely used to detect the location of PI(3)P in cells
(Lee et al., 2005). As shown in Figure 3A, all of the cell associated
red fluorescence is detected in punctate regions in a perinuclear
location (top), ascribed to a single fusion protein of the expected
size (Figure 3B) and recruited to the secretory pathway (as
proven by cleavage of the signal sequence, Figure 3C). A point
PI(3)P binding when introduced into the mCherry fusion, redis-
tributed red fluorescence to the parasite periphery (Figure 3A,
bottom), and was released into the supernatant when the red
Figure 1. Schematic of Intracellular Infection of Plasmodium and
Targeting Parasite Proteins to the Host Erythrocyte
A human erythrocyte (pink) infected by P. falciparum (blue). Invasion by the
extracellular merozoite stage leads to formation of a host derived PVM within
which the parasite resides and proliferates. Proteins (brown squares) secreted
by the parasite must cross the PVM to reach and mediate virulence and
structural changes in the erythrocyte. A consensus motif of RxLxE/D/Q at the
N terminus of parasite proteins is proteolytically cleaved after the RxL in the
ER, to generate proteins bearing xE/D/Q at their N terminus that are then
exported from the ER to the erythrocyte.
202 Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc.
cell and PVM were permeabilized with saponin (Figure 3D),
consistent with default secretion into the parasitophorous
vacuole (PV). A secretory fusion of a second PI(3)P binding
protein (p40-phox-PX) also showed perinuclear, punctate distri-
that abrogates PI(3)P binding is secreted to the PV (Figures S3A
% Protein bound
HRPII HT-GFP (RLLYE)36 ± 5 nM
HRPII ALLYE-GFP (ALLYE)> 5 µM
HRPII RLAYE-GFP (RLAYE)
HRPII RLLYA-GFP (RLLYA)
810 ± 140 nM
240 ± 40 nM
HRPII ALAYA-GFP (ALAYA)
HRPII HT-RFP (RLLYE)
> 5 µM
32 ± 4 nM
PfEMP1 (PFL0020w) KDVLE-GFP
PfEMP1 (PFL0020w) ADVLE-GFP
PfEMP1 (PFL1960w) KELLD-GFP
PfEMP1 (PFL1960w) AELLD-GFP
> 1 µM
Hsp40 (PFE0055c) RSLAE-GFP
Hsp40 (PFE0055c) ASLAE-GFP
> 5 µM
RIFIN (PFD0070c) RTLSE-GFP
RIFIN (PFD0070c) ATLSE-GFP
> 1 µM
STEVOR (PFA0090c) RLLAQ-GFP
STEVOR (PFA0090c) ALLAQ-GFP
> 5 µM
> 10 µM
μ μM HT-GFP :
0 500 1000 1500 2000 2500
Saturation Response Value (RU)
0 50 100150250 300
Response Value (RU)
GFP / RFP
40 amino acids
Figure 2. Lipid Binding Properties of HT Signals
proteins (recombinants purified from E. coli) contain the HT motif (RxLxE) and flanking sequences (that together comprise the vacuolar translocation sequence),
as shown in the schematic. Sequence information is in Table S2.
(B) Competition assay between HT-GFP and the p40-phox-PX domain. p40-phox-PX (5 mM) was incubated with 1 mM PI(3)P containing vesicles, and HT-GFP
was added at increasing concentrations from 0 to 20 mM. After 20 min, the supernatant fraction was resolved using SDS-PAGE following centrifugation.
M indicates total protein loaded into each reaction.
(C) SPR analysis demonstrates quantitative lipid binding of HT/RLLYE-GFP, ALLYE-GFP, RLAYE-GFP, and RLLYA-GFP for PI(3)P containing vesicles. One
micromolar of each construct was injected over a POPC:POPE:PI(3)P (75:20:5) surface at 30 ml/min using POPC:POPE (80:20) as a control. The control response
was subtracted from each active surface to yield the displayed sensorgrams.
(D) Equilibrium SPR binding analysis of HT/RLLYE-GFP (open circles) and RLLYA-GFP (filled circles). To determine Kdvalues, Reqvalues were plotted versus [P],
and the Kdvalue was determined by a nonlinear least-squares analysis of the binding isotherm using the following equation: Req= Rmax/(1 + Kd/P0). For proteins
withweakaffinity,dilutions weremade upto10mMtoprobe forlipid binding.Ifnobinding wasdetected thisallowedus toestimate theKdvaluetobegreater than
the highest protein concentration used for respective lipid vesicle.
(E) Kdvalues determined for each recombinant protein using the methods pictured and described in (D). Sequence logo is derived from HT signal of P. falciparum
secretory proteins. Amino acids are represented by one-letter abbreviations and color-coded as follows: blue, basic; red, acidic; black, hydrophobic; and green,
polar. Height of amino acids indicates their frequency at that position. Also see Table S2.
Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc. 203
Pl. V (ER)
Figure 3. PI(3)P Is Detected in P. falciparum Endoplasmic Reticulum
(A) Live P. falciparum-infected erythrocytes expressing the FYVE domain of EEA1 as a mCherry fusion recruited to the secretory pathway via an ER-type signal
sequence. The wild-type FYVE domain of EEA1 (EEA1WT), which binds PI(3)P with nanomolar affinity exhibits perinuclear staining (top). The point mutant FYVE
domain ofEEA1 (EEA1R1374A)that failstobindPI(3)P showsperipheral staining expectedforproteinsundergoing defaultsecretion tothePV(bottom). Leftpanels,
bright-field images with Hoechst 33342 nuclear staining (blue); right panels, fluorescence images. Dotted lines in the top panel and arrows (both panels) indicate
the location of the PV. Scale bars, 5 mm. Also see Figure S3A.
(B) Western blot of nontransfected 3D7 and transgenic parasites expressing EEA1WTor EEA1R1374Afused to mCherry. A single band at 42 kDa was detected
(arrow) for each fusion. Molecular weight standards in kilodaltons are shown in the left.
was digested with AspN and analyzed by LC-MS/MS. Mass spectra revealed that the most N-terminal peptide is Ac-AARSMEKLQTKVL.E, suggesting efficient
cleavage of the signal sequence by signal peptidase. Ac- indicates acetylation on N-terminal alanine. Observed b and y ions are shown on the peptide sequence
and the MS/MS spectra with neutral loss of water indicated by ‘‘o,’’ +1 ions shown in black and +2 ions shown in blue on the peptide sequence. When multiple
forms (different charge states or water loss) of an ion were observed, only one form is indicated on the peptide sequence. The observed and calculated masses
(M+H) are 1516.8542 and 1516.8515, respectively, indicating a 1.8 ppm mass error.
(D) Saponin lysis of transgenic parasites expressing secretory EEA1WTand EEA1R1374A. Western blots (top row) indicate that EEA1R1374Ais detected
into the supernatant (S) and thus released into the PV, whereas EEA1WTis retained within the parasite pellet (P). The parasite cytosolic protein PfFKBP
(middle row) is not released, confirming parasite integrity. PfHRPII (bottom row) exported to the erythrocyte and EEA1R1374Areleased in the PV are also
detected in parasite pellets (P) because they are continuously synthesized there. Molecular weight standards in kilodaltons are shown in the left. Also see
204 Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc.
and S3B), again suggesting that PI(3)P is present in highly local-
ized secretory regions within the parasite. These PI(3)P enriched
regions appear to reside in a subset of structures labeled by BiP,
a resident ER protein (Figures 3E and S3C) and also show over-
lap with plasmepsin V (another parasite ER protein) but their
distribution appears to be distinct from that of the early Golgi
lular parasite (Figure 3E). This suggested that PI(3)P-enriched
regions are likely to be present early in the secretory pathway,
in the ER, but are not concentrated in the Golgi or periphery of
the parasite. Immunoelectron microscopy studies revealed that
PI(3)P is detected in regions of reticular membranes closely
apposed to the nucleus (Figures 3F and S3D), and localizes to
perinuclear, tubovesicular structures containing the ER marker,
BiP, confirming its localization in the ER (Figure 3G).
An Endogenous, ER Form of PfHRPII Protein Containing
the HT Signal Recognizes PI(3)P prior to Cleavage
by Plasmepsin V
Since PI(3)P is found in the malarial ER and displays nanomolar
affinity for HT signal in in vitro assays, we investigated whether
the HT signal on the endogenous ER form of an effector protein
is associated with PI(3)P. Since plasmepsin V cleaves to sepa-
rate the sequences upstream of the HT signal from the rest of
the effector protein, we generated antibodies to recognize the
uncleaved precursor protein form but not the processed mature
As shown in Figures 4A and 4B, antibodies to the pre-HT
region of PfHRPII protein (also called anti-(pre-HT)) recognized
recombinant protein produced in Escherichia coli from con-
structs1-4, butnot 5-6that lackedpre-HT region. Inimmunoloc-
alization assays, pre-HT antibodies recognize protein only within
the parasite (Figure 4C, top). In contrast, antibodies to
sequences (YETQAHVDDVHHAHHADV) downstream of the HT
signal, also called post-HT-antibodies, detect PfHRPII exported
to the erythrocyte as well as within the parasite (Figure 4C,
endogenous protein band above the 50 kDa marker (asterisk in
Figure 4D, left) restricted to the parasite pellet upon saponin
treatment. In contrast, post-HT antibodies also recognize
a second exported form released into the supernatant (arrow-
head in Figure 4D, right). These data confirm the presence of
a precursor PfHRPII (pPfHRPII) protein containing the HT motif
located within the parasite. Further, pPfHRPII is in the ER and
serves as its marker, since plasmepsin V (which cleaves the
HT motif of pPfHRPII) resides in the ER.
It should be noted that these experiments were undertaken
with parasites ?12 hr in the intraerythrocytic infection cycle,
when PfHRPII is maximally synthesized, to facilitate detection
of endogenous precursors. Endogenous pPfHRPII was immuno-
purified using pre-HT antibodies, and found to contain ?10-fold
higher levels of PI(3)P relative to complexes immunopurified with
post-HT antibodies, on a per mole protein basis (Figures 4E and
S4). The extent of concentration was comparable to the level of
PI(3)P binding observed with secretory EEA1WTrelative to
binding by the EEA1R1374Amutant (Figures 4F and S4). SPR
analyses confirmed that the endogenous HT-signal bearing
precursor displayed high affinity for PI(3)P (Figure 4G). The inter-
action of the pPfHRPII with PI(3)P was specific since the interac-
tion with a second phosphoinositide, PI(4)P was undetectable
(Figure 4H). Together, these biochemical analyses firmly estab-
lish that endogenous HT signal can be detected on protein prior
to cleavage by plasmepsin V and thus resident in the ER. This
ER precursor binds PI(3)P with nanomolar affinity. Major sites
of PI(3)P/EEA1WTaccumulation, showed partial coassociation
with pPfHRPIIinindirect immunofluorescence assays(Figure4I).
It should be noted that in cells endogenous pPfHRPII will
compete with (potentially) hundreds of additional effectors for
PI(3)P in the ER. Nonetheless colocalization of pPfHRPII and
EEA1WTwasalsoseen inperinuclear regions byimmunoelectron
microscopy (Figure 4J), strongly supporting that pPfHRPII and
PI(3)P come together in the ER (or ER-derived membranes).
HT Signal-Dependent and -Independent Export
of Parasite Proteins to the Host Erythrocyte
in the Absence of Cleavage by Plasmepsin V
In the parasite ER, PI(3)P binding by the malarial HT signal must
precede HT signal cleavage by plasmepsin V. However, since
the relative importance of the individual residues in PI(3)P
binding are the same as those for protease cleavage, it is difficult
so, we turned to the oomycete HT signal that we have previously
shown to mediate protein export from the parasite to the host
erythrocyte (Bhattacharjee et al., 2006). Since the susceptibility
of the RQLR.DEER motif to the malarial protease was
unknown, we engineered a signal peptidase cleavage site by
insertion of AAAA, immediately after the signal sequence and
prior to the HT motif of the oomycete Phytophthora infestans
Procedures). Expression of this construct in P. falciparum, re-
sulted in export of GFP from the malaria parasite to the
erythrocyte (Figure 5A, top, and Figure 5B), which was blocked
by mutation of the RQLR motif (Figure 5A, bottom).
To investigate the relative contribution of plasmepsin V
cleavage to export, we undertook mass spectrometric analysis
of purified Nuk10-WT-GFP and mutant. Analysis of the most
(E) Localization of EEA1WT-mCherry as a marker for PI(3)P in the parasite ER. Transgenic parasites were fixed, permeabilized and probed with anti-mCherry as
well as antibodies to endogenous P. falciparum markers (green) of the ER, like BIP (top row) and plasmepsin V (Pl. V, second row); a marker of the Golgi (ERD2,
third row) or the PVM (Exp-2, bottom row). EEA1WT(red) is seen in punctuate ‘spots’ within the ER. Dotted circles show location of red cell membrane. Dotted
squares show regions magnified in the right panels. Parasite nuclei were stained with Hoechst 33342 (blue). Scale bars, 5 mm. Also see Figure S3C.
(F and G) Immunoelectronmicroscopy showing localization of EEA1WT-mCherry in perinuclear membranes and its coassociation with the ER marker BiP. (F) Thin
sections, probed with anti-mCherry and secondary antibody gold conjugates (10 nm) show label concentrated in membrane regions emerging from reticular
membrane apposed to the nucleus (arrows), consistent with localization in the ER. (G) Thin sections probed for EEA1WT-mCherry (10 nm gold) and BiP (15 nm
gold). Arrowheads show closed localization of BiP and EEA1WTsite. RBC, red blood cells; P, parasite. Scale bar, 0.5 mm. Control data for immunogold labeling is
shown in Figure S3D.
Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc. 205
Figure 4. Endogenous HT Signal on Precursor PfHRPII in the ER Associates with PI(3)P Both in Vitro and In Vivo
(A) Sequence of RFP fusions containing the first 40 amino acids present on pPfHRPII, and truncations, used in (B). The pentameric HT core in each sequence is
underlined. Peptide regions used to design anti-peptide pre-HT and post-HT antibodies are boxed in red and blue, respectively.
(B) Coomassie-stained gel of paired uninduced and IPTG-induced E. coli lysates expressing recombinant N-terminal fusions as indicated in (A) (top) were probed
with pre-HT antibody (bottom). Fusions 5 and 6 that lack the pre-HT sequence are not recognized by pre-HT antibody. Molecular weight standards (kDa) are
(C) Pre-HT antibodies recognize a PfHRPII form within the parasite (top). Post-HT antibodies recognize PfHRPII forms in the parasite and erythrocyte (bottom).
Nuclei were stained with Hoechst 33342 (blue). Bright-field images, left; fluorescent images, right. Scale bars, 5 mm.
(D) The pre-HT antibody recognizes pPfHRPII as a single protein band slightly above 50 kDa (asterisk in the left panel), present in the pellet (P) fraction of
fraction indicating exported and processed, mature HRPII (mPfHRPII). In the P fraction, post-HT antibody recognizes both mPfHRPII (indicated by arrowhead)
and pPfHRPII (asterisk in the right panel). Molecular weight standards (kDa) are shown.
(E) Quantitation of PI(3)P bound to pPfHRPII and mPfHRPII. pPfHRPII was immunoprecipitated and the amount of PI(3)P bound was detected by mass ELISA kit.
Lysates precleared of pPfHRPII were subjected to immunoprecipitation using post-HT antibodies and PI(3)P amount was determined. To determine nonspecific
206 Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc.
N-terminal peptide, suggested cleavage at the AAAA site for
both (Figures 5C and 5D), as expected for signal peptidase
action. Quantitative analysis of all released peptides confirmed
that the efficiency of signal peptide cleavage was comparable
for both (Figure 5E and Table S3). Further, intact RQLR contain-
ing peptide was recovered from immunopurified Nuk10 protein
(Figure 5F). This was observed even with the Nuk10 fraction ex-
ported to the erythrocyte (Figures S5A–S5D). These data estab-
lish that both the Nuk10-WT and the mutant are released from
the ER membrane by signal peptidase and that the oomycete
HT motif was not cleaved, likely because it is not a substrate
for malarial plasmepsin V. This was unexpected, since cleavage
by plasmepsin V was proposed as the export mechanism, yet
the oomycete HT signal appears to catalyze export in absence
of cleavage. SPR measurements confirm that Nuk10 HT signal
bound PI(3)P containing vesicles with 28 nM affinity, whereas
the mutant greatly reduced PI(3)P binding (Kd> 2 mM, Figures
5G and S5E), suggesting that PI(3)P binding is the mechanism
of export to the host cell.
The principal reason that plasmepsin V is thought to target
proteins for export, was the evidence that cleavage of a signal
anchor by signal peptidase failed to export protein to the eryth-
rocyte. To furtherinvestigate theidea, weengineered anefficient
signal peptidase cleavage site (AAAA) in two distinct malaria
protein chimeras in which the HT motif was abrogated but the
flanking sequences were kept intact (Figure 6). For PfHRPII, we
found that mutation of HT signal did not block export of the
GFP reporter to the erythrocyte (Figure 6A, middle). However,
replacement of all 9 downstream charged residues blocked
export (Figure 6A, bottom). Mass spectrometric analysis
confirmed all three chimeras were released from the signal
anchor either through cleavage of the HT signal or the AAAA
signal peptidase site (Figure S6). Hence, the block in export
seen in Figure 6A, bottom, is not due to failure to release protein
from the membrane. These data suggest that there are HT-inde-
pendent peptidic signals of parasite protein export to the eryth-
rocyte that may be dependent on charge.
For a second protein, we examined P. falciparum erythrocyte
membrane protein 3 (PfEMP3), utilized in earlier studies impli-
cating plasmepsin V as the export mechanism (Boddey et al.,
2010). As previously reported, replacement of RSLAQ motif
with A-AQ abrogated protein export (Figure 6B, top and middle)
by signal peptidase (Figure S6). However, we show that insertion
of the AAAA signal peptidase site followed by replacement of
RSLAQ by ASAAA, results in export of GFP to the erythrocyte
(Figure 6B, bottom). For this reporter, the primary site for
cleavage is within the AAAA sequence, suggesting that it too is
recognized by signal peptidase and released from the mem-
brane. Although we do not understand the basis of GFP export
in Figures 6A (middle) and 6B (bottom), the data affirm mutations
in the HT signal system may shift transport by an HT-indepen-
dent export pathway. Thus, the earlier conclusion (Boddey
et al., 2010) that failure to export in Figure 6B (middle) is due to
loss of the HT signal alone may have been premature.
The HTsignal functions in contextof an ?40 amino acid vacuolar
translocation signalneeded tomediateparasiteproteinexportto
the erythrocyte (Hiller et al., 2004; Lopez-Estran ˜o et al., 2003).
Thus, the HT signal may be one (likely the most important) region
necessary for selective PI(3)P recognition. In malarial HT signals,
PI(3)P binding is critically dependent on the high value R in the
plasmodial HT logo, providing one of the first clues that lipid
binding may play a major role in targeting malaria parasite
proteins to the host erythrocyte. The nanomolar affinity dis-
played by the HT signal of virulence determinants containing
the R/KxLxE motif, including the major virulence adhesin
lar phosphoinositide (PI) binding is primarily attributed to at least
four points of contact, it is likely that there are other cationic resi-
dues residing in the HT signal and flanking sequences that
contribute to PI(3)P recognition. Additionally, hydrophobic resi-
dues such as L or others within the HT signal may provide
further membrane anchorage through membrane penetration
(Lemmon, 2008), whereas the consensus E could form H-bonds
with the 5-OH and discriminate against other PIs as previously
tural and biophysical studies willbenecessary to fullyappreciate
this novel mechanism of PI(3)P recognition.
The presence of PI(3)P in the malarial ER and its affinity for
endogenous HT signals strongly suggests that HT-signal lipid
interactions occur early in the ER. Importantly, with the develop-
ment of specific antibody reagents, we are able to detect ER
state a detectable amount of endogenous precursor carrying the
HT signal has not been processed by plasmepsin V. Our finding
fails to cleave the oomycete HT signal, leads us to propose that
binding, lysates (without pre-clearing pPfHRPII) were incubated with beads and processed identically. Samples were quantitated using PI(3)P standard curve as
shown in Figure S4. Data represents mean ± SEM from triplicates.
out essentially as described in E. Samples were quantitated using PI(3)P standard curve as shown in Figure S4. Data represents mean ± SEM from triplicates.
(G) SPR sensorgrams showing PI(3)P binding for pPfHRPII, mPfHRPII (as described in E); and pre-HT antibody alone. Sensorgrams were also generated for
purified p40-phox-PX (226 nM).
(H) SPR sensorgrams of pPfHRPIIfor PI(4)P.Immunopurified pPfHRPII (as described in E)wasused to detect binding to PI(4)Pby SPR.Purified FAPP1-PH (1mM)
was used as a control for PI(4)P binding.
(I) Relative distribution of endogenous HT signal in pPfHRPII (green) and EEA1WT-mCherry (red) as detected by indirect immunofluorescence assays. Single
optical sectionsof20nmthicknessareshown.Dottedcirclesintheleftpanelsindicatered cellmembrane.Regionwithin dottedsquaresaremagnified intheright
panels. Yellow (and marked by white arrows) indicates sites of EEA1WT-mCherry/PI(3)P colocalization with endogenous pPfHRPII (merge). Scale bars, 5 mm.
(J) Immunoelectron micrograph showing sites of colocalization of EEA1WT-mCherry/PI(3)P (10 nm gold) with endogenous pPfHRPII (15 nm gold). Arrows show
close colocalization of pPfHRPII and EEA1WTin the perinuclear region. Scale bar, 5 mm.
Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc. 207
Nuk10 (WT)-GFP Nuk10 (Mut)-GFP
CSegrahCe d i tpeP
curve Charge SC
60+E0 6 . 3
60+E0 0 . 5
2+A.RHASLTGA - cA
Nuk10 DRQLRGF-GFP (RQLR)28 nM
Nuk10 ISAATAI-GFP (SAAT)> 2 μM
Figure 5. P. infestans Nuk10 HT Signal Shows PI(3)P Binding-Dependent Export and Is Not Cleaved by Plasmepsin V
(A) Live P. falciparum-infected erythrocytes expressing a secretory chimera of the HT signal of P. infestans Nuk10-WT-GFP (top) and corresponding
mutant Nuk10-Mut-GFP (bottom). Left, bright-field images with Hoechst 33342 nuclear staining (blue); right, fluorescence images. Scale bars represent
208 Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc.
(Figure 7) dependent on high-affinity binding to PI(3)P in the
sin V is expected to be restricted to an emerging and/or possibly
P binding and plasmepsin V cleavage, may suggest that associ-
However cleavage per se (whether by plasmepsin V or signal
peptidase) may not provide specificity for host targeting, rather
both release protein from the ER membrane.
Our data also suggest that HT-independent export of para-
site protein reporters to the host can occur. HT-independent
pathways of export have been proposed for major parasite pro-
teins such as P. falciparum Skeleton binding protein 1 (PfSBP1)
as well as other proteins, which lack the HT signal but that are
resident in the secretory structures called Maurer’s clefts in the
erythrocyte cytoplasm and are thought to promote protein
delivery to the host erythrocyte membrane and other destina-
tions in the erythrocyte (Cooke et al., 2006; Spielmann and
Gilberger, 2010). Our data suggest that non-HT-dependent
(B) Western blot of Nuk10-WT-GFP and Nuk10-Mut-GFP (indicated by arrows) immunopurified from parasites. Lower bands at ?27 kDa indicate free GFP.
Molecular weight standards (inkDa)are shown on theleft. The mobility of Nuk10-WT-GFP and Nuk10-Mut-GFPare identical tothat seen whentheseproteins are
expressed in E. coli indicating that mobility differences are not due to processing by malaria parasites (data not shown).
(C and D) Mass spectrometry analysis of N-terminal peptides, derived from Nuk10-WT-GFP (C) and Nuk10-Mut-GFP (D) chimeras in (A) are shown and indicate
that both are cleaved by signal peptidase. Ac- indicates acetylated N terminus and M* indicates dynamic oxidation of Methionine. Observed b and y ions are
shown on the peptide sequence and the MS/MS spectra and neutral loss of water is indicated by ‘‘o.’’ The +1 ions are shown in black, +2 ions shown in blue and
neutral loss of water in green on the peptide sequence. When multiple forms (different charge states or water loss) of an ion were observed, only one form is
indicated on the sequence. Observed and calculated M+H masses for (B) and (C) are: peptide B obs. 1785.8937, calc. 1785.8912 (1.4 ppm mass error) and
peptide C obs. 1785.8944 and calc. 1785.8912 (1.8 ppm mass error). Also see Figure S5.
(E) Comparison of top ten peptides obtained after AspN digestion and the area under the curve for each shows that signal peptidase processing is equivalent in
purified Nuk10-WT-GFPand mutantNuk10-Mut-GFP. Thepresence ofAAAA atthecleavagesite generates heterogeneity atthe Nterminus,but atlevelsthatare
comparable between the two chimeras. Peptides are represented in blue and the acetylated N termini are represented in gray. M* denotes dynamic oxidation of
Methionine and Ac- denotes N-terminal acetylation. ND, not detected. Also see Figure S5 and Table S3 for a complete list of peptides.
(F) The HT signal RQLR in Nuk10-WT-GFP chimera is not cleaved by plasmepsin V. Nuk10-WT-GFP was purified from transgenic parasites, digested with AspN
and analyzed by LC-MS/MS.Peptide yield of DRQLRGFYATEN*TDPVNNQ.D indicates that plasmepsin Vcleavage is not essential for export of Nuk10-WT-GFP.
N* denotes deamidation of Asparagine. Observed b and y ions are shown on the peptide and the MS/MS spectra with +1 ions shown in black, +2 ions shown in
is indicated on the peptide sequence. Spectrum shown is charge state +3. The observed and calculated masses (M+H) are 2239.0398 and 2239.0374,
respectively, with a resulting mass error of 1.1 ppm. Also see Figure S5.
(G) Sequence logo derived from HT signals of P. infestans secretory proteins and binding of P. infestans Nuk10 HT signal and corresponding mutant to PI(3)P.
Amino acids are represented by one-letter abbreviations and color-coded as follows: blue, basic; red, acidic; black, hydrophobic; and green, polar. Height of
amino acids indicates their frequency at that position. Kdvalues of Nuk10-WT and Nuk10 mutant were determined as described in Figure 2D.
SS ASAAA 65-83GFP
Protein Export to the Red Cell
(A) Four alanines were placed between the pre-
dicted signal sequence cleavage and the pen-
tameric HT core (RLLYE) or its mutant (ALAYA). In
live P. falciparum-infected erythrocytes express-
ing SS-AAAA-HT-GFP, GFP is exported to the red
cell (top) and the HT signal (RLLYE) is efficiently
cleaved by plasmepsin V (pV; for mass spec
analysis of peptides, also see Figure S6). Expres-
sion of SS-AAAA-ALAYA-GFP also results in GFP
export and cleavage by signal peptidase (SP) at
the AAAA site (also see Figure S6). Expression of
SS-AAAA-ALAYA-down-GFP with all charge resi-
fails to result in export GFP (bottom), despite
cleavage by SP at the AAAA site (also see Fig-
ure S6). SP shows heterogeneous cleavage at the
nuclear staining (blue), left; fluorescent images,
right. The scale bars represent 5 mm.
(B) Live P. falciparum-infected erythrocytes ex-
red cell (top), and the HT signal (RSLAQ) is
efficiently cleaved by pV (also see Figure S6).
PfEMP3xQ-GFP, with modified SP site and lack-
ing HT signal, does not export GFP (center) although it undergoes cleavage by SP at the AQ. However, placement of four alanines (SP cleavage site) upstream of
mutated motifin PfEMP3-A4-ASAAA-GFP results in export of GFP and cleavage by SP occurs at AAAA site. SP shows heterogeneous cleavage at the AAAA site.
Schematic representation of each construct and cleavage site is shown at the top. Bright-field images also show Hoechst 33342 nuclear staining (blue), and the
scale bars represent 5 mm. Also see Figure S6 for detailed LC-MS/MS analyses.
Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc. 209
export to the erythrocyte is based on charge. Chaperones
that interact with charged residues may well distinguish path-
ways of protein export to the erythrocyte relative to default sec-
retion of well-folded secretory proteins to the PV but these
need not be linked to chaperones reported to be associated
with plasmepsin V (Goldberg and Cowman, 2010; Russo
et al., 2010).
In summary, the parasite likely sorts newly synthesized
proteins into two, possibly three, different export pathways
that emerge from the ER, thus separating cargo for the erythro-
cyte from the PV (Figure 7), akin to early models of secretion
et al., 1997). Wiser et al. (1997) proposed a ‘‘secondary ER’’ for
kinetics of secretory protein exit (Crary and Haldar, 1992), but
evidence of specialized ER domains in parasite protein export
was lacking. The contribution of the Golgi to the PI(3)P/HT-signal
ER exit pathway remains unknown. A translocon-mediating
export of secretome proteins into the erythrocyte has been
proposed (de Koning-Ward et al., 2009). How it recognizes puta-
tive protein cargo is unclear, since the HT signal in most plasmo-
dial proteins is largely abrogated in ER exit.
Based on the steady-state distribution of secretory PI(3)P and
plasmepsin V, we propose that both recycle back to the ER once
HT signal sorting and cleavage are completed (Figure 7), at least
in early intraerythrocytic stages called ring and early trophozoite
stages investigated in this study. Recent studies have reported
the export of a PI3kinase into the erythrocyte and in association
with membrane structures in the red cell (Vaid et al., 2010), but
how this export occurs and whether PI3kinase is recruited in
the secretory pathway is not known. An alternate mechanism
of PI(3)P synthesized on the cytoplasmic face. A recent study
suggested that blood cell infection increases PI(3)P levels
(Tawk et al., 2010), although the overall ratio of PI(3)P in the ER
to total cellular PI(3)P is unknown.
The presence of PI(3)P in the lumen of ER regions and its
binding to HT signals is consistent with a sorting function for
this lipid in malaria parasites. Given that malarial PI(3)P is utilized
by the oomycete HT signal to target proteins to the host erythro-
cyte and malarial signals and oomycete signals are functionally
equivalent in both Plasmodium and Phytophthora, it is possible
that PI(3)P also functions in the P. infestans ER. Thus, PI(3)P
binding in the ER may be a generalized mechanism for
Figure 7. A Model for PI(3)P-Dependent Export from the ER of P. falciparum-Infected Erythrocytes
Proteins containing the malarial HT-signal (purple square) or the oomycete HT signal (gray square) are cotranslationally inserted via their signal anchor sequence
(blue square) into the ER membrane (step 1). The HT signals recognize the lipid PI(3)P (blue hexagons) enriched in regions of the ER (step 2) and may occur
cotranslationally (data not shown). Secretory proteins with a plasmepsin V-refractory HT signal, are cleaved by signal peptidase (red pac-man) but remain
associated toPI(3)P. Proteins withthemalarialHT signalare cleaved by plasmepsinV(orange pac-man, step3),whichalsodestroysthePI(3)P binding signaland
thus is likely to occur in anewly pinched off vesicle or one whose contents do notfreely diffuse with those of the ER. Plasmepsin V and PI(3)P are recycled back to
the ER (step 4), whereas cargo targeted to the erythrocyte moves forward across the parasite plasma membrane (PPM), and PVM (step 5). In default secretion,
secretory proteins are cotranslationally translocated into the ER (step 10), the signal sequence (blue square) is cleaved by signal peptidase (red pac-man; steps 20
and 30) and protein is delivered through vesicular intermediates to PPM, and released into the PV (steps 40and 50). PI(3)P/HT-independent export to the
erythrocyte may reflect a third sorting step in the ER, or later step of transport to PPM (red arrows) before further export (step 60). Steps 1–5 have ?400 predicted
cargo proteins exported to the erythrocyte and thus likely constitute the dominant pathway of protein export to the erythrocyte. The role of the Golgi in these
pathways is not known. A translocon has been proposed in export to the erythrocyte, but how it recognizes HT signals lost in the ER is unknown.
210 Cell 148, 201–212, January 20, 2012 ª2012 Elsevier Inc.
pathogenic secretion in eukaryotic pathogens, aspects of which
may be targeted to disrupt pathogen-host interactions that
All the primers used for vector construction are described in the Extended
Experimental Procedures and Table S1.
Expression and Purification of Recombinant Proteins in E. coli
All recombinant proteins were expressed in E. coli BL21 (DE3) cells. His-
tagged proteins were purified over Pro-Bond resin (Invitrogen), MBP-tagged
proteins were purified over Amylose resin (New England Biolabs), and GST-
fusions were purified over Glutathione resin (Clontech). The sequences of
recombinant proteins are shown in Table S2. See Extended Experimental
Procedures for additional details.
Generation of Antipeptide Antibodies
We used peptides NNLCSKNAKGLNLNKRLL (pre-HT) and YETQAHVDDVHH
AHHADV (post-HT), based on sequence N-terminal or C-terminal, respec-
tively, to the deduced plasmepsin V cleavage site of PfHRPII. Rabbit
polyclonal antibodies raised to each were designated as anti-(pre-HT) and
Parasite Culture and Live Cell Imaging
P. falciparum 3D7 parasites and transgenic parasites were propagated in
culture in A+ human erythrocytes in RPMI containing Albumax II (GIBCO)
and were imaged live in a biological chamber using DeltaVision Deconvolution
microscopy (Applied Precision). See Extended Experimental Procedures for
Immunoprecipitation and Western Blotting of Tagged
P. falciparum parasitized erythrocyte lysates were prepared from cultures at
8%–10% parasitemia. Proteins were extracted and immunopurified using
anti-GFP or anti-RFP beads (MBL), separated by SDS-PAGE and either
processed for LC-MS/MS or western blotting. See Extended Experimental
Procedures for details.
Selective Permeabilization of Infected Erythrocytes
Saponin was used to selectively permeabilize the infected erythrocyte
membrane and the PVM (but not the parasite plasma membrane) of
P. falciparum-infected erythrocytes. For selective permeabilization of just the
infected erythrocyte membrane, 100 U of tetanolysin was used. See Extended
Experimental Procedures for additional details.
Mass Spectrometry and Peptide Quantitation
Protein bands of interest were excised, digested with AspN and analyzed by
LC-MS/MS using reverse phase capillary HPLC with a Thermo Electron LTQ
OrbiTrap XL mass spectrometer. The peptide identities of integrated peaks
were verified based on retention times of matching MS/MS spectra. See
Extended Experimental Procedures for additional details.
Immunolocalization by Indirect Immunofluorescence Assays and
Immunofluorescence arrays (IFAs) were performed on parasites fixed with
glutaraldehyde/paraformaldehyde, using indicated primary and secondary
antibodies and slides were stained with Hoechst 33342. See Extended Exper-
imental Procedures for additional details.
For immunoelectron microscopy, infected erythrocytes were fixed and
embedded in LR white. Thin sections were probed with rabbit anti-mCherry
(single labeling) or mouse anti-mCherry and rabbit anti-BiP/ anti-pre-HT,
followed by gold conjugated secondary antibodies (10 nm and/or 15 nm).
See Extended Experimental Procedures for additional details.
Liposome Pelleting Assay
The liposome sedimentation assays were performed by generating liposomes
with POPC, POPE, and/or phosphoinositides. Liposomes were then mixed
with proteins to yield solutions containing 1 mM total lipid and 5 mM proteins.
Bound proteins were eluted with Laemmli buffer, resolved by SDS-PAGE, and
developed with Coomassie dye. See Extended Experimental Procedures for
SPR Binding Protein-Lipid Interactions
All SPR measurements were performed at 25?C in PBS. Sensor chip surfaces
were injected with POPC/POPE/Phosphoinositide (75:20:5) and POPC/POPE
(80:20) vesicles at 5 ml/min onto the active surface and the control surface,
respectively, to give the same resonance unit (6,000 RU) values. Equilibrium
SPR measurements were done at the flow rate of 5 ml/min. Each data set
was repeated three times to calculate the mean ± standard deviation. See
Extended Experimental Procedures for additional details.
Immunoprecipitation Using Pre-HT and Post-HT Antibodies and
Detection of PI(3)P by ELISA
P. falciparum 3D7-infected cells were cultured with 50 mM HIV protease inhib-
itor Lopinavir (Selleckchem), to inhibit plasmepsin V activity, for 7 hr and cell
lysates were subjected to immunoprecipitation. The relative amounts of
pPfHRPII (immunoprecipitated by pre-HT antibodies) and mPfHRPII (immuno-
precipitated by post-HT antibodies) were determined by western blotting with
a mouse anti-PfHRPII and densitometry (data not shown). Equal amounts of
pPfHRPII and mPfHRPII were used to detect PI(3)P binding by SPR and
estimate bound cellular PI(3)P. See Extended Experimental Procedures for
Supplementation Information includes Extended Experimental Procedures,
six figures, and three tables and can be found with this article online at
This project was conceived by K.H., R.V.S., and S.B., and the experiments
were planned by K.H., R.V.S., S.B., D.W.S., and K.D.S. S.B., R.V.S., and
K.D.S. conducted the experiments. K.H., R.V.S., and S.B. wrote the paper
with input from all authors. We thank Yi Xue and Jordan L. Scott for excellent
technical assistance, Andrew Osborne (University College of London) for help-
ful discussions, and Caroline Furtado Junqueira for help in figure representa-
tion. We would also like to thank Lennell Reynolds Jr., the Cell Imaging Facility
at NorthwesternUniversity, and William Archerat theUniversity of Notre Dame
for help with electron microscopy. This work was partially supported by NIH
grants HL069630, AI039071, HL078826 (K.H.); AI081077 (R.V.S.); HL038794
(D.W.S.) and CA10815 (Wistar Proteomics Core Facility).
Received: June 30, 2011
Revised: September 11, 2011
Accepted: October 28, 2011
Published: January 19, 2012
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