Invited review

Plasmodium falciparum signal sequences:simply sequences or special signals?q

Adela Nacera, Laurence Berrya, Christian Slomiannyb, Denise Matteia,*

aUnite de Biologie des Interactions Hote-Parasite, CNRS URA 1960, Institut Pasteur, 75724 Paris, FrancebLaboratoire de Physiologie Cellulaire, INSERM EPI 9938, Universite des Sciences et Technologies de Lille 1, 59655 Villeneuve D’Ascq, France

Received 16 February 2001; received in revised form 4 April 2001; accepted 4 April 2001

Abstract

The malaria parasite, Plasmodium falciparum, synthesises and exports several proteins inducing morphological and biochemical modi-

fications of erythrocytes during the erythrocytic cycle. The protein trafficking machinery of the parasite is similar to that of other eukaryotic

cells in several ways. However, some unusual features are also observed. The secretion of various polypeptides was inhibited when P.

falciparum-infected erythrocytes were incubated with Brefeldin A. Immunoelectron microscopy studies revealed substantial morphological

changes in the endoplasmic reticulum following exposure of parasitised erythrocytes to the drug. Immunofluorescence studies of Brefeldin

A-treated parasites suggest that polypeptide sorting to different intracellular destinations begins at the endoplasmic reticulum. The parasite

also secretes polypeptides by a Brefeldin A-insensitive route that bypasses the classical endoplasmic reticulum–Golgi complex pathway.

q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Keywords: Plasmodium; Protein trafficking; Brefeldin A; Endoplasmic reticulum

1. Introduction

The intra-erythrocytic cycle of Plasmodium falciparum

starts when merozoites, originating from liver schizonts,

are liberated into the bloodstream and invade erythrocytes.

The parasite then develops into ring, trophozoite and schi-

zont stages within a parasitophorous vacuole (PV). Once

released, the newly formed merozoites invade other erythro-

cytes, restarting the cycle. Within the host erythrocyte, the

parasite synthesises and exports several proteins: these

proteins are transported across the parasite plasma

membrane and the vacuolar membrane to the erythrocyte

cytoplasm and plasma membrane. Plasmodium thereby

modifies the morphological and biochemical properties of

the host cell. Mature erythrocytes are unable to synthesise

and transport proteins and lipids, and it is therefore likely

that the parasite induces a protein transport pathway in the

otherwise quiescent host cell. Several membranous struc-

tures, lipid free-aggregates and electron-dense material

have been observed in the erythrocyte cytoplasm and

seem to be involved in parasite-induced polypeptide trans-

port. The signals, mechanisms and pathways involved in

protein trafficking within the parasite and to the erythrocyte

remain poorly understood.

This review summarises recent data on parasite proteins

secreted to the cytosol of the host cell, focusing on transport

processes within the parasite. We consider the possible role

of the endoplasmic reticulum (ER) in the process of protein

sorting. Protein trafficking to organelles (rhoptries, micro-

nemes and dense granules), to the plastid, and to the erythro-

cyte, has been recently discussed and will not be addressed

in this review (Lingelbach, 1997; Foley and Tilley, 1998;

Haldar, 1998; van Dooren et al., 2000).

2. The secretory machinery

In eukaryotic cells, proteins that are targeted to the ER,

the initial step of the secretory pathway, carry a signal

sequence at the N-terminus (Rapoport et al., 1996). Signal

sequences have a positively charged n-region of variable

length; a hydrophobic core (h-region) of 6–15 amino acid

residues, followed by a short polar c-region which contains

the recognition site of the signal peptidase (von Heijne,

1985). Newly synthesised proteins are transported from

the ER to the intermediate compartment (ERGIC) and to

the Golgi complex. Proteins are also modified within the

International Journal for Parasitology 31 (2001) 1371–1379

0020-7519/01/$20.00 q 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

PII: S0020-7519(01)00253-3

www.parasitology-online.com

q Adela Nacer and Laurence Berry contributed equally to this work.

* Corresponding author. Tel.: 1 33-1-4568-8617; fax: 133-1-4568-

8348.

E-mail address: dmm@pasteur.fr (D. Mattei).

ER. The Golgi apparatus is organised as stacked flattened

cisternae — cis, medial, trans — and the trans-Golgi

network (TGN). These various compartments are distin-

guished according to the enzymatic activities that lead to

compartment-specific post-translational protein modifica-

tions. Protein sorting to different subcellular locations, like

secretory storage vesicles, lysosomes or plasma membrane

occurs in the TGN (Glick, 2000).

The Plasmodium trafficking machinery presents various

similarities to that of other eukaryotic cells. Bannister et al.

(2000), in a very well documented review, describes the

principal ultrastructural features of the intra-erythrocytic

asexual stages of P. falciparum. For instance, the free ribo-

somes and the rough ER, located close to the nucleus, prolif-

erate with the intensification of protein synthesis as the

parasite develops from ring to trophozoite and schizont

stages.

Many of the parasite-secreted polypeptides have a typical

eukaryote signal sequence at the N-terminus, e.g. Exp-1,

MSP-1 and serine-rich protein (SERP; Table 1). Other

proteins secreted by P. falciparum have atypical, putative

signal peptides: they contain more than 80 amino acid resi-

dues preceding the hydrophobic region (for review, see

Lingelbach, 1993). In addition, the variant antigen

PfEMP1 is exported from the parasite to the erythrocyte

membrane, although the only hydrophobic domain that

could qualify as a signal sequence is located near the C-

terminus of the polypeptide chain (Baruch et al., 1995).

Activities characteristic of ER-located enzymes, such as

disulphide isomerase (Ridley et al., 1990) and signal pepti-

dase (Kara et al., 1990) have been detected in the parasite.

Furthermore, genes encoding components of the trafficking

machinery have been isolated and characterised in P. falci-

parum, including those for the homologues of the chaperone

PfBiP (or GRP78, Kumar et al., 1988), the reticulocalbin

PfERC (La Greca et al., 1997) and PfSec61 (Couffin et al.,

1998).

The Golgi apparatus, however, does not share all the

morphological and functional properties of those in

mammalian cells. The typical stacks are not observed in

the parasite’s cytoplasm (reviewed in Bannister et al.,

2000) and some enzymatic activities that define the different

compartments are absent or almost undetectable (Dieck-

mann-Schuppert et al., 1992). Nevertheless, genes encoding

PfERD2, homologous to the XDEL receptor (Elmendorf

and Haldar, 1993), Rab homologues involved in vesicular

trafficking, e.g. Rab 6 (de Castro et al., 1996), Rab 11

(Langsley and Chakrabarti, 1996) and Arf1 (Stafford et

al., 1996), have been found in P. falciparum. The presence

of these genes suggests the existence of a functional Golgi-

like complex, perhaps in the form of a single cisternum (van

Wye et al., 1996; Trelka et al., 2000).

Although the parasite’s secretory machinery shares

several characteristics with that of other eukaryotic cells,

some unusual features are again observed. Apparently, Plas-

modium seems to secrete some components of the traffick-

ing machinery beyond its plasma membrane to the

erythrocyte cytosol. For example, the P. falciparum homo-

logue of Sar1p, a small GTP-binding protein, has been

located in a compartment near the periphery of the tropho-

zoite and in association with membranous structures, prob-

ably the Maurer’s clefts, in the erythrocyte cytoplasm

(Albano et al., 1999). Similarly, the parasite sphingomyelin

synthase activity, usually a marker of the Golgi apparatus,

has been detected in the tubulovesicular network in the

cytoplasm of the host cell (Elmendorf and Haldar, 1994).

The P. falciparum polypeptide Pf41-2 is homologous to

the yeast Bet3p, which is required for vesicular transport

between the ER and Golgi apparatus (Jiang et al., 1998).

The Pf41-2 signal peptide carries information for N-termi-

nus-dependent post-translational translocation, charac-

terised by low hydrophobicity and the presence of amino

acids with small side chains or which are hydroxylated, like

alanine, serine and threonine (Ng et al., 1996). It is interest-

ing that, like PfSar1p (Albano et al., 1999), Pf41-2 has been

localised both within the parasite and in association with

Maurer’s clefts in the erythrocyte cytoplasm (Knapp et al.,

1989a). This observation further supports the idea that P.

falciparum might export components of the secretory

machinery to the host cell.

Brefeldin A inhibition experiments (D. Mattei, unpub-

lished results) suggest that the variant antigen PfEMP1

seems to be secreted through the classical ER–Golgi path-

way, despite the absence of an N-terminal signal peptide.

Insertion of the hydrophobic C-terminus of the human

synaptobrevin into the ER membrane has been described

(Kutay et al., 1995). The transmembrane region of

PfEMP1 may be similarly inserted post-translationally

into the ER membrane. Studies are in progress to address

this question.

3. Experimental approaches to study intracellulartrafficking

3.1. Cell-free systems

The translation and translocation of eukaryotic polypep-

tides can be studied using cell-free systems, for example

wheat germ or rabbit reticulocyte lysates supplemented

with canine pancreatic microsomal membranes. In such

systems, both Exp-1 and SERP are translocated into

mammalian microsomes as assessed by the cleavage of

their typical signal sequences (Ragge et al., 1990; Gunther

et al., 1991). However, this in vitro approach is not always

appropriate for analysing polypeptide secretory processes in

Plasmodium. GBP-130 and Pf41-2, both have atypical

signal sequences, and are not translocated into microsomal

membranes (Knapp et al., 1989a; Benting et al., 1994);

however, their transport is blocked by Brefeldin A in

vivo, suggesting that they are secreted through the ER–

Golgi pathway (Benting et al., 1994; Mattei et al., 1999a).

A. Nacer et al. / International Journal for Parasitology 31 (2001) 1371–13791372

A. Nacer et al. / International Journal for Parasitology 31 (2001) 1371–1379 1373

Tab

le1

Sig

nal

seq

uen

ced

iver

sity

inP

lasm

odiu

mfa

lcip

aru

ma,

b

Pro

tein

Loca

tion

Sig

nal

sequen

ceR

efer

ence

PfH

RP

IIS

ecre

ted

cM

VS

FS

KN

KV

LS

AA

VF

AS

VL

LL

DN

NN

SA

FN

NN

LC

Wel

lem

san

dH

ow

ard,

1986

PfE

MP

1R

BC

surf

ace

No

ne

Bar

uch

etal

.,1

99

5

PfH

RP

IR

BC

MM

KS

FK

NK

NT

LR

RK

KA

FP

VF

TK

ILL

VS

FL

VW

VL

KC

Tri

gli

aet

al.,

19

87

ME

SA

RB

CM

ME

NE

GN

KV

KK

VY

NN

SS

LK

KY

MK

FC

LC

TII

CV

FL

LD

IYT

NC

Co

pp

elet

al.,

19

86

RE

SA

RB

CM

MR

PF

HA

YS

WIF

SQ

QY

MG

TK

NV

KE

KN

PT

IYS

FD

DE

EK

RN

EN

KS

FL

KV

LC

SK

RG

VL

PII

GIL

YII

LN

GN

LG

YN

GF

aval

oro

etal

.,1

98

6

Pf4

1-2

RB

CC

yt

MD

KS

KS

SIE

KE

LN

RIK

QD

VS

LS

AF

SIL

FS

EM

VQ

YC

Kn

app

etal

.,1

98

9a

GB

P130

RB

CC

yt

MR

LS

KV

SD

IKS

TG

VS

NY

KN

FN

SK

NS

SK

YS

LM

EV

SK

KN

EK

KN

SL

GA

FH

SK

KIL

LIF

GII

YV

VL

LN

AY

ICG

DK

YE

KA

VD

YG

FK

och

anet

al.,

19

86

SE

RP

PV

MK

SY

ISL

FF

ILC

VIF

NK

Kn

app

etal

.,1

98

9b

S-a

nti

gen

PV

MN

RIL

SV

SF

YL

FF

LY

LY

IYK

TY

GK

VK

NT

DH

EL

SN

Nic

ho

lls

etal

.,1

98

8

Ex

p-1

PV

M,

RB

CC

yt

MK

ILS

VF

FL

AL

FF

IIF

NK

ES

LA

ES

imm

on

set

al.,

19

87

MS

P-1

Par

asit

em

emb

ran

eM

KII

FF

LC

SF

LF

FII

NT

QC

Pet

erso

net

al.,

19

88

BiP

ER

MK

QIR

PY

ILL

LIV

SL

LK

FIS

AK

um

aret

al.,

19

88

aE

R,

end

opla

smic

reti

culu

m;

RB

C,

ery

thro

cyte

;R

BC

M,

eryth

rocy

tem

emb

ran

e;R

BC

Cyt,

eryth

rocy

tecy

top

lasm

;P

V,

par

asit

op

horo

us

vac

uo

le;

PV

M,

par

asit

op

ho

rou

sv

acu

ole

mem

bra

ne;

ME

SA

,m

atu

re-

par

asit

e-in

fect

eder

yth

rocy

tesu

rfac

ean

tig

en;

RE

SA

,ri

ng

-in

fect

eder

yth

rocy

tesu

rfac

ean

tog

en;

BiP

,im

mu

no

glo

bu

lin

-bin

din

gp

rote

in.

bH

yd

roph

ob

icco

res

of

the

sign

alse

qu

ence

sar

ein

bo

ld.

cS

ecre

ted

toth

eex

tern

alm

ediu

m.

It is possible that atypical signal sequences are modified

within the parasite, such that they become compatible

with the parasite’s translocation machinery. Alternatively,

the translocons at the ER membrane of Plasmodium and

mammalian cells may have different specificities. The fail-

ure to reconstitute the translocation process in vitro may

thus be due to incompatibilities with components of the

heterologous system. For example, the mammalian signal

recognition particle may have only a low affinity for the

parasite signal sequences. Another possibility is that the

insertion of parasite polypeptides, e.g. Pf41-2, into the ER

membrane is a post-translational event, not compatible with

the heterologous system.

3.2. Brefeldin A and low temperature

Brefeldin A, a heterocyclic lactone of fungal origin, is a

powerful tool for studying intracellular traffic. Brefeldin A

treatment of mammalian cells results in the transfer of Golgi

apparatus components into the ER, leading to the inhibition

of protein transport. The morphological changes due to

Brefeldin A are not limited to the cis-Golgi/ER: the TGN

also mixes with the endosomal system. Other cellular

processes, including protein synthesis, are affected only

after prolonged treatment. The effects of Brefeldin A are

rapidly and fully reversible upon removal of the drug (for

review, see Klausner et al., 1992). The mechanism of action

of Brefeldin A in P. falciparum remains unknown. Previous

studies have shown that the secretion of several P. falci-

parum proteins is inhibited when synchronised cultures

are incubated in the presence of Brefeldin A (Elmendorf

et al., 1992). However, the transport of some polypeptides,

like PfHRPI, seems to be insensitive to the drug, suggesting

the existence of an alternate route, independent of the ER–

Golgi apparatus pathway (Mattei et al., 1999a).

In mammalian cells incubated at 158C, secreted polypep-

tides accumulate in the ERGIC, whereas at 208C, the trans-

port is blocked at the late trans-Golgi compartment (Saraste

and Kuismanen, 1984). Likewise, protein transport inhibi-

tion is observed when P. falciparum cultures are incubated

at either temperature (Benting et al., 1994; Mattei et al.,

1999a).

4. The effects of Brefeldin A on Plasmodium falciparum

Although Brefeldin A has been used to study the secretion

of various Plasmodium proteins, the morphological changes

induced by the drug in the parasite ER–Golgi complex have

not been documented. To address this issue, we used immu-

noelectron microscopy to investigate Brefeldin A-treated

parasites labelled with a rabbit anti-BiP serum (a plasmodial

ER marker) and with secondary anti-rabbit antibodies

conjugated to 18-nm gold particles (Griffiths et al., 1983).

In untreated young trophozoites, both the ER and the

nuclear envelope were labelled by the anti-BiP antibodies:

gold particles lined these organelles (Fig. 1A). Extremely

swollen, labelled ER cisternae were observed in the Brefel-

A. Nacer et al. / International Journal for Parasitology 31 (2001) 1371–13791374

Fig. 1. Morphological immunoelectron microscopy analysis of the effects of Brefeldin A on Plasmodium falciparum trophozoite stages. Ring stage cultures

were incubated in the absence (A) or presence (B) of Brefeldin A for 12 h and prepared for electron microscopy. Parasite sections were incubated with rabbit

anti-BiP serum and then labelled with anti-rabbit gold-conjugated IgG. (A) Untreated trophozoite stage showing gold particles lining the endoplasmic

reticulum and the nuclear envelope. Note the connection between these organelles. (B) Brefeldin A-treated trophozoite stage. Compared with the control,

the Brefeldin A-treated cells present a swollen endoplasmic reticulum. The inset shows the intense labelling of a cistenum with gold particles. N, nucleus; P,

pigmented digestive vesicle; (*), endoplasmic reticulum. Scale bar represents 0.5 mm.

din A-treated trophozoites (Fig. 1B). Thus, in analogy to

mammalian cells, Brefeldin A treatment probably leads to

an accumulation of secreted polypeptides in a compartment

that, by its reaction with the BiP antiserum, was identified as

the parasite ER. This experiment, however, does not discri-

minate between protein accumulation resulting from retro-

grade transport and inhibition of anterograde transport.

Inhibition of protein synthesis by cycloheximide followed

by Brefeldin A treatment might allow to distinguish

between these possibilities.

Protein trafficking is essential for P. falciparum survival

within the erythrocyte. However, the parasite’s protein

targeting and sorting processes remain poorly understood.

It is possible that sorting of polypeptides targeted to differ-

ent intracellular locations starts in the ER.

To investigate if polypeptides secreted to various loca-

tions within parasitised erythrocytes are sorted to different

subcompartments within the parasite ER–Golgi complex

before being exported, we performed a series of immuno-

fluorescence experiments. Ring stage (8–12 h after inva-

sion) synchronised cultures were incubated for 18 h in the

presence of Brefeldin A and processed for immunofluores-

cence as described previously (Hinterberg et al., 1994). The

Brefeldin A-treated parasites were double-labelled with

antibodies directed against proteins located within the para-

site and antibodies against proteins secreted to the PV,

erythrocyte cytoplasm or membrane (Table 2).

Brefeldin A-treated P. falciparum cultures were labelled

with both rabbit anti-Pf332 (red) and mouse anti-MESA

(green) antibodies. Immunofluorescence analysis showed

that these proteins accumulate within the same cytoplasmic

compartment, mostly adjacent to the nucleus (blue) (Fig. 2).

A more diffuse and extended labelling pattern near the para-

site nucleus was observed with mouse anti-Exp-1 and rabbit

anti-SERP antibodies. The compartment where the polypep-

tides accumulated was identified as ER by double-labelling

experiments of Brefeldin A-treated parasites with the anti-

BiP serum, and Pf332 or Exp-1 antibodies (Fig. 2). Pf332

and Exp-1 gave different patterns of labelling consistent

with these polypeptides being located in distinct subcom-

partments or domains of the ER. Although the hypothesis of

protein sorting at the parasite’s ER is attractive, the under-

lying mechanisms remain unknown. It is possible that sort-

ing in the ER is mediated by the different properties of the

polypetides — for instance, Pf332 is soluble whereas Exp-1

is an integral membrane protein — or by post-translational

modifications. This latter alternative is supported by recent

work by Muniz et al. (2001) showing that in Saccharomyces

cerevisiae, glycosylphosphatidylinositol (GPI)-anchored

proteins are sorted from other polypeptides in the ER.

Immunoelectron microscopy studies of double-labelled

Brefeldin A-treated parasites are in progress to elucidate

the process of polypeptide sorting at the ER.

Note that immunofluorescence images similar to those

observed with Brefeldin A-treated parasites were obtained

when cultures were incubated at 158C (Fig. 3).

Immunofluorescence studies of Brefeldin A-treated para-

sites led Wiser et al. (1997) to propose the existence of a

secondary ER-like compartment in Plasmodium. In this

model, polypeptides destined to the parasite membrane

and intracellular organelles are secreted through the classi-

cal ER. In contrast, polypeptides are exported beyond the

parasite membrane to the PV, and to the erythrocyte cyto-

plasm and membrane transit via a compartment distinct

from the ER, termed the secondary endoplasmic reticulum

of Apicomplexa (sERA). These studies were carried out in

the rodent parasites, Plasmodium chabaudi and Plasmodium

berghei. As the Brefeldin A was added during very late

schizogony and release of merozoites, it is possible that

these proteins were carried into newly infected erythrocytes

as it is the case for the P. berghei 21 kDa MSP-1 fragment.

In other words, they may be proteins still associated with the

membrane of ring stages rather than proteins secreted in the

ring stage (Blackman et al., 1990). There is still no formal

demonstration of the presence of a sERA in P. falciparum

(Mattei et al., 1999b).

5. Non-classical route to the parasite membrane: thePfHRPI alternative secretory pathway

Treatment of parasite cultures with Brefeldin A does not

inhibit the transport of all secreted proteins (Elmendorf et

al., 1992). The secretion of PfHRPI, one of the major struc-

tural knob components (Taylor et al., 1987), is not affected

by either Brefeldin A treatment or by low temperature

(Mattei et al., 1999a). The N-terminal sequence of PfHRPI

presents an atypical signal sequence consisting of eight

positively charged amino acid residues followed by 15

hydrophobic residues (Triglia et al., 1987). In vitro, the

translated polypeptide does not seem to be translocated

into heterologous (mammalian) microsomal membranes.

These data strongly suggest that an alternative pathway,

different from that involving the ER–Golgi complex,

secretes PfHRPI (Mattei et al., 1999a). The C-terminal

moiety of the PfHRPI contains the sequence -GCCG, a

prenylation motif (Biermann et al., 1996), immediately

preceded by the polybasic sequence -KKKKKR. In eukar-

A. Nacer et al. / International Journal for Parasitology 31 (2001) 1371–1379 1375

Table 2

Mouse monoclonal antibodies and rabbit seraa

Protein Location Antibody Reference

PfBIP ER r GRP (BiP) John Adams, MR4

Exp-1 PVM, RBC Cyt m Mab5.1 Simmons et al., 1987

SERP PV r SERP Knapp et al., 1989b

Pf332 Maurer’s clefts, RBCM r EB200 Hinterberg et al., 1994

MESA RBCM m Mab8B7.4 Howard et al., 1987

a ER, endoplasmic reticulum; m, mouse; r, rabbit; RBC, erythrocyte;

RBCM, erythrocyte membrane; RBC Cyt, erythrocyte cytoplasm; PV,

parasitophorous vacuole; PVM, parasitophorous vacuole membrane;

GRP, glucose-related protein.

A. Nacer et al. / International Journal for Parasitology 31 (2001) 1371–13791376

Fig. 2. Immunofluorescence microscopy of Brefeldin A-treated parasites. Synchronised ring stage cultures were incubated in the presence of 5 mg/ml Brefeldin

A for 18 h and processed for immunofluorescence. Air-dried monolayers were sequentially incubated with mouse sera, anti-mouse fluorescein isothiocyanate-

conjugated IgG (first column of panels) rabbit sera and anti-rabbit tetramethyl rhodamine isothiocyanate-conjugated IgG (second column). Nuclei were stained

with 4 0,6 0-diamidino-2-phenylindole (DAPI; third column). The fourth column shows the fluorescein isothiocyanate, tetramethyl rhodamine isothiocyanate

and 4 0,6 0-diamidino-2-phenylindole images superimposed. (A) Control parasites showing the location of mature-parasite-infected erythrocyte surface antigen

(MESA) in the red blood cell membrane and Pf332 in the erythrocyte cytoplasm. Anti-SERP and anti-Exp-1 sera label the parasitophorous vacuole. (B)

Brefeldin A-treated parasites showing accumulation of the polypeptides in the parasite cytoplasm. Labelling with anti-BiP antibodies identifies the endo-

plasmic reticulum. Scale bar represents 5 mm.

yotes, prenylation involves the attachment of farnesyl or

geranylgeranyl (15- or 20-carbon, respectively) isoprenoids

to cysteine-containing motifs at the C-terminus of proteins.

One of the main roles of this class of lipid modification is to

promote protein–membrane and protein–protein interac-

tions, and the polybasic motif may act as a membrane-

targeting signal (reviewed in Sinensky, 2000). The prenyla-

tion and the polybasic domain of PfHRPI may allow the

protein to be transported to the parasite plasma membrane

without entering the classical secretory pathway (L. Berry

and D. Mattei, unpublished data).

6. Concluding remarks

The diverse data available suggest that Brefeldin A

blocks the transport of most secreted polypeptides, and

these polypeptides then accumulate within the ER. The

exception is the PfHRPI protein for which a post-transla-

tional modification seems to direct the polypeptide away

from the ER–Golgi complex pathway to an alternative

route. Co-localisation studies of several secreted proteins

in Brefeldin A-treated parasites suggest that the ER is

composed of different domains. It is possible that the prop-

erties of the polypeptides, such as their solubility or affinity

for membranes, protein–protein interactions or post-transla-

tional modifications, might determine their ‘compartmenta-

lisation’. In view of our results, we hypothesise that P.

falciparum protein targeting to the different compartments

of the infected erythrocyte begins within the ER. It is clear

that the parasite employs different mechanisms and path-

ways to achieve its main goal: to survive within the vacuole

in the host erythrocyte.

Acknowledgements

The authors would like to thank Genevieve Milon, P.

David and A. Scherf for critical reading of the manuscript

and useful discussions. The authors would like to thank

Shirley Longacre, R. Howard, B. Knapp and K. Lingelbach

for providing antibodies. GRP(BiP) rabbit antiserum and

preimmune serum, from John H. Adams, were obtained

through the Malaria Research and Reference Reagent

Resource Center, Division of Microbiology and Infectious

Diseases, NIAID, NIH. LB was supported by a Ministere de

l’Education Nationale, de la Recherche et Technologie

fellowship. This work was supported by the Institut Pasteur

and CNRS URA1960.

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