protein trafficking in malaria-infected erythrocytes

International Journal for Parasitology 17 "0887# 0560Ð0579

9919!6408:87:,08[99 Þ 0887 Australian Society for Parasitology[ Published by Elsevier Science Ltd[ All rights reserved[PII] S9919!6408 "87#99021!4

Protein tra.cking in malaria!infected erythrocytesMichael Foley\ Leann Tilley�

Department of Biochemistry\ La Trobe University\ Bundoora 2972\ Victoria\ Australia

Received 7 May 0887^ received in revised form 2 June 0887^ accepted 3 June 0887

Abstract

The malaria parasite invades the human erythrocyte and converts this simple {{sack of haemoglobin|| back into afunctional eukaryotic cell[ Parasite!encoded proteins are tra.cked to the red blood cell membrane where they modifyits properties to meet the needs of the intracellular parasite[ Tra.cking of proteins within the parasite probably occursvia a {{classical|| vesicle!mediated secretory pathway^ however\ the transit of proteins from the parasite plasma membraneto the erythrocyte membrane appears to involve both a novel vesicle!mediated pathway and a direct protein!translocationsystem[ The polypeptide signals that direct parasite proteins into these novel export pathways may include an unusual{{internal|| hydrophobic sequence\ as well as a series of basic motifs[ Þ 0887 Australian Society for Parasitology[Published by Elsevier Science Ltd[ All rights reserved[

Keywords] Malaria^ Protein tra.cking^ Plasmodium^ Signal sequences

0[ Introduction

The classical protein secretory pathway in euka!ryotic cells involves the transport of proteinsbetween intracellular organelles by the budding andfusion of small vesicles[ Exported proteins transitthrough the endoplasmic reticulum "ER# and theGolgi apparatus\ prior to release\ by exocytosis\ atthe cell surface[ The polypeptide signal whichdirects proteins to the secretory pathway is a hydro!phobic segment near the N!terminus of the proteinsequence ð0Ł[ Additional targeting signals are usedto divert proteins to other compartments "e[g[\ tothe lysosome or to a regulated secretory com!partment^ see ð1Ł\ for review#[ Protein tra.cking in

� Corresponding author[ Fax] 50!2!83681356^ e!mail]L[TilleyÝlatrobe[edu[au[

malaria!infected erythrocytes has an added levelof complexity in that the parasite exports proteinsbeyond the con_nes of its own plasma membrane\to achieve extensive modi_cations of both the cyto!plasm and the plasma membrane of the host r[b[c["Fig[ 0# ð2\ 3Ł[

In the mature stages of the intra!erythrocyticcycle\ the membrane of the erythrocyte becomesdistorted with knobby protrusions[ These knobs areinvolved in cytoadherence of infected erythrocytesto the vascular endothelium ð4Ł[ The adhesive struc!tures are formed by deposition of parasite proteinsunderneath the erythrocyte membrane and inser!tion of proteins into the erythrocyte membranebilayer "Fig[ 1#[ Two major components of these{{knobs|| are a peripheral membrane protein\ theknob!associated histidine!rich protein "KAHRP#\which forms the major structural element of theknob ð5Ł and an integral membrane protein\ the P[falciparum erythrocyte membrane protein!0

M[ Foley\ L[ Tilley:International Journal for Parasitolo`y 17 "0887# 0560Ð05790561

Fig[ 0[ Diagrammatic representation of putative tra.cking pathways in malaria!infected erythrocytes[ Proteins such as ring!infectederythrocyte surface antigen\ mature parasite!infected surface antigen and knob!associated histidine!rich protein are exported by themalaria parasite to interact with the host r[b[c[ cytoskeleton[ Plasmodium falciparum erythrocyte membrane protein!0 is inserted intothe erythrocyte membrane[ Tra.cking of these proteins to the parasite plasma membrane appears to involve elements of a {{classical||vesicle!mediated tra.cking pathway^ however\ transit of these proteins from the parasite plasma membrane to the r[b[c[ membraneoccurs by a largely uncharacterised mechanism[

"PfEMP!0#\ which is inserted into the erythrocytemembrane and acts as the ligand for binding toendothelial cell receptors ð6Ð8Ł[ To target proteinsto the r[b[c[ membrane\ the parasite has to transportthem past its own plasma membrane "PM# acrossthe parasitophorous vacuolar membrane "PVM#and then out to the r[b[c[ membrane "Fig[ 0#[Although the molecular machinery involved in theexternal part of this transport system is largelyuncharacterised\ it presumably involves unusual\parasite!speci_c components[

1[ Traf_cking of proteins within the malaria

parasite

Elements of the {{classical|| vesicle!mediatedsecretory pathway for the export of proteins arepresent within the cytoplasm of the malaria parasite

and appear to be involved in the transport of pro!teins to the parasite PM[ Homologues of a numberof tra.cking components have been found "see Fig[0#\ including the ER molecular chaperone\ Pfgrpð09Ł\ the P[ falciparum ER calcium!binding protein"PfERC# which is a reticulocalbin homologue ð00Ł\a homologue of the KDEL!binding protein\PfERD1 ð01Ł and homologues of the tra.cking!associated GTPases\ PfRab5 ð02\ 03Ł and PfRab 00ð04Ł[ Moreover\ brefeldin A\ a drug that speci_callyblocks vesicle!mediated tra.cking\ blocks thesecretion of some proteins ð05Ð07Ł[

The secretory pathway within the malaria para!site cytoplasm appears\ however\ to be unusual inthat a morphologically identi_able Golgi has notbeen observed in trophozoite!stage intra!ery!throcytic parasites "although there appears to be aGolgi!like compartment in the developing mero!zoite ð08Ł#[ Indeed\ in trophozoite!stage parasites\

M[ Foley\ L[ Tilley:International Journal for Parasitolo`y 17 "0887# 0560Ð0579 0562

Fig[ 1[ Putative mechanisms for the sorting of proteins destined for di}erent compartments in malaria!infected erythrocytes[ Proteinssuch as S!antigen "squares# are probably secreted into the parasitophorous vacuole "Steps 0 and 1# where they remained trapped[Proteins such as knob!associated histidine!rich protein "circles#\ that are destined for the red cell cytosol\ may be secreted into theparasitophorous vacuole then speci_cally translocated across the parasitophorous vacuolar membrane "Step 2#[ Alternatively\ theymay be sorted into a specialised secretory organelle "Step 3# and secreted directly into the r[b[c[ cytoplasm at a region of hemi!fusionbetween the parasite plasma membrane and the parasitophorous vacuolar membrane "Step 4#[ Plasmodium falciparum erythrocytemembrane protein!0 appears to be tra.cked to the r[b[c[ membrane by a vesicle!mediated pathway which may involve budding ofvesicles from the parasitophorous vacuolar membrane "Step 6# or from a region of hemi!fusion "Step 5#[

the cis!Golgi marker\ PfERD1 ð01Ł\ has been shownto be spatially separated from the trans!Golgimarker\ PfRab5 ð02\ 03Ł[ Recently\ Wiser et al[ ð19Łidenti_ed a brefeldin A!induced compartmentlocated near the periphery of the parasite cytoplasmin Plasmodium berghei and Plasmodium chabaudi[These authors proposed that this compartmentmight represent a secondary ER for the export ofproteins to the erythrocyte cytosol ð19Ł[ In anotherapicomplexan organism\ Toxoplasma gondii\ thedefault pathway for export of proteins involvesexocytosis from organelles referred to as densegranules ð10Ł[ A dense!granule compartment is pre!sent in the merozoite stage of the malaria parasite\where it functions as a storage compartment for theregulated secretion of the ring!infected erythrocytesurface antigen "RESA#\ which is released andtra.cked to the r[b[c[ membrane just after mero!

zoite invasion ð11\ 12Ł[ A related organelle may beinvolved in protein export in mature stage parasite!infected r[b[c[

2[ Export of proteins to different destinations

A major unanswered question with regard to pro!tein export in malaria!infected erythrocytes is howare proteins\ that are destined for external com!partments\ tra.cked across the two membranesthat separate the parasite and erythrocyte cyto!plasms[ Some potential pathways for the export ofproteins to the parasitophorous vacuole "PV#\ theerythrocyte cytoplasm and the erythrocytemembrane\ respectively\ are shown dia!grammatically in Fig[ 1[ Proteins\ such as S!antigen\the merozoite surface antigen!0 "MSA!0#\ exp!0


and the serine rich protein "SERP#\ which are des!tined for the parasite plasma membrane\ the PV\ orthe PVM are probably exported through a {{classi!cal|| secretory route[ They are presumably thenreleased into the PV by exocytosis "Steps 0 and 1in Fig[ 1#[ Proteins\ such as KAHRP\ RESA\ thefalciparum erythrocyte serineÐthreonine kinase"FEST# and the glycophorin!binding protein"GBP#\ that are destined for sites within the r[b[c[cytoplasm presumably require additional machin!ery[ They may enter the ER and be secreted intothe PV via a {{classical|| vesicle!mediated pathwayand then be speci_cally translocated across thePVM "Step 2 in Fig[ 1#[ Indeed\ there is evidencethat GBP is secreted in a {{two!step|| pathway\ withthe PV acting as an intermediate compartment ð13Ł[A second possibility is that the exported proteinsare directed to an {{alternative|| secretory organelle"{{secondary ER||# and then released into a regionof hemi!fusion between the PVM and the PM\ aspostulated Elmendorf and Haldar ð01Ł "Steps 3 and4 in Fig[ 1#[ In this context\ it is interesting tonote that export of KAHRP does not appear to beinhibited by brefeldin A "ð07Ł\ D[ Mattei\ personalcommunication#[

The exported proteins presumably di}use to their_nal destinations in the r[b[c[ cytoplasm rather thantaking an additional vesicle!mediated route "whichwould deposit them outside the r[b[c[ surface#[Indeed\ KAHRP and the mature parasite!infectedsurface antigen "MESA# have been shown to bepresent in structures that are separate from themembrane!bound compartments in the r[b[c[ cyto!plasm ð14\ 15Ł[ By contrast\ the integral membraneprotein\ PfEMP!0\ presumably does require a ves!icle!mediated tra.cking route from the PVM to ther[b[c[ membrane[ PfEMP!0 may be incorporatedinto an external vesicular compartment at the PVMsurface "Step 6# or in an area of hemi!fusion of thePM and PVM "Step 5# and eventually be deliveredto the r[b[c[ membrane by fusion of this vesicle withthe r[b[c[ membrane[ Some of the proteins in ther[b[c[ cytoplasm may be involved in the formationand delivery of these PfEMP!0!containing vesicles[It is interesting to note that\ in parasitised eryth!rocytes in which KAHRP has been {{knocked out||using transfection technology\ PfEMP!0 was shownto accumulate in membranous structures beneath

the r[b[c[ membrane ð16Ł[ These data suggest thatKAHRP may be required to facilitate the fusion ofthe membrane vesicles with the r[b[c[ surface[

3[ Traf_cking machinery in the erythrocyte

cytoplasm

Ultrastructural studies reveal a variety of mem!branous structures in the r[b[c[ cytoplasm "see ð17Ł\for review#[ Some of these may be involved in pro!tein tra.cking[ The most prominent structure isthe complex tubulovesicular network "TVN# whichappears to be a series of blind {{appendices|| extend!ing out from and wholly connected to the PVM ð18Ð20Ł[ Interestingly\ sphingomyelin synthase activity\which is found mainly in the Golgi of other euka!ryotic cells ð21Ł\ appears to be at least partiallylocated within the TVN ð01Ł[ This has led to thesuggestion that some Golgi functions or perhaps anadditional rudimentary Golgi compartment mightbe exported to the r[b[c[ cytoplasm[ Extensive elab!oration of the TVN\ does not\ however\ appear tobe necessary for protein export\ as treatment of cellswith a sphingomyelin synthase inhibitor\ which lim!its the TVN development\ does not prevent proteinexport ð22Ł[

In addition to the TVN\ at least three types ofmembranous structures have been observed in thecytoplasm of parasitised r[b[c[ These are {{Maurer|sclefts|| ð23Ł\ membranous whorls that are somewhatreminiscent of Golgi compartments ð14Ł and small\single membrane!bound vesicles ð14\ 24Ł[ There isevidence for the association of some exportedproteins\ such as FEST\ GBP and 30!1\ with mem!branous structures in the r[b[c[ cytoplasm ð25Ð28Ł[However\ it is not known whether these proteinsare associated with the lumen or the exterior ofthese vesicles[

Until recently\ there was no evidence for the pres!ence of proteins from the {{classical|| secretory path!way in the cytosol of the erythrocyte[ However\ wehave now identi_ed and characterised a plasmodialhomologue "PfSar0p# of the small GTP!bindingprotein\ Sar0p\ which appears to be present in ther[b[c[ cytosol "F[ Albano\ A[ Berman\ A[R[ Hibbs\M[ Foley\ L[ Tilley\ unpublished data#[ Yeast Sar0phas been shown to play an essential role in the


tra.cking of vesicles between the ER and the Golgið39Ł[ We have found that PfSar0p is located in ade_ned compartment adjacent to the parasite per!iphery "possibly a {{secondary ER||#\ as well asbeing associated with vesicular structures within theerythrocyte cytosol\ where it may be involved intra.cking of proteins to the erythrocyte membrane"Albano et al[\ unpublished data#[ Clearly\ furtherstudies are required to delineate all of the com!ponents of the unusual external tra.cking pathwayof the malaria parasite and to _nd a means ofspeci_cally interfering with this pathway[

4[ Polypeptide signals that determine traf_cking

destinations

The other major unanswered question withregard to protein tra.cking in malaria!infectederythrocytes is the nature of the polypeptidesecretory signals[ An analysis of the proteinsequences of the parasite proteins that are exportedto the r[b[c[ cytosol reveals some rather unusualmotifs[ A hydropathy plot of the _rst 099 aminoacids "aa# of some secreted parasite proteins isshown in Fig[ 2[ Proteins such as S!antigen\ thatare transported to the PV\ have a {{classical|| hydro!phobic N!terminal signal sequence\ i[e[ a stretch of09Ð04 hydrophobic aa commencing 2Ð06 aa fromthe N!terminus ð0Ł[ Proteins destined for the plasmamembrane\ such as MSA!0\ or for the rhoptries\such as the apical membrane antigen!0 "AMA!0#\also have classical N!terminal signals "Table 0#[Exp!0\ which is thought to be present in the PVMð30\ 31Ł\ also has a classical N!terminal signal "Fig[2\ Table 0#[ The secretory signal of exp!0 has beenshown to be recognised by the mammaliansecretory apparatus ð31Ł and is cleaved in vivo ð32Ł[Our analysis reveals\ however\ that proteins thatare directed past the PVM to the r[b[c[ cytosol donot have classical N!terminal signals[ Instead\ pro!teins such as KAHRP\ MESA and RESA have ahydrophobic stretch of aa starting 19Ð49 aa fromthe N!terminus Figs[ 2 and 0[ This {{internal||hydrophobic signal does not appear to be recog!nised by the eukaryotic translocation machinery\ asit has been shown that neither KAHRP nor protein30!1 "see Table 0# are translocated across the ER

membrane in cell!free systems using mammalianmicrosomes "ð2Ł\ D[ Mattei\ personal communi!cation#[

The lack of {{classical|| signal sequences suggeststhat the export of proteins to the r[b[c[ cytosolinvolves a novel and unusual secretory pathway[This novel pathway probably does involve transitthrough a membrane!bound compartment as\ atleast in the case of RESA\ delivery of the protein toa membrane!bound storage organelle "the densegranules# has been shown to occur prior to releaseð11Ł[ It seems likely\ therefore\ that the {{internal||hydrophobic signals do function to direct these pro!teins into the plasmodial ER[ The translocationmachinery of the parasite has presumably evolvedto recognise these unusual signal sequences[ It isinteresting to note that internal signal sequences arefound in some proteins of higher eukaryotes\ mostnotably ovalbumin ð33Ł[ The internal hydrophobicsignal sequence of ovalbumin directs the trans!location of the entire protein into the ER ð33Ł[

PfEMP!0 does not have a hydrophobic regionnear the N!terminus ð6Ð8Ł and\ thus\ appears to bea type!II integral membrane protein ð34Ł[ For type!II membrane proteins the transmembrane!span!ning region also functions as a {{start transfer|| sig!nal and the protein would be inserted into the ERwith its N!terminus facing the parasite cytoplasm[The pathway for export of PfEMP!0 to the r[b[c[membrane presumably re!orients the PfEMP!0molecule so that its N!terminus eventually faces theexterior of the r[b[c[ "see Fig[ 1#[

Proteins destined for the r[b[c[ cytosol pre!sumably contain a polypeptide {{signal|| whichallows sorting into a specialised compartment des!tined for export beyond the PVM[ The sorting pro!cess could take place either at the level of diversioninto a specialised secretory organelle "Step 3 in Fig[1# or at the level of a specialised protein trans!location machinery at the PVM "Step 2 in Fig[ 1#[ Itis possible that the unusual {{internal|| hydrophobicsignal sequence also acts as a signal for the furtherexport of this class of proteins[ Alternatively\ theseproteins may contain additional signal motifs[ Wehave analysed a number of members of this classof secreted proteins\ using the Psort program forprediction of protein sorting and localisation sitesð35Ł[ This analysis reveals the presence of a series of


Fig[ 2[ Hydropathy plots of the _rst 099 amino acids of a number of secreted proteins of P[ falciparum[ Plots were prepared using theDNASIS program "Hitachi Corp[#[ The plots were obtained using a window of 19 amino acids and the hydropathy scale of Kyte andDoolittle ð58Ł[ Values represent the average hydropathy per residue[ Regions which may act as hydrophobic signal sequences areshaded[

basic motifs within the polypeptide sequences "seeTable 0#[ For example\ FEST has 01 motifs thatmatch a four!residue basic motif "containing threeLys or Arg plus His or Pro#\ and two motifs thatmatch the 09!residue Robbins:Dingwell basic motifð36Ł[ PfEMP!0 has eight motifs that match the four!residue pattern and six motifs that match the 09!residue pattern[ All of the proteins destined for ther[b[c[ cytosol "except GBP and 30!1# have basicmotif {{scores|| of 9[4 or greater\ while all the pro!

teins that do not cross the PVM have very lowscores "Table 0#[ Basic motifs of these types arenormally found in proteins that are destined for thenucleus ð36Ł^ however\ they do not appear to havethis function in these exported proteins of themalaria parasite[ Presumably\ the exported parasiteproteins are co!translationally translocated into theER[ Therefore\ the basic motifs would never havethe opportunity to interact with the apparatusinvolved in nuclear targeting[ It seems possible\


Table 0Putative signalling motifs in secreted proteins of Plasmodium falciparuma

Protein Location N!terminal {{Internal|| Basic amino!acid Referencesignal signal motifs "signal scoreb#

Exp!0 PM:PVM Yes No No "9[90# ð49\ 40ŁMSA!0 PM Yes No No "9[94# ð41ŁS!Antigen PV Yes No No "9# ð42ŁABRA PV Yes No No "9[0# ð43ŁSERP PV Yes No No "9# ð26ŁRAP!0 Rhoptries Yes No No "9[0# ð44ŁEBA!064 Micronemes Yes No No "9[93# ð45ŁAMA!0 Rhoptries Yes No No "9# ð46ŁHRPII Food vacuole:secreted Yes No No "9# ð47ŁHRPIII Food vacuole Yes No No "9# ð48ŁGBP r[b[c[ cytoplasm No Yes No "9# ð59\ 50Ł30!1 r[b[c[ cytoplasm No Yes No "9# ð51Ł00[0 r[b[c[ cytoplasm No Yes Yes "9[5# ð52ŁFIRA r[b[c[ cytoplasm No Yes Yes "9[4# ð53ŁFEST r[b[c[ cytoplasm No Yes Yes "9[84# ð28ŁKAHRP r[b[c[ cytoskeleton No Yes Yes "9[7# ð5\ 54ŁMESA r[b[c[ cytoskeleton No Yes Yes "9[6# ð55ŁRESA r[b[c[ cytoskeleton No Yes Yes "9[5# ð56ŁPfEMP0 r[b[c[ membrane No No Yes "9[8# ð7\ 57Ł

a The Psort program "ð35Ł^ http]::psort[nibb[ac[jp:# for locating signal motifs was used to search for hydro!phobic N!terminal signal sequences and basic nuclear localisation signals[

b The Psort program searches for XXXX "2 K or R\ plus H or P#\ PXXXX "2 K or R#\ BB 09 spacer XXXX"2 K or R# and a high overall ratio of basic amino acids and uses an algorithm to determine a basic signalscore[

however\ that these motifs could be recognised bythe tra.cking mechanism"s# that directs these pro!teins into the r[b[c[ cytosol[

5[ The future for protein traf_cking studies in

malaria

Much of our current knowledge of the nature ofthe polypeptide signals that control tra.cking todi}erent compartments in higher eukaryotes comesfrom very elegant studies involving the constructionof chimeric proteins\ in which a putative {{signal||is appended to a reporter protein[ The gene forthe chimeric protein is transfected into the cell ofinterest and the fate of the reporter protein is moni!tored[ Until recently\ it has not been possible toperform similar experiments in malaria!infected

erythrocytes\ as a system for transfection has notbeen available[ Recently\ however\ foreign geneshave been successfully introduced into malariaparasites ð37\ 38Ł and studies can now be under!taken that will allow us to identify the importantcomponents and signals of the parasite proteintra.cking pathways[ It is likely that some of thetra.cking components that are involved in exportof proteins to the r[b[c[ cytosol will be found to beunique to the malaria parasite[ If so\ they may proveuseful targets for novel antimalarial strategies[

Acknowledgements

We are very grateful to Dr Alan Hibbs\ EpworthHospital\ Richmond\ Australia\ for useful dis!


cussions and to Dr Denise Mattei\ Pasteur Institute\France\ for sharing unpublished data[

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protein trafficking in malaria-infected erythrocytes

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