Characterization and cloning of the gene encoding the vacuolar membrane protein EXP-2 from Plasmodium falciparum1
Introduction
Most apicomplexan parasites reside and develop within a parasitophorous vacuole that forms during invasion of the host cell [1]. The outer limits of this vacuole are defined by the parasitophorous vacuolar membrane (PVM) acting as an interface between the parasite and its host cell. The biological function of the PVM, and its molecular composition, are thus poorly understood. Initial data indicate that the PVM plays an important role in the recruitment and transport of metabolites and solutes, which is facilitated by small proteinaceous pores 2, 3, 4. The PVM also prevents fusion with the endomembrane system of the host cell, as observed in cells infected with Toxoplasma gondii [5]. In erythrocytes infected with the malaria parasite Plasmodium falciparum, the PVM is maintained and enlarged by the biosynthetic activity of the parasite. It eventually protrudes into the cytoplasm of the host erythrocyte where it forms a network of tubovesicular membranes which extends to the erythrocyte membrane [6]. Inhibitors which prevent the formation of the network severely compromise the parasite’s ability to take up ions and metabolites, including nucleotides and amino acids from the extracellular, i.e. extraerythrocytic, medium [7]. These data indicate that the network acts as a transport system which allows direct access of extracellular small molecules to the intracellular parasite, a phenomenon previously described as ‘metabolic window’ [8]. While the uptake of proteins into the network and the vacuole is still a matter of debate 9, 10, 11, it is clear from many studies that proteins are transported from the parasite, across the PVM, into the erythrocyte cytoplasm (for reviews see 11, 12). This transport pathway is clearly selective, in that some parasite proteins remain within the vacuole whereas others are transported beyond the PVM [13]. The PVM of the growing parasite apparently lacks proteins derived from the erythrocyte plasma membrane 14, 15but it contains proteins which are synthesized by the parasite and subsequently inserted into the PVM 16, 17, 18. A prerequisite for a better understanding of the biological functions of the PVM and the tubovesicular network is the identification of proteins associated with this endomembrane system and the molecular cloning of the corresponding genes. Recently we have described a protein, designated exported protein-2 (EXP-2), which is associated with the PVM [17]. EXP-2 does not expose a major protein domain on the erythrocytic face of the PVM and thus differs from other known proteins of the PVM, such as EXP-1 which is an integral transmembrane protein [19]. Here we describe the biochemical purification of EXP-2, the isolation and characterization of its cDNA and genomic sequences, as a contribution to define the biological function of this protein.
Section snippets
Antibodies
The monoclonal antibody McAb7.7 was kindly provided by Dr J. McBride, University of Edinburgh. Its specificity has been described previously 17, 20. For the purification of EXP-2 by affinity chromatography, the antibody was coupled to cyanogen bromide activated Sepharose-4B (Pharmacia). The monoclonal antibody McAbN1 recognizes the N-terminal half of EXP-1 [21]. In order to raise an antiserum against recombinant EXP-2, amino acids 53–127 encoded by the open reading frame were expressed in pJC40
Determination of N-terminal amino acid sequences
It has recently been shown that EXP-2 is tightly associated with the PVM and that it resists extraction of a membrane fraction from infected erythrocytes using high salt buffers [17]. We exploited this property as the first step of the purification protocol. Infected erythrocytes were lysed by freezing and thawing. The membrane fraction was subsequently extracted with 500 mM KCl. The extracted membranes contained less than 1% of the total cellular protein, and EXP-2 was enriched more than a
Discussion
The vacuolar membrane in erythrocytes infected with P. falciparum acts as an interface between the host cell cytoplasm and the parasite surface. During parasite development, the PVM extends and forms a network of tubovesicular membranes. The PVM that surrounds the parasite and the extended tubovesicular membranes belong to the same endomembrane system [43]but it is unknown whether they represent regions of distinct functions. The biological properties of this membrane system include the
Acknowledgements
We thank L. Kremp and K. Paprotka for excellent technical assistance, M. Leippe and E. Tannich for many helpful discussions, and F. Seeber for critical reading of the manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft to K.L.
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2022, Trends in ParasitologyCitation Excerpt :Following sporozoite invasion of a hepatocyte, the parasite establishes a PVM, the membrane that separates the parasite from the host cells, as it is required to hijack the host to mediate nutrient exchange and other processes to facilitate successful development. The identified proteins localized to the PVM include the exported protein-1 (EXP1) [48,88–90], EXP2 [89,91], upregulated in infectious sporozoites-4 (UIS4) [88], and liver-specific protein-2 (LISP2) [92]. EXP1 is abundant only in the PVM of late schizonts after day 6 postinfection, while EXP2 is expressed in developing schizonts on days 3–5 postinfection.
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2015, Biochemical and Biophysical Research CommunicationsCitation Excerpt :EXP2, PTEX150 and HSP101/ClpB2 components associate into a detergent-resistant core complex that can be extracted from parasites membranes [6]. The membrane-associated subunit EXP2 (exported protein-2) [7,8] is a possible candidate for the trans-membrane protein-conducting pore and may structurally resemble hemolysins, bacterial pore-forming cytolytic α-helical toxins [9–11]. Proteins destined for export harbor a vacuolar secretion signal or PEXEL (Plasmodium export element) [12,13].
Remodeling of human red cells infected with plasmodium falciparum and the impact of PHIST proteins
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2013, Parasitology InternationalCitation Excerpt :The clinical manifestations of malaria are caused by the asexual blood stages of the parasite undergoing cycles of invasion into, replication in, and egress from red blood cells (RBCs) [3]. To survive within its host RBC, a metabolically inert cell devoid of all protein trafficking and transport machinery, the parasite must export hundreds of proteins across both its parasite plasma membrane (PPM) and the surrounding parasitophorous vacuole membrane (PVM), which serves as a selective semi-permeable barrier between the parasite and the parasite-infected RBC (iRBC) cytosol [4,5]. This has led to a significant interest in the membranous structures that the parasite establishes in the iRBC cytosol.
Plasmodial ortholog of Toxoplasma gondii rhoptry neck protein 3 is localized to the rhoptry body
2011, Parasitology InternationalCitation Excerpt :To investigate PfRON3 localization after the parasite invades erythrocytes, we performed IFA on ring stage parasites. PfRAP1 and PfEXP2 were used as parasitophorous vacuolar (PV) markers because they have been demonstrated to be present in ring stages and to associate with the PV [28–30]. We found that PfRON3 colocalized with the PV markers, PfRAP1 and PfEXP2 in a discrete compartment surrounding the ring stage parasites (Fig. 6A).
Towards an understanding on how RxLR-effector proteins are translocated from oomycetes into host cells
2010, Fungal Biology ReviewsCitation Excerpt :Proteomic analysis of parasite membranes combined with specific criteria were used to predict potential translocon proteins, including a secreted heat shock chaperone protein (HSP101/ClpA/B) containing AAA+ ATPase domains, a novel protein called PTEX150, and the previously characterised vacuolar membrane protein EXP2 (Fischer et al., 1998), as well as two other potential accessory proteins thioredoxin (TRX2) and PTEX88. Despite the lack of a predicted transmembrane domain, there is evidence that EXP2 is associated with the PVM (Fischer et al., 1998), and evidence suggests EXP2 to be the channel forming element of the translocon complex (de Koning-Ward et al., 2009). Other components of this machinery are likely to be involved in the recognition of PEXEL-protein cargo. de Koning-Ward et al. (2009)
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Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession number AJ000652.
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Present address: European Molecular Biology Laboratories, Heidelberg, Germany.