Characterization and cloning of the gene encoding the vacuolar membrane protein EXP-2 from Plasmodium falciparum1

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Abstract

As a contribution to the characterization of the parasitophorous vacuolar membrane from Plasmodium falciparum we have begun the identification of vacuolar membrane proteins. Exported protein-2 (EXP-2) is a vacuolar membrane protein exposed into the vacuolar space. To further characterize EXP-2, it was purified, and the 45 N-terminal amino acids were determined by micro-sequencing. Based on this information, partial cDNA and genomic fragments were amplified by PCR and used as probes for the isolation of complete cDNA and genomic DNA clones. The single copy gene is located on chromosome 14, and is transcribed during the ring stage of parasite development. The open reading frame encodes an N-terminal signal sequence which is cleaved from the mature protein. The amino acid composition of EXP-2 is characterized by charged amino acids, with a high abundance of aspartate residues in the C-terminal portion of the protein. In contrast to EXP-1, an integral protein of the vacuolar membrane, EXP-2 lacks a typical hydrophobic transmembrane domain. We suggest that EXP-2 may associate with the vacuolar membrane via an amphipathic helix located in the N-terminal half of the protein.

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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    1

    Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the accession number AJ000652.

    2

    Present address: European Molecular Biology Laboratories, Heidelberg, Germany.

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