A point mutation in an unusual Sec7 domain is linked to brefeldin A resistance in a Plasmodium falciparum line generated by drug selection.

The malaria parasite Plasmodium falciparum has an unusual organization of its secretory compartments. As an approach to a functional identification of auxiliary proteins involved in secretion, a parasite line was generated by drug selection that is resistant to brefeldin A, an inhibitor of the secretory pathway. In the resistant line, neither protein secretion nor parasite viability were affected by the drug. The analysis of a sec7 domain, a conserved structure of guanine nucleotide exchange factors (ARF-GEF) required for the activation of ADP-ribosylation factors, revealed a single methionine-isoleucine substitution in the resistant parasite line. ARF-GEFs are key molecules in the formation of transport vesicles and the main targets of brefeldin A. The methionine residue in this position of sec7 domains is highly conserved and confers brefeldin A sensitivity. Unlike other eukaryotes that have multiple ARF-GEFs, the plasmodial genome encodes a single sec7 domain. This domain shows a distinct structural difference to all sec7 domains analysed so far; two conserved subdomains that are essential for protein function are separated in the plasmodial protein by an insertion of 146 amino acids.

Luciferase, when fused to an N-terminal signal peptide, is secreted from transfected Plasmodium falciparum and transported to the cytosol of infected erythrocytes.

Plasmodium falciparum, a unicellular parasite that causes human malaria, infects erythrocytes where it develops within a vacuole. The vacuolar membrane separates the parasite from the erythrocyte cytosol. Some secreted parasite proteins remain inside the vacuole, and others are transported across the vacuolar membrane. To identify the protein sequences responsible for this distribution we investigated the suitability of the green fluorescent protein and luciferase as reporters in transiently transfected parasites. Because of the higher sensitivity of the enzymatic assay, luciferase was quantified 3 days after transfection, whereas reliable detection of green fluorescent protein required prolonged drug selection. Luciferase was confined to the parasite cytosol in subcellular fractions of infected erythrocytes. When parasites were transfected with a hybrid gene coding for the cleavable N-terminal signal peptide of a secreted parasite protein fused to luciferase, the reporter protein was secreted. It was recovered with the vacuolar content and the erythrocyte cytosol. The results suggest that no specific protein sequences are required for translocation across the vacuolar membrane. The high local concentration of luciferase within the vacuole argues against free diffusion, and thus transport into the erythrocyte cytosol must involve a rate-limiting step.

Eukaryotically encoded and chloroplast-located rubredoxin is associated with photosystem II.

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.

Analysis of recombinant merozoite surface protein-1 of Plasmodium falciparum expressed in mammalian cells.

Synthetic chimeric DNA constructs with a reduced A + T content coding for full-length merozoite surface protein-1 of Plasmodium falciparum (MSP1) and three fragments thereof were expressed in HeLa cells. To target the recombinant proteins to the surface of the host cell the DNA sequences coding for the N-terminal signal sequence and for the putative C-terminal recognition/attachment signal for the glycosyl-phosphatidyl-inositol (GPI)-anchor of MSP1 were replaced by the respective DNA sequences of the human decay-accelerating-factor (DAF). The full-length recombinant protein, hu-MSP1-DAF, was stably expressed and recognised by monoclonal antibodies that bind to the N-terminus or the C-terminus of the native protein, respectively. Its apparent molecular mass is higher as compared to the native protein and it is post-translationally modified by attachment of N-glycans whereas native MSP1 is not glycosylated. Immunofluorescence images of intact cells show a clear surface staining. After permeabilization hu-MSP1-DAF can be detected in the cytosol as well. As judged by protease treatment of intact cells 25% of recombinant MSP1 is located on the surface. This fraction of hu-MSP1-DAF can be cleaved off the cell membrane by phosphatidylinositol-specific phospholipase C indicating that the protein is indeed bound to the cell membrane via a GPI-anchor. Human erythrocytes do not adhere to the surface of mammalian cells expressing either of the constructs made in this study.

Novel secretory pathways in Plasmodium?

The secretion of proteins from intraerythrocytic stages of Plasmodium falciparum into the infected host cell is still poorly understood. A recent proposal that two distinct, mutually exclusive, secretory compartments may exist within the parasite cell has received much attention. Denise Mattei, Gary Ward, Gordon Langsley and Klaus Lingelbach here critically discuss the data on which this model is based, and then they address a more general question: to what extent are unusual aspects of protein secretion in Plasmodium unique among eukaryotic cells?

Reconstitution of protein transport across the vacuolar membrane in Plasmodium falciparum-infected permeabilized erythrocytes.

The parasite Plasmodium falciparum induces morphological and biochemical alterations of its host erythrocyte. Some of these changes are mediated by parasite proteins that are transported to specific destinations within the erythrocyte or to the erythrocyte plasma membrane. The pathways underlying this transport are still unknown. We anticipate that at least some aspects of these pathways may be biologically unique and therefore potential targets for chemotherapeutic intervention. We have utilized bacterial pore-forming proteins to establish an experimental system that allows selective permeabilization of the erythrocyte plasma membrane, without affecting the integrity of the vacuolar membrane and the parasite plasma membrane, in order to study protein transport from the parasite into the host erythrocyte. Physiological properties of the parasite within permeabilized erythrocytes, such as the ability to synthesize proteins, will be described. The permeabilization of infected erythrocytes has allowed the dissection of individual steps in protein transport from the parasite surface across the vacuolar membrane. Possible pathways involved in the trafficking of parasite proteins within the erythrocyte cytosol, i.e. in a cell that normally has no need to transport proteins, will be discussed.

The parasitophorous vacuole membrane surrounding Plasmodium and Toxoplasma: an unusual compartment in infected cells.

Plasmodium and Toxoplasma belong to a group of unicellular parasites which actively penetrate their respective mammalian host cells. During the process of invasion, they initiate the formation of a membrane, the so-called parasitophorous vacuolar membrane, which surrounds the intracellular parasite and which differs substantially from endosomal membranes or the membrane of phagolysosomes. The biogenesis and the maintenance of the vacuolar membrane are closely related to the peculiar cellular organization of these parasites and are unique phenomena in cell biology. Here we compare biological similarities and differences between the two parasites, with respect to: (i) the formation, (ii) the maintenance, and (iii) the biological role of the vacuolar membrane. We conclude that most differences between the organisms primarily reflect the different biosynthetic capacities of the host cells they invade.

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

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.

Protein trafficking in the Plasmodium-falciparum-infected erythrocyte--from models to mechanisms.

Plasmodium falciparum is a eukaryotic single cell which invades human erythrocytes. Within the host cell, the parasite is surrounded by the membrane of the parastiophorous vacuole. Parasite proteins are secreted either into the vacuolar space or are exported, by an as yet unknown mechanism, across the vacuolar membrane into erythrocyte cytoplasm. In recent years, several groups have devised experimental approaches to follow the transport pathways of proteins from the parasite into the host cell. The concepts underlying these approaches and the peculiarities of the transport pathways are discussed and compared with protein transport in higher eukaryotes.

Protein sorting in Plasmodium falciparum-infected red blood cells permeabilized with the pore-forming protein streptolysin O.

Plasmodium falciparum is an intracellular parasite of human red blood cells (RBCs). Like many other intracellular parasites, P. falciparum resides and develops within a parasitophorous vacuole which is bound by a membrane that separates the host cell cytoplasm from the parasite surface. Some parasite proteins are secreted into the vacuolar space and others are secreted, by an as yet poorly defined pathway, into the RBC cytosol. The transport of proteins from the parasite has been followed mainly using morphological methods. In search of an experimental system that would allow (i) dissection of the individual steps involved in transport from the parasite surface into the RBC cytosol, and (ii) an assessment of the molecular requirements for the process at the erythrocytic side of the vacuolar membrane, we permeabilized infected RBCs with the pore-forming protein streptolysin O using conditions which left the vacuole intact. The distribution of two parasite proteins which served as markers for the vacuolar space and the RBC cytosol respectively was analysed morphologically and biochemically. In permeabilized RBCs the two marker proteins were sorted to the same compartments as in intact RBCs. The protein which was destined for the RBC cytosol traversed the vacuolar space before it was translocated across the vacuolar membrane. Protein transport could be arrested in the vacuole by removing the RBC cytosol. Translocation across the vacuolar membrane required ATP and a protein source at the erythrocytic face of the membrane, but it was independent of the intracellular ionic milieu of the RBC.

A minimalist view of the secretory pathway in Plasmodium falciparum.

Would nature accept a eukaryotic cell that lacks a Golgi complex during a major part of its life cycle? Here, George Banting, Jürgen Benting and Klaus Lingelbach review recent morphological and biochemical data on the asexual intraerythrocytic stages of the protozoan parasite Plasmodium falciparum. They argue that these data may indicate that some stages of the life-cycle of this highly specialized organism lack a 'classical' Golgi complex.

Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin.

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.

Chemical and thermal inhibition of protein secretion have stage specific effects on the intraerythrocytic development of Plasmodium falciparum in vitro.

The intraerythrocytic stages of the human malaria parasite Plasmodium falciparum induce a variety of physiological changes of the host erythrocyte. Many proteins are secreted from the parasite and are subsequently found at specific locations within the host cell. To elucidate the importance of protein secretion for parasite survival, infected red blood cells (IRBC) were subjected to the fungal metabolite brefeldin A (BFA) and to incubation at 15 degrees C, treatments that inhibit protein secretion and parasite development. Evidence is provided that retardation of parasite development in the presence of BFA correlates with an inhibition of protein secretion. Incubation at 15 degrees C and BFA reversibly arrest parasite development at the ring stage. Arrested ring stages loose 50% of their competence to develop to trophozoites after 1.5 days of treatment with BFA and after approximately 4 days at 15 degrees C. BFA affects development of trophozoites at concentrations similar to those required to arrest rings. In contrast to rings, the viability of trophozoites cultured at 15 degrees C or in the presence of BFA is completely abolished within 24 h.

Characterization of membrane proteins exported from Plasmodium falciparum into the host erythrocyte.

Plasmodium falciparum is an intracellular parasite of the red blood cell. During development it exports proteins which are transported to specific locations within the host erythrocyte. We have begun to identify and characterize exported membrane proteins of P. falciparum in order to obtain specific marker molecules for the study of the mechanisms involved in the distribution of parasite-derived proteins within the host cell. In this report we describe the characterization of a 35 kDa protein which is recognized by a monoclonal antibody. The protein is tightly associated with membranes isolated from infected erythrocytes; it is resistant to extraction with alkali and soluble after treatment with detergents. It is located at the membrane of the parasitophorous vacuole and in membrane-bound compartments which appear in the cytoplasm of the infected erythrocyte. The protein co-localizes with the previously described exported protein-1 (exp-1). Considering its localization and physical similarities to exp-1, we name the 35 kDa protein the exported protein-2 (exp-2).

Brefeldin A inhibits transport of the glycophorin-binding protein from Plasmodium falciparum into the host erythrocyte.

Plasmodium falciparum, a protozoan parasite of the human erythrocyte, causes the most severe form of malaria. During its intraerythrocytic development, the parasite synthesizes proteins which are exported into the host cell. The compartments involved in the secretory pathway of P. falciparum are still poorly characterized. A Golgi apparatus has not been identified, owing to the lack of specific protein markers and Golgi-specific post-translational modifications in the parasite. The fungal metabolite brefeldin A (BFA) is known to inhibit protein secretion in higher eukaryotes by disrupting the integrity of the Golgi apparatus. We have used the parasite-encoded glycophorin-binding protein (GBP), a soluble protein found in the host cell cytoplasm, as a marker to investigate the effects of BFA on protein secretion in the intracellular parasite. In the presence of BFA, GBP was not transported into the erythrocyte, but remained inside the parasite cell. The effect caused by BFA was reversible, and the protein could be chased into the host cell cytoplasm within 30 min. Transport of GBP from the BFA-sensitive site into the host cell did not require protein synthesis. Similar observations were made when infected erythrocytes were incubated at 15 degrees C. Incubation at 20 degrees C resulted in a reduction rather than a complete block of protein export. The relevance of our findings to the identification of compartments involved in protein secretion from the parasite cell is discussed.

A novel short consensus repeat-containing molecule is related to human complement factor H.

We have identified a novel factor H-related cDNA, which was isolated from a human liver cDNA library. The DOWN16 clone is 1269 base pairs in size and hybridized to a mRNA of 1.4 kilobases. Similar to the previously described factor H-related proteins, the predicted translation product of 331 amino acids contains a hydrophobic signal sequence followed by a stretch of five short consensus repeats (SCRs). These five SCRs display homology to SCRs of factor H: SCRs1-3 (DOWN16) are homologous to SCRs6-8 of factor H, while SCRs4 and -5 are related to SCRs19 and -20. In vitro translation demonstrated that the DOWN16 cDNA encodes a primary translation product of an apparent molecular mass of 37,500 Da which is directed to the secretory pathway and is glycosylated. Thus, we propose that the protein will be present in human serum. The relatedness of structural elements between this novel gene and factor H may suggest common functions of these proteins not yet determined.