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Doktorarbeit / Dissertation, 2014
153 Seiten, Note: 1.3
1.2 The complex life-cycle of Plasmodium falciparum
1.3 The intraerythrocytic stage
1.3.1 P. falciparum -infection leads to extensive modification of the red blood cell !
1.3.2 Novel structures and compartments in P. falciparum- infected red blood cell "
1.3.3 Protein secretion mechanisms in the P. falciparum -infected red blood cell $
1.4 Unconventional protein secretion
1.5 Role of fatty acid acylation of proteins in plasma membrane binding
1.5.1 Protein N -myristoylation #
1.6 Acylated proteins as candidates of an alternative secretory pathway in P. falciparum ?
1.6.1 P. falciparum ADP-ribosylation factor
1.6.2 P. falciparum adenylate kinase (2)
2 Materials and Methods
2.1 Materials and Chemicals
2.1.1 Appliances !
2.1.2 Materials "
2.1.3 Chemicals "
2.1.4 Cell Culture Materials $
2.1.5 Molecular Biological Kits %
2.4 Solutions and buffers
2.5 Vectors and oligonucleotides
2.5.3 Plasmids designed for this work
2.6 Cells and Organisms
2.8 Cell culture techniques
2.8.1 In-vitro cultivation of Plasmodium falciparum"
2.8.2 Synchronization of Plasmodium falciparum with Sorbitol #
2.8.3 Enrichment of trophozoite-stage parasites via Gelafundin flotation #
2.8.4 High enrichment of late-stage parasites using a high gradient magnetic field #
2.8.5 Transfection and Co-transfection of Plasmodium falciparum $
2.8.6 Cryopreservation of Plasmodium falciparum -infected erythrocytes %
2.8.7 Thawing of cryopreserved Plasmodium falciparum -infected erythrocytes %
2.9 Molecularbiological methods
2.9.1 Cultivation of Escherichia coli
2.9.2 Preparation of electrocompetent bacterial cells (E. coli strain TOP10)
2.9.3 Mini- and Maxipreparation for isolation of plasmid DNA
2.9.4 Transformation of E. coli cells
2.9.5 Isolation of genomic DNA from Plasmodium falciparum
2.9.6 Isolation of mRNA from Plasmodium falciparum
2.9.7 Quantification of nucleic acid
2.9.8 Reverse transcriptase PCR
2.9.9 Polymerase chain reaction
2.9.10 In-vitro site-directed mutagenesis !
2.9.11 Agarose gel electrophoresis "
2.9.12 Purification of DNA "
2.9.13 Ethanol precipitation of DNA "
2.9.14 Restriction of DNA #
2.9.15 Ligation of DNA #
2.9.16 Screening for positive clones with colony PCR #
2.9.17 Sequencing of DNA $
2.9.18 Generation of plasmid constructs for transfection $
2.10 Biochemical methods
2.10.1 Cell fractionation of Plasmodium falciparum- infected red blood cells !
2.10.2 Streptolysin O permeabilization of Plasmodium falciparum -infected erythrocytes !
2.10.3 Saponin lysis of Plasmodium falciparum -infected erythrocytes !
2.10.4 Protease protection assay !
2.10.5 SDS-PAGE !
2.10.6 Semi-Dry-Immunoblotting !!
2.11 Fluorescence microscopy
2.11.1 Live cell imaging !"
2.11.2 Immunofluorescence assay !"
2.11.3 Image processing with Image J !#
2.12 Experimental design
3.1 Selected candidate proteins
3.1.1 Pf ARF1 shows a different subcellular localization upon removal of the N - myristoylation site in P. falciparum -infected red blood cells "
3.1.2 Pf ARF1 shows co-localization with marker proteins of the compartments of the secretory pathway "!
3.1.3 Pf ARF1 is not secreted into the PV in the blood-stage according to biochemical analyses "$
3.2 Is Pf AK2 secreted beyond the parasite plasma membrane?
3.2.1 A multiple sequence alignment #$
3.2.2 A putative palmitoylation site at the N-terminus of Pf AK2 $
3.2.3 The Pf AK2G2 AC4 A expressing parasites localize to the parasite cytosol $
3.2.4 Is a third motif involved in the secretion process of Pf AK2? $"
3.2.5 Is the N-terminus of Pf AK2 - containing a N -myristoylation site, a putative palmitoylation site and a polybasic cluster of amino acids - sufficient for protein secretion? $$
3.2.6 The ARF-AK2/GFP chimera is targeted to the parasite plasma membrane %
3.3 The mDHFR fusion system
4.1 The secretion hypothesis is based on the result of a preceding PV proteome analysis
4.1.1 Does the 'Met-Gly...' motif at the N-terminus of Pf Prefoldin (PF3D7_0904500), Pf CDPK4 (PF3D7__0717500) and Pf ARF1 (PF3D7_1020900) affect their subcellular localization?
4.1.2 Analysis of the subcellular localization of Pf ARF1 in P. falciparum -infected RBC
4.2 Is Pf AK2 a candidate protein of an alternative secretory pathway?
4.2.1 How much of the N-terminus of Pf AK2 is required for targeting other myristoylated proteins like Pf ARF1 to the PPM and beyond? %
4.2.2 An analysis about the folding state of Pf AK2 as it translocates from the parasite cytosol into the PV
4.2.3 A model for Pf AK2 protein anchoring to the PPM and secretion
4.3 Concluding remarks on the analysis of Pf AK2 as a candidate protein of an alternative secretory pathway in P. falciparum
7.1 Coding sequences (PlasmoDB, version 10.0)
7.2 Multiple sequence alignment (Clustal W: T-coffee)
7.3 Expression profile of Pf AK2 (PlasmoDB, version 10.0)
7.4 Potential proteins of the P. falciparum genome as candidate proteins of an alternative secretory pathway
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The malaria parasite P. falciparum invades human red blood cells (RBCs). During invasion a compartment surrounding the parasite, the so-called parasitophorous vacuole (PV), is formed. The parasite resides and develops within the PV, which protects the parasite from the host cell cytosol. During its intraerythrocytic growth the parasite exports a vast number of proteins to the host cell in order to maintain its survival within the RBC. Proteins, which are directed to the host cell cytosol and host cell membrane, respectively, therefore are challenged to cross the parasite plasma membrane, the PV and the parasitophorous vacuolar membrane (PVM). However, the secretion and export mechanisms of many parasite proteins are still not understood.
The current study focuses on the discovery of an alternative secretory pathway to the ER/Golgi route in the malaria parasite P. falciparum in infected RBCs. Two proteins appeared to be promising candidates of an alternative secretory pathway: the Pf ADP- ribosylation factor 1 (ARF1) and the Pf adenylate kinase 2 (AK2). Both proteins contained a N -myristoylation site at their N-terminus, which is indicative for N - myristoylation. N -myristoylation is a co-translational modification of a protein, whereby a fatty acid (myristate) is irreversibly attached to the glycine residue at the N-terminus of a protein via the PfN -myristoyltransferase (NMT). A preceding proteomic analysis of the parasitophorous vacuole and a reporter construct study proposed for both Pf ARF1 (determined by a proteomic study) and Pf AK2 (determined by a reporter construct study) PV localization although both proteins lacked a signal peptide. That’s why it was hypothesized whether or not N -myristoylation would drive protein secretion across the parasite plasma membrane (PPM). The subcellular localization of the Pf ARF1/GFP parasites and the Pf AK2/GFP parasites, respectively, were analyzed via epifluorescence microscopy and biochemical methods. In parallel, another batch of reporter constructs were generated and analyzed, where the N -myristoylation site of Pf ARF1 (this study) and Pf AK2 (Ma et al., 2012), respectively, was removed (Pf ARF1G2 A/GFP and Pf AK2G2 A/GFP). Live cell imaging showed that the fusion protein ARF1/GFP was localized as dot-like structures in the parasite. In contrast, the phenotype of the fusion protein of the Pf ARF1G2 A/GFP parasites showed an evenly distributed signal in the parasite cytosol. Further analysis of the subcellular localization of the Pf ARF1 strongly supports its localization to compartments of the early secretory pathway of the parasite, but no localization in the PV. In contrast, the fusion protein Pf AK2/GFP localized to a ring-like structure around the parasite indicating PV localization. The Pf AK2G2 A/GFP parasites showed a cytosolic localization of the fusion protein (Ma et al., 2012). Biochemical analyses revaled that the fusion protein Pf AK2/GFP was secreted into the PV when the N -myristoylation site was present. Furthermore, it could be shown that the N-terminus of the Pf AK2 protein is sufficient for parasite plasma membrane targeting, stable membrane anchoring and subsequent protein translocation across the PPM. A possible role of the early secretory pathway in Pf AK2 trafficking and the folding state of Pf AK2 prior to translocation across the PPM was also examined. However, the exact mechanism how Pf AK2 is translocated across the PPM still remains elusive.
Der Malariaerreger P. falciparum befällt rote Blutkörperchen im Menschen. Bei der Invasion der Erythrozyten formt der Parasit eine sogenannte parasitophore Vakuole (PV), die ihn dann umgibt. Der Parasit verbleibt und entwickelt sich in dieser PV, die ihn vom Zytosol der Wirtszelle schützt. Während des intraerythrozytären Wachstums exportiert der Parasit eine hohe Anzahl seiner eigenen Proteine in die Wirtszelle um sein Überleben in der Wirtszelle zu sichern. Proteine, die entweder in das Zytosol oder der Membran der Wirtszelle exportiert werden, müssen zunächst die Plasmamembran des Parasiten (PPM), die PV und die anliegende parasitophore Vakuolenmembran (PVM) passieren. Der Sekretions- und Exportmechanismus vieler Parasitenproteine ist jedoch noch immer unbekannt.
Ziel dieser Arbeit ist es alternative Sekretionswege zum klassichen ER/Golgi Sekretionsweg im Malariaparasiten P. falciparum aufzudecken. Zwei Proteine schienen geeignete Kandidaten eines alternativen Sekretionsweges zu sein: der Pf ADP- Ribosylierungsfaktor 1 (ARF1) und die Pf Adenylat kinase 2 (AK2). Beide Proteine besitzen eine N -myristoylierungsstelle am N-terminus was auf eine N -myristoylierung des jeweiligen Proteins hindeutet. N -myristoylierung ist eine ko-translationale Modifizierung eines Proteins, wobei eine Fettsäure (Myristat) irreversibel am Glycinrest am N-terminus eines Proteins durch die PfN -myristoyltransferase (NMT) angehängt wird. Eine vorangegangene Proteomuntersuchung der parasitophoren Vakuole und eine Untersuchung mit Reporterkonstrukten ergab für Pf ARF1 (Proteomuntersuchung) und Pf AK2 (Analyse der Reporterkonstrukte) eine PV Lokalisation, obwohl beiden ein Signalpeptid fehlt. Deshalb wurde die Hypothese aufgestellt, dass N -myristoylierung womöglich an der Proteinsekretion über die Plasmamembran des Parasiten beteiligt sein könnte. Demnach wurde die subzelluläre Lokalisation der Pf ARF1/GFP Parasiten und der Pf AK2/GFP Parasiten mithilfe von Epifluoreszenzmikroskopie und biochemischen Methoden untersucht. Parallel dazu wurden Reporterkonstrukte generiert und analyisert, bei denen die N -myristoylierungsstelle von Pf ARF1 (diese Arbeit) und Pf AK2 (Ma et al., 2012) entfernt wurden (Pf ARF1G2 A/GFP und Pf AK2G2 A/GFP). Beim live cell imaging war das Fusionsprotein ARF1/GFP als punktförmige Struktur im Parasiten erkennbar. Der Phänotyp des Fusionsproteins der Pf ARF1G2 A/GFP Parasiten dagegen zeigte ein zytosolisches Signal im Parasiten. Weitere Analysen im Hinblick auf die subzelluläre Lokalisation des Pf ARF1 deuten auf eine Lokalisation dieses Proteins mit Kompartimenten des frühen Sekretionsweges des Parasiten hin jedoch auf keine Lokalisation in der PV. Im Gegensatz dazu war das Fusionsprotein Pf AK2/GFP als ringförmige Struktur sichtbar was auf eine PV Lokalisation hindeutet. Die Pf AK2G2 A/GFP Parasiten zeigten hingegen eine zytosolische Lokalisation des Fusionsproteins (Ma et al., 2012). Biochemische Untersuchungen konnten zeigen, dass das Fusionsprotein Pf AK2/GFP in Anwesenheit der N- myristoylierungsstelle in die PV sekretiert wurde. Des Weiteren konnte gezeigt werden, dass der N-terminus von Pf AK2 das Protein zur Plasmamembran führt und eine stabile Membranverankerung hervorruft bevor die Translokation über die Plasmamembran des Parasiten erfolgt. Eine mögliche Rolle des frühen Sekretionsweges im Transport von Pf AK2 und der Faltungszustand von Pf AK2 vor der Translokation über die Parasitenplasmamembran wurden ebenfalls untersucht. Dennoch ist der genaue Mechanismus der Proteintranslokation über die Plasmamembran des Parasiten nicht bekannt.
Since ancient times a disease today referred to as malaria ('mal' 'aria' meaning 'bad air') has been noted to have a detrimental effect on people’s life quality, impeding population growth and affecting settling patterns throughout human history (Carter and Mendis, 2002; Sallares et al., 2004). Today malaria is recognized as one of the largest, life-threatening, infectious diseases in the world, caused by a eukaryotic parasite of the genus Plasmodium and transmitted by a bite of an infected female Anopheles mosquito. Although the causative agent of this disease was identified in the late 19th century malaria continues to be a major health problem in tropical and subtropical parts of the world, where billions of people are still exposed to this deadly disease (Fig. 1) (WHO 2011; cdc). In 2010 approximately 216 million clinical cases were reported by the World Health Organisation with 80 % occurring in African regions alone. Also 90 % of the 655 000 cases of deaths by malaria were registered in Africa (WHO 2011). In malaria-endemic regions pregnant women and children under the age of five (86 % of malaria deaths) succumb to this lethal disease more frequently than other groups of people as stated by the WHO in 2011. Countries, in which malaria is prevalent are also the ones suffering from a high poverty rate and a low economic growth making malaria prevention control and treatment more difficult (Gallup and Sachs, 2001; Sachs and Malaney, 2002).
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Figure 1.1 Malaria distribution
To date five different species of the genus Plasmodium - P. vivax, P. ovale, P. malariae, P. falciparum and P. knowlesi - are known to cause malaria in humans. While P. vivax and P. ovale, the causative agents of tertian malaria, and P. malariae, the causative agent of quartian malaria, are less likely to lead to severe forms of malaria outbreaks in humans, P. falciparum infections are mostly responsible for the high morbidity and mortality rates in endemic regions of Africa (cdc). Only recently P. knowlesi, a Plasmodium species known to infect macaque monkeys with malaria, was discovered to cause malaria in humans as well. (Singh et al., 2004; Cox-Singh et al., 2008).
Since the early 20th century many projects emerged to fight this deadly disease involving the use of the synthetic insecticide dichlorodiphenyl-trichloroethane (DDT) to prevent transmission by mosquitoes and the chemical compound chloroquine, known to inhibit the development of the blood-stage parasite. However, the increasing resistance in the mosquitoes and the Plasmodium -species, respectively, and the adverse effect of DDT on the environment made these attempts over the period of time unfruitful (cdc). Nevertheless, many malaria eradication programmes, especially in the early 21st century, are determined to reduce the high malaria casualties by the use of bed-nets treated with insecticide, artemisin-based combination therapies, etc. In fact the combination of these various methods has reduced the number of malaria cases of deaths by around 33 % since 2000 as registered in African regions, which are monitored by the WHO (WHO 2011). However, the number of malaria infections is still high and strains resistant to the available current anti-malarial drugs are already occurring. That is why continuous study on the biology of the parasite, to eventually develop an efficient vaccine and developments of new drugs against malaria, still needs to be continued.
Plasmodium falciparum has a very complex life-cycle involving mosquitoes as vectors and humans as hosts for its survival. The transmission of P. falciparum into a human occurs when an infected female Anopheles mosquito bites a human for a blood-meal thereby injecting saliva into the human. The saliva of an infected mosquito contains asexual forms of the parasite, the so-called sporozoites, which after entering the circulatory blood system travel to and invade liver cells for invasion (exo-erythrocytic stage). After entering liver cells the sporozoites multiply asexually and re-differentiate into thousands of merozoites in a process referred to as schizogony (Shortt, 1951). These thousands of merozoites are initially released in so-called merosomes (detached membrane-bound structures from the host cell) into the blood stream to escape the immune system (Sturm et al., 2006). Once the merozoites are completely released into the blood stream they target and invade red blood cells (erythrocytic stage). Following invasion a single merozoite multiplies into 20-30 daughter cells in an asexual process which takes about 48 hours: Initially the merozoite (now called the ring-stage) increases in size (0-12 hours post invasion) resulting into the more mature trophozoite form (12- 30 hpi) before the nucleus of the trophozoite form divides into many nuclei forming the so-called multinucleated schizont (30-48 hpi). After division of the multinucleated schizont into many individual merozoite forms the red blood cell ruptures releasing the newly formed parasites into the circulatory system to invade new erythrocytes (Wenk and Renz, 2003; Cowman and Crabb, 2006). However, a late trophozoite-stage occasionally also differentiates into a sexual form of the parasite, the gametocytes, which remain in the erythrocyte. Only when the erythrocytes containing these gametocytes are taken up via a blood-meal by a mosquito do they transform into gametes - male and female - in the gut of the mosquito. Fusion of the male and female gametes leads to a diploid zygote, the motile ookinete, which forms eventually an oocyst in the midgut of the mosquito. There the haploid sporozoite-forms are produced after many rounds of mitotic division (Wenk and Renz, 2003; Beier et al., 1998; Matuschewski, 2006). Eventually, these asexual sporozoites travel through the mosquito body to the salivary glands of the mosquito, where they can enter the host, once the infested mosquito bites a human re-starting the cycle (Fig. 1.2) (Matuschewski, 2006).
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Figure 1.2 Life-cycle of P. falciparum
A bite of a female Anopheles mosquito leads to the transmission of the malaria parasite into the human. Initial asexual multiplication takes place within the liver cells, before the infective merozoite form invades and multiplies within the red blood cells. The gametocytes, the sexual forms of the parasite, are produced in the human; however, the sexual development of P. falciparum occurs in the midgut of the mosquito (according to Ménard, 2005).
The first symptoms caused by P. falciparum infection emerge after a week or even longer, when the infected individual suffers from headache, fever or chills. This is usually a result of the intraerythrocytic growth of the parasite, which is followed by rupture of the erythrocyte and the release of new merozoites. If the P. falciparum infection is not treated within the next 24 hours, it can lead to severe illness due to rapid spreading of infected red blood cells in the circulatory system. This affects several organs, for example the nervous system (cerebral malaria) and can lead to metabolic acidosis and anemia as a consequence of hemolysis, resulting in coma and death of the infected person (Miller et al., 2002; reviewed in WHO 2013).
Plasmodium belongs to the phylum Apicomplexa - a large and diverse group of obligate intracelluar parasitic protists - which has one feature in all of their invasive stages in common: the apical complex. The apical complex, containing three secretory organelles - the micronemes, the rhoptries and the dense granules - enables parasites like Plasmodium to actively invade their host cells distinguishing this group from other pathogens regarding their mode of invasion (Aikawa, 1971; Cowman and Crabb, 2006). Interestingly, the merozoites - the invasive forms of P. falciparum produced during infection in humans - choose to invade erythrocytes, terminally differentiated cells, which lack a nucleus, subcellular organelles and do not perform any lipid- and protein synthesis (Mohandas and Gallagher, 2008). To reside and asexually multiply within this kind of a host cell therefore demands extensive modifications of the erythrocyte by the parasite. Already the invasion process, which comprises 1) the initial binding of the parasite to the host cell, 2) its repositioning and 3) the formation of a tight junction with the host cell (Dvorak et al., 1975; Bannister and Dluzewski, 1990) accompanied by the discharge of various proteins from the secretory organelles (Kats et al., 2008; Dowse et al., 2008; Cowman and Crabb, 2006) leads to a disruption of the red blood cell membrane and also affecting its cytoskeletal protein composition (McPherson et al., 1993; Roggwiller et al., 1996). However, the major alterations of the host cell takes place once the parasite has gained entry into the red blood cell and starts exporting a large number of its own proteins into the host cell cytosol and membrane (Maier et al., 2009; Marti et al., 2005). One of the prominent protein, which is exported to the red blood cell membrane, is the so-called P. f alciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 is a large protein of the size of 200-350 kDa and is responsible for antigenic variation. These proteins are the major cause of the pathogenicity of P. falciparum -infection due to their characteristics to adhere to receptors of the endothelium preventing destruction of infected red blood cells (RBCs) by the immune system (Su et al., 1995; Baruch et al., 1995). Their presentation on the surface of the RBC is supported by another kind of protein, the knob-associated histidine rich protein (KAHRP), which causes knob-like protrusions in the erythrocyte membrane (Culvenor et al., 1987; Pologe et al., 1987; Trager et al., 1966; Crabb et al., 1997). The interaction between PfEMP1 proteins and KAHRP not only increases the chances of receptor binding and sequestration of infected cells under the flow conditions of the circulatory system (Crabb et al., 1997), but also leads to increased rigidity of the erythrocyte membrane (Glenister et al., 2002). Another key aspect in the survival of the parasite is the nutrient acquisition throughout its intraerythrocytic development. Therefore
P. falciparum not only modifies host cell transporters for constitutive nutrient uptake, but was also found to create new permeability pathways (NPPs) in the membrane of the host cell (Kutner et al., 1985; Kirk, 2001).
1.3.2 Novel structures and compartments in P. falciparum- infected red blood cell Apart from the already mentioned secretory organelles at the apical complex harbouring different lipids and proteins, which mainly play a role in the invasion process of the merozoite, other structures and compartments formed by P. falciparum during its intraerythrocytic development are also only found in the phylum Apicomplexa and in particular in Plasmodium (Fig. 1.3).
An ultrastructural analysis of the infected RBC supported the long-standing notion of the existence of parasite produced membranous structures in the erythrocyte cytosol: the Maurer’s clefts. These thin structures are composed of an electron-dense coat with a translucent lumen most likely located in close proximity to the erythrocyte membrane, possibly supporting the trafficking of parasite proteins to the host cell membrane (Aikawa et al., 1986; Wickert and Krohne, 2007; Lanzer et al., 2006). Another set of membranous network also located in the red blood cytosol and found to play a role in nutrient acquisition is the so-called tubovesicular network (TVN) (Atkinson and Aikawa, 1990; Lauer et al., 1997). These tubular and vesicular membranous structures seem to be connected with the membrane of the parasitophorous vacuole (PV) (van Ooij et al., 2008), a compartment which is formed by the parasite during host cell invasion and which surrounds the parasite throughout its entire development inside the RBC (Lingelbach and Joiner, 1998). The PV is a compartment existing in some of the Apicomplexan parasites like Toxoplasma and Plasmodium and acts as a barrier between parasite and host cell cytosol. Although the PV itself is topologically different from the parasite and the host cell, the parasitophorous vacuolar membrane (PVM) seems to consist of proteins and lipids of the host cell (Lauer et al., 2000; Murphy et al., 2004), but is also proposed to be of parasite origin (Bannister and Dluzewski, 1990), this still being a matter of debate. The cellular function of the PV is not fully understood, however, it is suggested to play a role in the protection of the parasite from detrimental substances of the host cell cytosol and enables nutrient uptake for parasite growth (Lingelbach and Joiner, 1998). According to two independent screening studies of parasite exported proteins to the host cell by Marti and colleagues and Hiller and colleagues more than 250 proteins were discovered to be secreted through the PVM (Marti et al., 2004; Hiller et al., 2004), many of them being responsible for the virulence of P. falciparum infection. However, a proteomic approach to study the PV content revealed not only a large number of proteases and chaperones possibly playing a role in the lysis process during parasite release, but also revealed many proteins of unknown function. In general, the role of the PV can be attributed to a sorting compartment for distinguishing between resident PV proteins and proteins en route to the host cell, leaving the latter ones to pass through the PVM (Nyalwidhe and Lingelbach, 2006).
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Figure 1.3 Compartments of P. falciparum -infected red blood cell
Left side: Trophozoite stage of P. falciparum -infected red blood cell displaying the nucleus, the ER, the Golgi-complex, the mitochondrion (M), the food (digestive) vacuole (DV) and the apicoplast (A) within the parasite cytosol. The parasite is surrounded by the PV, from which the TVN is formed and found located inside the erythrocyte cytosol similar to the Maurer’s clefts (MCs). At the erythrocyte membrane knob-like protrusions are shown (K). Right side: Schizont-infected RBC with the apical organelles: rhoptries (R), micronemes (MN) and dense granules (D) (modified according to Deponte et al., 2012).
Apart from the PV two other compartments are also present in the parasite: the food vacuole and the apicoplast (apicomplexan plastid). The food vacuole is formed by a cytosomal system, membrane-enclosed structures of parasite origin, which take up hemoglobin in portion - hemoglobin makes up 95 % of the red blood cell cytosol- during parasite growth. Inside the food-vacuole hemoglobin is degraded, and heme is crystallized to the dark-pigment hemozoin (Francis et al., 1997). The apicoplast is a non-photosynthetic four-membrane-bound plastid located in the cytosol of the parasite and found to be crucial for the parasite’s survival. Its function is not fully understood but it seems to play a role in fatty acid, isoprenoid and heme synthesis (Waller and McFadden, 2005). Although Plasmodium contains some compartments characteristic only of Apicomplexan parasites, the following common eukaryotic compartments are also found in the parasite: Plasmodium falciparum has a nucleus harbouring a haploid genome (Gardner et al., 2002), a single mitochondrion containing its own, but highly reduced genome (Bender et al., 2003) and the common compartments of the secretory system found in all eukaryotic cells. These are the endoplasmic reticulum (ER) and an unstacked Golgi-complex. Both compartments, however, show some differences in their morphology to their counterparts in higher eukaryotes (Couffin et al., 1998; Van Wye et al., 1996).
Being a eukaryotic organism, protein secretion in P. falciparum via the early secretory pathway is most likely to resemble that of other eukaryotes. However, there are many exceptions to the rule since Plasmodium harbours compartments, which are missing in other eukaryotic cells and to which proteins are directed to. Before focusing on the protein secretion mechanism during the intraerythrocytic stage of P. falciparum the general model for the early secretory pathway - keeping in mind that there are many variations to the rule across eukaryotes - in eukaryotes is described:
The early secretory pathway in eukaryotes: Early studies on intracellular protein trafficking in eukaryotes included morphological, genetic and biochemical analyses mainly on yeast in order to identify the components of the secretory membrane system and to understand the underlying molecular mechanisms of its regulation (Palade, 1975; Rothman, 1994). In brief: secretory protein synthesis begins in the cytosol at ribosomes, which are associated with the endoplasmic reticulum (rough ER) - a large intracellular compartment spread throughout the cytoplasm, consisting of many membranous layers interconnected with each other and mainly specialized in protein- and lipid biosynthesis. The pre-proteins contain a N-terminal signal peptide (SP) of mostly 15-30 hydrophobic amino acids, which is produced during translation and is recognized and bound to a so- called signal recognition particle (SRP). The SRP stops protein translation, then binds to the SRP-receptor in the ER membrane that directs the entire SRP-ribosome complex to a translocator residing in the ER membrane - the Sec61 complex, which is known to be highly conserved in bacteria and eukaryotic cells - where protein translation proceeds once the SRP/SRP-receptor is released. The proteins are co-translationally transferred across the ER membrane, where soluble proteins are fully released into the lumen of the ER after translation, whereas integral membrane proteins become embedded in the ER membrane. The signal peptide is cleaved off by a signal peptidase. Many of the soluble and integral membrane proteins either become resident ER proteins or are further trafficked to the plasma membrane or other compartments. The forward trafficking of these proteins is dependent on another compartment, the Golgi-complex, which consists of tubular membranous structures, divided into functionally distinct cis -, medial and trans -Golgi stacks. The proteins leave the ER once they are packaged into vesicles termed COPII-coated-vesicles. These vesicles then fuse with the cis -Golgi and progressively move through the medial- and trans -Golgi (anterograde transport) in a budding and fusion process, before they are released from the Golgi-complex to the cell surface or to other compartments inside the cell. During the vesicular transport through the Golgi-complex proteins are post-translationally modified by resident Golgi-enzymes specialized in performing different kinds of glycosylation reactions. Retrograde transport from the cis -Golgi towards the ER via COPI-coated vesicles occurs when ER resident proteins accidentally enter the anterograde secretion pathway. They are recognized via their XDEL ER-retrieval sequence by specialized receptors located at the cis -Golgi and are trafficked back to the ER (Fig. 1.4) (Blobel and Dobberstein, 1975a; Blobel and Dobberstein, 1975b; Palade, 1975; Rothman, 1994; Alberts et al., 2008).
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Figure 1.4 Simplified model of the classical secretory pathway of the eukaryotic cell
Protein translation begins in the cytosol at ribosomes associated with the ER. Proteins destined to be secreted to the extracellular space contain a signal peptide (SP) at their N-terminus, which is recognized and bound to a signal recognition particle (SRP). Translation stops until the SRP-ribosome complex is bound to a SRP- receptor, which then trafficks the entire SRP-ribosome complex to a translocon in the ER-membrane (Sec 61 complex), where the protein is co-translationally translocated across the ER membrane. This protein is then trafficked via vesicles generated from the ER to the Golgi-complex and is released from the Golgi-complex via vesicles into the extracellular space. The trafficking of proteins from the ER towards the plasma membrane is referred to as anterograde trafficking. However, ER-proteins, which accidentally escape the ER, are transferred back from the Golgi to the ER, in a process called retrograde transport.
Early secretory pathway in P. falciparum: Many different in vitro and in silico studies on the intraerythrocytic stage of P. falciparum revealed the existence of some components playing a role in the early secretory pathway. These are for example the gene homologue of the mammalian α-subunit of the Sec61 translocon PfSec61 (Couffin et al., 1998) and the lumenal proteins of the ER PfERC2 and PfBIP (Kumar et al., 1991; La Greca et al., 1997) supporting the existence of an ER in the parasite cytosol. The existence of a Golgi apparatus was initially doubted since typical Golgi stacks could not be found by electron microscopy (Aikawa, 1988) and parasite proteins were not subjected to any kind of N-linked glycosylation (Dieckmann-Schuppert et al., 1992). However, other studies revealed the existence of typical Golgi proteins like the ERD2- receptor, which binds to proteins with a KDEL-retrievel sequence (Elmendorf and Haldar, 1993), the GTPase Rab 6 (van Wye et al., 1996) and the Golgi re-assembly stacking proteins 1 and 2 (Struck et al., 2005; Struck et al., 2008a; Struck et al., 2008b) indicating the existence of a possible rudimentary Golgi-complex. Furthermore, the gene of the ADP-ribosylation factor 1, a small GTPase important for vesicular trafficking in the early secretory pathway, was isolated, expressed and characterized from P. falciparum and more recently its crystal structure was also determined (Stafford et al., 1996; Cook et al., 2010) . These findings are in accordance with an in silico analysis by Gardner and colleagues in 2002. They identified potential homologue gene candidates of proteins involved in the early secretory pathway like for example the SRP, a signal peptidase and proteins involved in the budding and fusion of secretory vesicles (Gardner et al., 2002). Furthermore, studies with the fungal metabolite Brefeldin A (BFA), a known inhibitor of the secretory pathway, was shown to inhibit the secretion of a number of parasite proteins (Benting et al., 1994; Hinterberg et al., 1994; Baumgartner et al., 2001; Lippincott-Schwartz, 1989).
Many of the proteins secreted from P. falciparum not only contain the 'classical' signal sequence found in other eukaryotic organisms, but also revealed a modified version of the SP: a prolonged stretch of hydrophobic amino acids of up to 80 amino acids and recessed from the N-terminus of the protein (Lingelbach, 1993; Cooke et al., 2004). In addition, it could be shown that proteins containing the classical signal sequence are directed towards the parasite plasma membrane (PPM), the PV and the PVM, respectively, while those containing the recessed signal are found transported beyond the PVM en route to the host cell (Albano et al., 1999; Lingelbach, 1993). Here, too, many exceptions to the rules exist, like in the case of STEVOR (Przyborski et al., 2005) and the histidine-rich protein 2 (HRP2) (Howard et al., 1986), both containing a classical signal sequence, but are found exported to the host cell. Further trafficking of exported proteins, after entering the classical secretory pathway, is postulated to occur in a vesicle-mediated process (two-step model): vesicles containing the exported proteins fuse with the PPM and release the content into the lumen of the PV, before the protein is trafficked to the host cell across the PVM (Ansorge et al., 1996; Charpian and Przyborski, 2008).
Protein secretion beyond the PVM: Compared to protein secretion in higher eukaryotes, in which many secretory proteins are subsequently secreted to the extracellular milieu via exocytosis by just crossing one membrane - the plasma membrane of the respective cell - many malarial proteins are found and predicted to be exported beyond the PVM (Marti et al., 2004; Hiller et al., 2004). This calls for additional targeting signals in the amino acid sequence of these proteins. Indeed a conserved motif consisting of five amino acids (RxLxE/Q/D), referred to as P lasmodium export element (PEXEL) or host targeting signal (HT), was found in two independent in silico and reporter construct studies. They are present at the N-terminus of the exported proteins in close proximity to the recessed signal peptide (~ 20 amino acids downstream). These studies enabled the identification of a large number of proteins - soluble and transmembrane proteins - coded in the parasite genome and containing the PEXEL motif (~ 8 % of proteins), which are ever since collectively called the 'exportome' of P. falciparum (Marti et al., 2004; Hiller et al., 2004). Furthermore, the predicted genes of the malarial 'exportome', containing the PEXEL motif are found to be localized at subtelomeric regions of the parasite genome, where proteins responsible for host cell modifications are normally found (Maier et al., 2008). Interestingly, more recent studies revealed that the first three residues of the PEXEL motif (RxL) are actually cleaved during the early secretory pathway in the ER by an aspartic protease, which has been identified as Plasmepsin V, followed by N-acetylation of the cleaved protein (Chang et al., 2008; Boddey et al., 2010; Russo et al., 2010). Although different models for further trafficking towards the PV involving the remaining PEXEL residues and Plasmepsin V dependent export, respectively, have been proposed, none of it has been verified (Boddey et al., 2010; Crabb et al., 2010). Another model, although heavily debated, suggests the role of Phosphatidylinositol(3,4,5)-triphosphate (PIP3) in PEXEL-protein delivery to the PV (Bhattacharjee et al., 2012). Whatever the mode of protein secretion in the late secretory pathway (Golgi PPM PVM) might be, it seems like that exported proteins are first secreted into the PV before being directly translocated across the PVM in an ATP- dependent process as shown in two independent localization studies with exported proteins (Ansorge et al., 1996; Wickham et al., 2001). This corresponds with the earlier mentioned two-step model in protein secretion (Charpian and Przyborski, 2008) assigning the PV as a transit compartment. This finding coincides with experiments using the dihydrofolate reductase system (DHFR) showing that exported proteins need to be unfolded before passing the PVM (Gehde et al., 2009). This observation strongly suggest the existence of a translocation system in the PVM. In fact, an ATP-driven translocon, composed of five different Plasmodium proteins, was found in the membrane of the PV and appears to be a possible candidate responsible for protein translocation of exported proteins across the PVM. Since PEXEL proteins seem to be unique to the Apicomplexan Plasmodium the translocon termed P lasmodium translocon of exported proteins (PTEX) also consists of parasite proteins restricted to the Plasmodium genome (de Koning-Ward et al., 2009; Bullen et al., 2012). However, this transport model system only seems to fit for the export of soluble parasite protein, since proteins containing a 'transmembrane domain' would rather end up in the parasite plasma membrane, when secreted via the secretory pathway. One solution to this dilemma would be the way these proteins are synthesized meaning that they are initially synthesized as 'soluble' proteins, which take up their membrane topology only after entering their destination in the host cell. This hypothetical model actually coincides with solubility studies of different transmembrane containing exported proteins (Papakrivos et al., 2005; Przyborski et al., 2005; Saridaki et al., 2009).
However, some well-known exported proteins of P. falciparum lack the typical PEXEL motif and they are generally termed PEXEL-negative exported proteins (PNEPs). These are for example the skeletal-binding protein 1 (SBP1), the membrane-associated histidine-rich protein 1 (MAHRP1) and the ring exported proteins 1 and 2 (REX 1 and 2). These proteins are resident proteins of the Maurer’s clefts, which not only lack a distinguishable PEXEL motif, but also a typical signal sequence for ER entry. However, localization and solubility studies revealed the existence of a single transmembrane region in each of these proteins - except for REX 1, which contains a recessed signal peptide - which appears to be responsible for entering the secretory pathway. This is furthermore supported by studies revealing their sensitivity to BFA (Saridaki et al., 2009; Spycher et al., 2008; Dixon et al., 2008; Haase et al., 2009). However, the further transport of these proteins, which pass the PV and PVM until reaching their final destination in the host cell cytosol, still remains enigmatic. The transport process possibly involves other, not yet identified atypical signals/regions in the amino acid sequence of these proteins and possibly also other kind of translocons in the PVM. Since the number of exported PNEPs in the malaria genome might be greater than known to date, the chance to discover alternative secretory pathways in P. falciparum is quite high.
As described in the previous chapter, secretory proteins are released into the extracellular milieu via the conserved secretory pathway universal to almost all eukaryotic organisms: the 'classical secretory pathway' involving the ER/Golgi- complex. In contrast, many studies in the past years have revealed the secretion of a small number of proteins, which lack a signal peptide to enter the classical secretory pathway and were not affected in their secretion to the cell surface in the presence of BFA (Rabouille et al., 2012). Thus, these proteins were found to be secreted in a mostly ER-to-Golgi independent manner into the extracellular space (Fig. 1.5). That is why this mode of secretion is referred to as 'unconventional protein secretion' and is used by a small number of proteins involved in cell survival, angiogenesis and in inflammatory responses (Nickel, 2005).
In general, two mechanisms for unconventional protein secretion were discovered: vesicular pathways versus non-vesicular pathways. A well-studied candidate of the non- vesicular pathway is the Fibroblast growth factor 2 (FGF2) - a protein involved in angiogenesis - which was found to be directly translocated across the plasma membrane (Schäfer et al., 2004). The translocation process requires the interaction with phosphoinositide phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2) - a component located at the inner leaflet of the plasma membrane - and heparan sulfates found at the outer leaflet of the plasma membrane (Temmerman et al., 2008; Zehe et al., 2006). Another candidate secreted in a non-vesicular mechanism is for example the yeast mating factor α, which is translocated via the membranous ABC transporter Step6 (McGrath and Varshavsky, 1989). Other unconventional secretion pathways involve vesicle-mediated secretion, as in the case of interleukin 1β (IL1β) (Rubartelli et al., 1990). Although the mechanism of the processing pattern of IL1β and the components involved are mostly understood (Franchi et al., 2009), the nature of the vesicles responsible for trafficking IL1β to the plasma membrane is still not clear. One model proposes the secretion of IL1β via secretory lysosmes, a compartment assigned to have a dual function: degradation of proteins, but also storage of secretory proteins before regulated release to the extracellular space upon external stimuli (Andrei et al., 1999; Griffiths, 1996). However, two further mechanisms were also suggested to be involved in the secretion of IL1β: microvesicle shedding at the external side of the plasma membrane (MacKenzie et al., 2001) and the formation of multivesicular bodies (MVB), respectively, which are vesicles formed inside endosomes and are afterwards released as vesicles into the extracellular space, then referred to as 'exosomes' (Stoorvogel et al., 2002). Just recently, other studies discovered an unusual vesicle-mediated secretion pattern of the acyl-CoA-binding protein A (AcbA) from Dictyostelium discoideum involving proteins responsible for the formation of autophagosomes. The autophagosomes then fuse with the plasma membrane to release AcbA (Cabral et al., 2010; Duran et al., 2010; Manjithaya et al., 2010). In addition, some studies imply the role of endosomes, which form multivesicular bodies to fuse with the plasma membrane. This hypothesis is supported by the findings of proteins, which are characteristic of this pathway (Duran et al., 2010; Manjithaya et al., 2010). Furthermore, the yeast ortholog to the mammalian GRASP1 is proposed to play a role in secretion of AcbA as well (Kinseth et al., 2007). Last but not least, a pathway termed the 'Golgi bypass' is used by transmembrane proteins. These proteins initially enter the classical secretory pathway and are eventually released at the plasma membrane, but on their way avoid the Golgi-complex. This for instance is characterized by their insensitivity to BFA (Grieve and Rabouille, 2011).
The appearance of the increasing number of unconventionally secreted proteins and the different mechanisms involved raises the question, why some proteins are differently secreted from the cell, compared to the huge majority of secretory proteins, which uses the classical ER-to-Golgi pathway. So far, two hypotheses to address this question exist: First, it is assumed that in the case of FGF2, this protein, while trafficked through the classical secretory pathway, would bind at a very early stage to glycoproteins, leading to aggregation of the protein and non-secretion of FGF2. The second model, however, proposes the secretion of a non-functional protein due to posttranslational modification, caused while trafficking via the ER/Golgi pathway. Indeed, the latter model was supported by experiments with FGF2. A signal peptide was fused to FGF2 directing this protein into the classical secretory pathway, however, the protein was not secreted into the extracellular space in its functional form (Nickel, 2010; Wegehingel et al., 2008). Since many of the proteins involved in unconventional protein secretion seem to be biomedically relevant, they appear to be suitable drug targets, if their mode of secretion and the components involved would be totally independent of the ER-to-Golgi route.
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Figure 1.5 Unconventional protein secretion mechanisms
Four different types of unconventional protein secretion mechanisms are illustrated. Type1: Vesicular trafficking via lysosomal secretion. Type 2: Non-vesicular trafficking via plasma membrane resident transporters. Type 3: Vesicular trafficking via formation of multivesicular bodies. Type 4: Membrane blebbing is the microvesicle shedding at the external side of the plasma membrane. Missing in this illustration: Golgi-bypass pathway (according to Nickel, 2005).
Interestingly, mechanisms of unconventional protein secretion also appear in protozoan parasites as described for the Leishmania hydrophilic acylated surface protein B (HASPB) (Denny et al., 2000) and the Calcium-dependent protein kinase 1 (CDPK1) of P. falciparum (Möskes et al., 2004) . Both proteins show the same mode of unconventional protein secretion: dual acylation of the N-terminus of the respective proteins, which mediates export to the extracellular surface and the parasitophorous vacuole, respectively. The role of acylation-dependent export is discussed in the next chapter.
N -myristoylation is a co- and post-translational modification of proteins found in all eukaryotic cells. Proteins are characterized as N -myristoylated, when a 14-carbon saturated fatty acid (myristate) is irreversibly attached to the N-terminal glycine residue of the target protein. The glycine residue at the N-terminus of a protein sequence is a prerequisite for N -Myristoylation to take place. At first, methionine - the initiating amino acid in the protein sequence 'Met-Gly-...' - is removed by a methionine aminopeptidase during translation, leaving the glycine residue at the 2nd position of the N-terminus exposed. The myristate from Myristoyl-CoA is then linked to the glycine residue via an amide bond by the N -myristoyltransferase (NMT) (Fig. 1.6). NMT is an enzyme present in all eukaryotic cells and was discovered to be essential for the viability of different eukaryotic organisms (Resh, 1999; Wright et al., 2010). Importantly, NMT protein substrates require in addition to the glycine residue at the N- terminus specific amino acids downstream of the protein sequence - serine or threonine are usually found at position 6 and lysine and arginine, respectively, are found at position 7/8 - to be recognized by the NMT (Resh, 1999). However, more recent studies showed that N -myristoylation also posttranslationally takes place in cells undergoing apoptosis involving caspase cleavage (Zha et al., 2000).
Figure 1.6 Co-translational N -myristoylation
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During translation the initiating methionine is removed by a methionine aminopeptidase (MetAP). Then the 14-carbon saturated fatty acid (myristate) is irreversibly attached to the glycine residue at the second position of the protein sequence via the Myristoyl-CoA by the N -myristoyltransferase (NMT) (according to Wright et al., 2010).
N -myristoylation is commonly found in proteins involved in signal transduction (protein kinases and phosphatases), Gα proteins, calcium-binding proteins, ADP-ribosylation factors (ARF) and also MARCKS (membrane and cytoskeletal-bound proteins) as known for mostly animal and fungal cells. Most of these proteins play a role in signalling processes and in the case of the ARFs in vesicular shuttling revealing their functional importance in these organisms (Resh, 2006). Furthermore, bioinformatic studies, using prediction models, revealed that about 0.5 % of the proteome in eukaryotes appear to be substrates of the NMT (Maurer-Stroh et al., 2002). It appears that apart from viral proteins, bacterial proteins can also be subjected to N -myristoylation by the N -myristoyltransferase of their respective eukaryotic host cell (Maurer-Stroh and Eisenhaber, 2004). The myristoyl moiety enables a reversible binding of the protein to the plasma membrane and to other intracellular membranes in a eukaryotic cell, respectively. However, due to the low binding energy of myristate to the phospholipids of a membrane (approximately 10-4 M K d) a myristoylated protein cannot efficiently anchor to the plasma membrane (Peitzsch and McLaughlin, 1993). In order to achieve a sufficient binding to the phospholipid bilayer, a second signal within the amino acid sequence of the myristoylated protein is required. This hypothesis is referrred to as the 'two-step model'. The second signal can either be another fatty acid group like palmitate (16-carbon saturated fatty acid) or a polybasic cluster of amino acids, located in proximity to the N -myristoylation site. The binding of the respective protein to the membrane occurs when ten of the fourteen carbon atoms of the myristate insert into the phospholipid bilayer and the polybasic cluster of amino acids interacts with the acidic phospholipids of the cellular membrane (electrostatic interaction). The dual binding property induced by the myristate and the polybasic domain synergize leading to a stable anchoring of the protein to the membrane (Murray et al., 1997; Murray et al., 1998; Sigal et al., 1994; Buser et al., 1994). A similar mechanism to establish a strong membrane attachment is achieved by dual acylation of a protein with a myristate and a palmitate moiety (Resh, 1999), which will be discussed in more detail in the next section. Interestingly, the attachment of a myristate to a respective protein also plays a role in membrane targeting to the right target membrane (Murray et al., 1998), although the mechanisms involved here are not yet understood. Further, the membrane anchoring of N -myristoylated proteins can be characterized as a dynamic process, since the myristate moiety on the respective protein switches between two different conformations: it is either exposed on the outside of the protein enabling membrane attachment, or it is segregated into a hydrophobic groove of the protein leading to detachment from the membrane. This mechanism known as the 'myristoyl switch' can be induced by e.g. a ligand or electrostatic interaction (Fig. 1.7) (McLaughlin and Aderem, 1995). A ligand induced myristoyl switch has been characterized for the ADP-ribosylation factor (ARF) proteins, which are regulated by the binding of GTP, which induces the exposure of the myristoyl moiety - initially located in a hydrophobic pocket in its GDP-bound form - and subsequent binding to the membrane (Amor et al., 1994). MARCKS proteins, on the other hand, are regulated by protein kinase C (PKC), which phosphorylates the stretch of the N-terminal polybasic serine residues - the second motif required for membrane binding in the two-step model - leading to an increased negative charge at the polybasic domain. This diminishes the electrostatic interaction with acidic phospholipids leading to the dissociation of the respective protein from the plasma membrane (Thelen et al., 1991).
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Figure 1.7 Two-signal model of myristate-mediated protein binding to the membrane
The low binding energy of the myristoyl-group on the protein promotes reversible binding to the membrane. A second signal is required for stable membrane anchoring: either a polybasic cluster of amino acids to interact with the negatively charged phospholipid groups of the membrane via electrostatic interaction or a second saturated fatty acid like palmitate. Blue circle (protein), red (myristate), pink (palmitate) (according to Wright et al., 2010).
Similar to N -myristoylation protein palmitoylation is a lipid modification, whereby usually a 16-carbon saturated fatty acid is attached to a cysteine residue via a thioester bond. Compared to the irreversible attachment of myristate to a protein, palmitoylation was found to be a reversible modification (Linder and Deschenes, 2003). This is due to the labile thioester linkage between protein and palmitate. The dynamic feature of protein palmitoylation is regulated by palmitoylation and depalmitoylation in regard to the function and localization of the respective protein (Zacharias et al., 2002; Wedegaertner and Bourne, 1994). So far, two different classes of enzymes responsible for palmitoylation and de-palmitoylation have been discovered: Protein acyltransferases (PATs) catalyze palmitate transfer to the respective protein, while the protein acylthioesterases are in charge of removing palmitate from the protein (Resh et al., 2006; Mitchell et al., 2006). Interestingly, no consensus sequence for palmitoylation exists except for the presence of the cysteine residue and in addition in vitro studies revealed a non-enzymatic addition of palmitate from palmitoyl-CoA to proteins (Bañó et al., 1998). However, proteins which are subjected to palmitoylation are usually peripherally attached membrane proteins or proteins containing a transmembrane domain. Proteins containing a transmembrane domain are usually palmitoylated at the junction between cytoplasm and membrane or at the C-terminus located in the cytoplasm. In contrast, the peripherally associated membrane proteins can be dually acylated or are just palmitoylated by adjacent cysteine residues. A cysteine residue at the N-terminus often exists in close proximity to a lipidation site like a preceding N -myristoylation site. Upon palmitoylation a protein becomes more hydrophobic, which leads to a strong protein-membrane anchoring (two-step model). Dually acylated proteins with palmitoylation and preceding N -myristoylation are found among Gα proteins and tyrosine kinases and frequently exhibit the motif 'Met-Gly-Cys' at their N-terminus (Smotrys and Linder, 2004; Nadolski and Linder, 2007). Mutational analyses revealed that substitution of either of the amino acids responsible for lipid modification leads to a decrease or even loss of the protein binding capacity to the plasma membrane (Resh, 1999). More intriguingly, some studies claim that these dually fatty acid acylated proteins are targeted to specific membrane regions of the plasma membrane, the so-called rafts. Rafts are microdomains in the plasma membrane, which are enriched in cholesterol and sphingolipids. However, the mechanism responsible for this observation has not been verified to date (Smotrys and Linder, 2004). In summary, palmitoylation is an important feature of proteins in regard to protein-membrane attachment, trafficking of transmembrane proteins and regulation of intracellular signalling processes of many proteins (Linder and Deschenes, 2003).
Like other eukaroytic organisms, the malaria parasite Plasmodium falciparum also contains genes encoding the ADP-ribosylation factor (ARF), a small GTP binding protein, which plays a crucial role in vesicular trafficking (Stafford et al., 1996; Boman and Kahn, 1995). ARF proteins belong to the superfamily of Ras proteins - a small group of GTPases - and are involved in a number of cellular processes, like for example in vesicular biogenesis and trafficking processes of the secretory pathway (Boman and Kahn, 1995). To date six highly conserved members of the ARF protein family are known for mammalian cells (Kahn et al., 1991). However, so far, only one of the two genes encoding ARF proteins, the gene arf1, in P. falciparum, is isolated, expressed and characterized (Stafford et al., 1996) and structurally determined (Cook et al., 2010). One of the functions attributed to ARF proteins is their role in the COPI pathway (retrograde transport from cis -Golgi to the ER), where they recruit other coat proteins and initiate vesicle formation upon activation. Activation of the soluble GDP-bound ARF to the GTP-bound form, which is able to bind to the membrane is catalyzed by the guanine nucleotide exchange factors (GEFs). The attachment to the membrane occurs, when the N -myristoylation site at the N-terminus of the protein is exposed in the GTP bound conformation of ARF. However, once the GTP on the ARF protein is hydrolyzed to GDP by GTPase activating proteins (GAPs) ARF is released from the membrane and the myristoyl moiety is covered in a hydrophobic groove of the protein. The release of ARF from the vesicle also leads to the detachment of other coat proteins from the vesicle, whereby ARF functions as a trigger. The vesicle formation and cargo trafficking in e.g. the anterograde pathway depends on the cycling of ARF between its soluble GDP-bound state and the membrane-associated GTP-bound state (Boman and Kahn, 1995; Kirchhausen, 2000).
Although some components of the classical secretory pathway are gradually being identified within the parasite cytosol, some studies also report the export of these components and the presence of secretory vesicles in the host cell. These findings implicate the existence of a vesicle-mediated secretion pathway for some parasite proteins from the parasite cytosol to their host cell and host cell membrane, respectively (Trelka et al., 2000; Taraschi et al., 2001). Further studies, apart from electronmicroscopical evidence, supporting the model of a dual operating system in the parasite cytoplasm and the host cell cytosplasm, are missing. On the contrary, a new study with GFP reporter constructs proposed the localization of the components involved in the secretory pathway exclusively to the parasite cytosol (Adisa et al., 2007). Another interesting aspect observed by Stafford and colleagues is that a high mRNA level of Pf ARF1 is reached during merozoite formation, shortly before a high level of msp1 mRNA - merozoite surface protein 1 (MSP1) is located on the merozoite surface - is expressed. They hypothesize a role of Pf ARF1 in MSP1 shuttling to the merozoite surface via the ER-to-Golgi pathway (Stafford et al., 1996). A different observation regarding the localization of ARF was made by a recent proteome analysis of the PV, where Pf ARF1 was found to be located in the PV of the parasite (Nyalwidhe et al., manuscript in preparation), this hypothesis, however, still needs to be reviewed.
Adenylate kinases are ubiquitous enzymes, which play an important role in energy- dependent and nucleotide signalling processes. This enzyme catalyzes the following magnesium dependent reversible reaction: ATP + AMP 2 ADP maintaining the ratio between AMP and ATP in response to the cellular energy need. To date eight different isoforms of adenylate kinases have been discovered in mammalian cells showing distinct intracellular compartmentalization, a localization in different tissues and a developmentally regulated gene expression (Dzeja and Terzic, 2009). In contrast, two adenylate kinases (Pf AK1 and Pf AK2), a GTP:AMP phosphotransferase (Pf GAK) and two adenylate kinase-like proteins (Pf AKLP1 and Pf AKLP2) have been characterized in the malaria parasite P. falciparum to date (Ulschmid et al., 2004; Rahlfs et al., 2009; Ma et al., 2012). These findings reveal the need of the parasite for a high level of adenylate kinase activity to deal with the increased ATP-turnover rate due to the high energy consumption for example during invasion, but also for the biosynthesis of macromolecules. Indeed, infected erythrocytes compared to non-infected cells reveal an increased glucose uptake, a substrate of the glycolytic pathway, responsible for producing high amounts of ATP (Roth, 1990). Differences in the kinetic properties of adenylate kinases might be a result of the distinct subcellular localization and in case of the mammalian adenylate kinase isoenzymes also might be due to tissue-specificity. The mammalian AK1, for example, is mostly found in brain and muscle cells revealing its high activity in these cells and also in erythrocytes, whereas mammalian AK2 is predominantly found in the intramembrane space of the mitochondria in liver, kidney, heart and spleen. Mammalian AK3, however, is largely found in the mitochondrial matrix of the liver and the heart and is actually a GTP:AMP phosphotransferase showing a high substrate specificity to GTP rather than ATP (Khoo and Russell, 1972; Wilson et al., 1976; Tomasselli et al., 1979; Dzeja and Terzic, 2009). P. falciparum AK1 exhibits a higher substrate specificity (75 U/mg) compared to AK2 (10 U/mg), which might be a result of the distinct subcellular localization (Ulschmid et al., 2004). In fact, immunofluorescence analyses with reporter constructs revealed the localization of Pf AK1, Pf AKLP1 and Pf AKLP2 in the parasite cytosol. The Pf GAK, however, was assigned to the mitochondrion of the parasite being a possible homologue to the mammalian GAK. More intriguingly, the phenotype of Pf AK2 was different, insofar, that the observed signal of the chimera was a ring-like structure around the parasite with a knob-like protrusion formed towards the PV and the host cell (Ma et al., 2012). The amino acid sequence of Pf AK2 has a N -myristoylation site and studies on expressed recombinant Pf AK2 and Pf NMT revealed Pf AK2 to be a substrate of Pf NMT (Rahlfs et al., 2009). Further, the group of Becker could also show that substitution of the glycine residue by alanine at the N-terminus of Pf AK2 changes the localization of adenylate kinase 2 to a rather cytosolic signal as observed for Pf AK1 (Ma et al., 2012). These results, taken together, identify Pf AK2 as a N -myristoylated membrane-bound protein.
Over the years many studies on the intraerythrocytic stage of the malaria parasite P. falciparum have already revealed a great amount of information on protein secretory pathways different from the ER/Golgi route as a result of the parasite’s development within the red blood cell. However, the secretory pathway of many parasite proteins, especially those involved in the pathogenesis, still remain obscure.
This study focuses on the existence of alternative secretory pathways to the classical secretory pathway during the intraerythrocytic stage of P. falciparum. A preceding proteomic analysis of the parasitophorous vacuole revealed the secretion of parasite proteins into the PV, which lack any known signal sequence (Nyalwidhe et al, manuscript in preparation). A small percentage of the proteins identified in the PV contained a putative myristoylation site, including the P. falciparum ADP-ribosylation factor 1 (ARF1) - a protein known to be myristoylated in eukaryotic cells. Since it is known that the myristoylation site of ARF1 can anchor this protein to membranes, it was hypothesized that this protein might bind to the inner leaflet of the parasite plasma membrane and is subsequently flipped over into the PV. In order to validate these results on the subcellular localization of Pf ARF1 reporter construct studies, co- localization studies, fluorescence analyses and biochemical analyses were performed. In parallel to Pf ARF1, a different study revealed the secretion of another protein into the PV, the so-called Pf adenylate kinase 2. Similar to Pf ARF1, this protein also lacks a signal sequence, but contains a N -myristoylation site and was found to be a substrate of Pf NMT (Ma et al., 2012; Rahlfs et al., 2009). To further validate and investigate the subcellular localization of Pf AK2 mutagenesis analyses, fluorescence analyses, biochemical analyses and a translocation study with the mDHFR fusion system were carried out. Finally, the sequences of the N-terminus of Pf ARF1 and Pf AK2 were compared to each other to identify possible key differences in their amino acid sequences. Potential motifs in the sequence of Pf AK2, which might play a role in the translocation process of this protein across the parasite plasma membrane, which were missing in the sequence of Pf ARF1, were sought. Subsequently, a chimeric reporter construct of the N-terminus of both proteins was designed and analyzed via fluorescence and biochemical methods.
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