Traffic to the Malaria Parasite Food Vacuole
2007; Elsevier BV; Volume: 282; Issue: 15 Linguagem: Inglês
10.1074/jbc.m610974200
ISSN1083-351X
AutoresMichael T. McIntosh, Ankush Vaid, H. Dean Hosgood, Justin Vijay, A. Bhattacharya, Mayurbhai H. Sahani, Pavlina Baevova, Keith A. Joiner, Pushkar Sharma,
Tópico(s)Toxoplasma gondii Research Studies
ResumoPhosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV. Phosphatidylinositol 3-phosphate (PI3P) is a key ligand for recruitment of endosomal regulatory proteins in higher eukaryotes. Subsets of these endosomal proteins possess a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family. We have identified a single FYVE domain-containing protein in Plasmodium falciparum which we term FCP. Expression and mutagenesis studies demonstrate that key residues are involved in specific binding to PI3P. In contrast to FYVE proteins in other organisms, endogenous FCP localizes to a lysosomal compartment, the malaria parasite food vacuole (FV), rather than to cytoplasmic endocytic organelles. Transfections of deletion mutants further indicate that FCP is essential for trophozoite and FV maturation and that it traffics to the FV via a novel constitutive cytoplasmic to vacuole targeting pathway. This newly discovered pathway excludes the secretory pathway and is directed by a C-terminal 44-amino acid peptide domain. We conclude that an FYVE protein that might be expected to participate in vesicle targeting in the parasite cytosol instead has a vital and functional role in the malaria parasite FV. Malaria parasites inflict a tremendous global burden on the health and productivity of human kind. Among the species predominantly infective to humans, Plasmodium falciparum stands out as responsible for more than half of all infections and for causing the most severe forms of the disease resulting in the death of more than 1.5 million people annually (1Danis M. Gentilini M. Rev. Prat. 1998; 48: 254-257PubMed Google Scholar). Malaria parasites circulate in the human host by repeated invasion and development within the human erythrocyte. During the 48-h cycle of development, parasites endocytose 80-90% of the host cell cytosol and hemoglobin. Specialized proteases are believed to traffic to vesicles containing hemoglobin to initiate the digestion of hemoglobin on route to its final destination, the lysosomal compartment, or parasite food vacuole (FV) 6The abbreviations used are: FV, food vacuole; EEA1, early endosomal autoantigen-1; AD, activation domain; BD, DNA binding domain; ER, endoplasmic reticulum; BFA, brefeldin A; Cvt, cytoplasmic to vacuole trafficking; TGN, trans-Golgi network; HRPII, histidine-rich protein II; PI3P, phosphatidylinositol (PI) 3-phosphate; GFP, green fluorescent protein. (2Klemba M. Beatty W. Gluzman I. Goldberg D.E. J. Cell Biol. 2004; 164: 47-56Crossref PubMed Scopus (112) Google Scholar). Although the endocytic process is characterized by morphologically distinct parasite structures including specialized structures for the uptake of hemoglobin (cytostomes), hemoglobin-containing vesicles, and a single FV (3Atkinson C.T. Aikawa M. Blood Cells. 1990; 16: 351-368PubMed Google Scholar), the contribution of protein components to the endocytic pathway required for membrane trafficking, molecular signaling, effector protein recruitment, and formation of these structures remains unknown. Phosphatidylinositol 3-phosphate (PI3P) plays a fundamental regulatory role in endocytic systems of higher eukaryotes (4Pfeffer S. Cell. 2003; 112: 507-517Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 5Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar, 6Gillooly D.J. Raiborg C. Stenmark H. Histochem. Cell Biol. 2003; 120: 445-453Crossref PubMed Scopus (85) Google Scholar). Rab5, a universally conserved component of early endosomes, recruits the type III PI 3-kinase in mammals and yeast. Hence, the product of this kinase, PI3P, is found in the early endosomes of mammals and is likewise restricted to the endocytic pathway in yeast (6Gillooly D.J. Raiborg C. Stenmark H. Histochem. Cell Biol. 2003; 120: 445-453Crossref PubMed Scopus (85) Google Scholar, 7Gillooly D.J. Morrow I.C. Lindsay M. Gould R. Bryant N.J. Gaullier J.M. Parton R.G. Stenmark H. EMBO J. 2000; 19: 4577-4588Crossref PubMed Scopus (863) Google Scholar, 8Christoforidis S. Miaczynska M. Ashman K. Wilm M. Zhao L. Yip S.C. Waterfield M.D. Backer J.M. Zerial M. Nat. Cell Biol. 1999; 1: 249-252Crossref PubMed Scopus (507) Google Scholar). Various other endosomal regulatory proteins bind PI3P and thereby localize to endocytic compartments in yeast and mammals (9Birkeland H.C. Stenmark H. Curr. Top. Microbiol. Immunol. 2004; 282: 89-115PubMed Google Scholar). Many among these including the early endosomal autoantigen-1 (EEA1) contain a highly selective PI3P binding zinc finger motif belonging to the FYVE domain family (conserved in Fab1, YOTB, Vac1, and EEA1) (9Birkeland H.C. Stenmark H. Curr. Top. Microbiol. Immunol. 2004; 282: 89-115PubMed Google Scholar). Although P. falciparum appears limited with regard to Rab5 effectors and other endocytic machinery, it does possess a phosphatidylinositol biosynthetic pathway (10Elabbadi N. Ancelin M.L. Vial H.J. Mol. Biochem. Parasitol. 1994; 63: 179-192Crossref PubMed Scopus (35) Google Scholar). We, therefore, asked if P. falciparum possesses functional FYVE domain proteins. Using a bioinformatics approach we identified a single FYVE domain-containing protein encoded in the parasite genome. Surprisingly, this protein did not localize to endocytic structures as would be predicted for an FYVE domain family protein but, rather, localized discreetly to the lumen of the FV, a lysosomal compartment characterized by the presence of hemozoin (a crystallized heme byproduct of hemoglobin digestion). By identifying the domain responsible for targeting to the FV, we not only explain this unexpected result but identify a new direct trafficking pathway between the parasite cytosol and the parasite FV. In Vitro Cultivation of P. falciparum—P. falciparum strain 3D7 (obtained from MR4) was employed in experiments contributing to Figs. 4 and 5, whereas strain FCR3 (a gift from Dr. Kasturi Haldar) was used to obtain Figs. 6, 7, 8 and 9. Parasites were maintained in 5% CO2 (for FCR3) or 90% N2, 5% CO2, and 5% O2 for (3D7) in leukocyte-free erythrocytes of blood group A+ (American Red Cross) at 5% hematocrit and cultured in RPMI 1640 medium (Invitrogen) supplemented with 25 mm HEPES, sodium bicarbonate (2 g·liter-1), gentamicin (1 μg·ml-1), 92 μm hypoxanthine and contained 10% human serum, type AB as described (11Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6219) Google Scholar). Parasite synchronization was achieved by isolation of late stage trophozoites on Percoll sorbitol gradients as described (12Kutner S. Breuer W.V. Ginsburg H. Aley S.B. Cabantchik Z.I. J. Cell. Physiol. 1985; 125: 521-527Crossref PubMed Scopus (143) Google Scholar) followed by reintroduction into culture with fresh red blood cells. Four hours after the first detection of ring stage parasites, cultures were treated with 5% sorbitol (13Lambros C. Vanderberg J.P. J. Parasitol. 1979; 65: 418-420Crossref PubMed Scopus (2855) Google Scholar) to lyse all remaining late stage parasites. This yielded highly synchronous cultures.FIGURE 5FCP localizes to the FV but does not traffic via the classical or alternate secretory pathways. A, immunofluorescence using anti-FCP revealed a discrete localization to the parasite FV. Ring stage parasites were treated either with 5 μg·ml-1 BFA (anti-FCP + BFA) or solvent as control (anti-FCP) for 12 h before immunofluorescence microscopy. BFA treatment did not alter FV localization of FCP. B, as a control experiment, parasites treated with BFA (anti-HRPII + BFA) or without BFA (anti-HRPII) were stained using antisera against a known secreted protein, HRPII. HRPII was localized predominantly to the cytoplasm of the host infected erythrocyte (anti-HRPII). BFA treatment resulted in a significant loss in erythrocyte localization of HRPII and a concomitant increase in the parasite cytoplasm (anti-HRPII + BFA). C, trophozoite-infected erythrocytes were treated with saponin to separate the erythrocyte and parasitophorous vacuole contents (Sup) from the parasite material (Pellet), and immunoblotting was performed for FCP. FCP was found in the pellet fraction rather than the concentrated supernatant fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6FCP localizes to the lumen of the FV. A, cryo-immunoelectron micrograph showing a close-up of a folded FV with immunogold-stained FCP in the lumen of the parasite FV in a late trophozoite. Various structures are indicated as host red blood cell cytosol (RBC Cytosol), parasite body (parasite), parasite nucleus (Nu), FV, and hemozoin (Hz). B shows a more complete parasite with a cross-section through the FV indicating a lack of immunogold staining outside the FV. C, three parasites from a different region of the same ultrathin section that was immunogold-stained for anti-FCP in A and B. Note the lack of concentrated gold particles on all visible parasite structures including ER, parasite plasma membrane (PPM), parasitophorous vacuolar membrane (PVM), and hemoglobin containing vesicles (HV). As apparent from A and C, less than 1% of immunogold appeared outside of the parasite FV.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 7Localization of GFP-FCP and domain deletion mutants in P. falciparum. A, stage synchronous P. falciparum was transfected with either GFP-wild type FCP (WT-FCP) yielding a discrete localization to the FV characterized by the presence of hemozoin crystals (arrow). B, a schematic diagram shows the various GFP gene fusions and deletion constructs employed. aa, amino acids. C, various domain deletions of FCP as indicated in B were transfected into P. falciparum. Deletion of the FYVE domain (GFP-FCP-ΔFYVE) produced morphologically stunted parasites but did not appear to alter localization of the protein. In contrast, C-terminal deletion including deletion of the coiled-coil domain (GFP-FCP-Δ-c-coil) or deletion of the C-terminal 44-amino acid domain alone (GFP-FCP-Δc44) each displayed altered localization to the cytoplasm. DIC, differential interference contrast.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 8C-terminal peptide directs traffic to the FV. A, differential interference contrast (DIC) and fluorescence images of P. falciparum transfected with an expression plasmid encoding GFP with a C-terminal fusion of the 44 amino acid peptide (GFP-44aa) deemed necessary for trafficking of GFP-FCP to the FV. In trophozoite stage parasites fluorescence appeared localized only to the FV (arrow), indicating this peptide is sufficient for trafficking to the FV. B, control studies with the GFP expression vector lacking the targeting peptide (GFP-Control) demonstrated a cytoplasmic fluorescence pattern that excluded the FV (arrow). In the composite image, brightness and contrast of individual image layers were adjusted. Pseudo green color was applied to the fluorescence layer and superimposed onto the DIC image using Adobe Photoshop 6.0.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 9Morphology of the ΔFYVE mutant. Transfection of parasites with GFP-FCP-ΔFYVE revealed a profound dominant negative effect. A, bright field and fluorescence images of a late stage trophozoite expressing GFP-FCP- ΔFYVE. Note the stunted size of the parasite, nominal FV, and negligible amount of hemozoin as compared with the control GFP-transfected late stage trophozoite in panel B. B, control studies with the GFP expression vector lacking FCP (GFP-Control) demonstrated a cytoplasmic fluorescence pattern that excluded the FV (arrow).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Cloning of FCP and Plasmid Constructs—Using open reading frame-specific primers with appropriate 5′ end restriction sites, FCP cDNA was reverse transcription-PCR amplified from total asexual stage P. falciparum RNA using RT-Superscript II (Invitrogen) and Kod hot start DNA polymerase (Novagen Inc.). For yeast 2-hybrid analysis, FCP cDNA was appropriately digested and ligated into yeast two-hybrid vectors pACT-2 and pGBT-9 (Invitrogen) between the BamHI and XhoI sites of pACT-2 and EcoRI and BamHI sites of pGBT-9. Ligations were made in-frame and downstream of the GAL4 activation domain (AD) or DNA binding domain (BD) to obtain pAD-FCP and pBD-FCP. The cDNA sequence of cloned FCP was identical to that of the putative gene (Pf14_0574) represented in the 3D7 P. falciparum genome data base. For GFP expression studies in Plasmodium, the eGFP gene of pEGFP-C2 (Invitrogen) was replaced with the GFP-M2 gene of pHDGFP (14Kadekoppala M. Kline K. Akompong T. Haldar K. Infect. Immun. 2000; 68: 2328-2332Crossref PubMed Scopus (29) Google Scholar). This yielded the plasmid pGFP-M2-C2. Subsequently FCP was subcloned from the yeast expression vector pBD-FCP into the EcoRI and SalI sites of pGFP-M2-C2 such that the gene fusion was in-frame and downstream of GFP-M2. Domain deletions were constructed from pBD-FCP and converted to GFP-M2 gene fusions by ligation between the EcoRI and SalI sites of pGFP-M2-C2. FCP-Δ-c-coil was obtained by digestion of pBD-FCP with BglII and BamHI followed by blunt ending and self-ligation. This removed the entire C terminus including the complete coiled-coil domain. FCP-Δ-FYVE was obtained by digestion of pBD-FCP with KpnI and BglII followed by blunt ending and self-ligation. This deleted only the FYVE domain and kept the N terminus and remainder of FCP inframe. FCP-Δ-c44 was obtained by digestion of pBD-FCP with StyI and BamHI followed by blunt ending and self-ligation. This removed the C-terminal conserved domain encoding the final 44 amino acids of FCP. Transfer to pGFP-M2-C2 yielded the following plasmid constructs: pGFP-F-Δ-c-coil, pGFP-F-Δ-FYVE, and pGFP-F-Δ-c44. To explore the function of the C-terminal 44 amino acids, pGFP-44aa was constructed by PCR amplification of the C-terminal 132 bp encoding the 44-amino acid peptide using forward and reverse primers bearing EcoRI and SalI sites, respectively, and cloning into the EcoR1 and SalI sites of pGFP-M2-C2. Primers (GFPM2-ATTB1, 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTAAAAATGAGTAAAGGAGAAGAACTTTTC-3′, and GFP-ATTB2, 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTGATCAGTTATCTAGATCCGG-3′) flanking the GFP gene fusion cassettes in the above plasmids, which contained 5′ attB1 and attB2 recombination sites, respectively, were used to PCR amplify all GFP-gene fusions such that they were suitable for cloning into pDONR-21 (Invitrogen) using the BP reaction and Gateway™ recombination cloning system (Invitrogen). Subsequent LR reactions (Invitrogen) with the Plasmodium Gateway™ destination vector pHH1-DR0.28-DEST (15Skinner-Adams T.S. Hawthorne P.L. Trenholme K.R. Gardiner D.L. Trends Parasitol. 2003; 19: 17-18Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar) (a kind gift from Prof. Tina Skinner-Adams, Queensland Institute of Medical Research, Australia) resulted in the following Plasmodium GFP expression vectors: EXP-GFPM2, EXP-GFP-FCP, EXP-GFP-FCPΔ-c-coil, EXP-GFP-FCPΔ-c44, EXP-GFP-FCPΔ-FYVE, EXP-GFP-44aa. These expression vectors all utilize the Plasmodium HSP86 5′-untranslated region as a constitutive promoter for the expression of the GFP gene fusions. For protein expression studies, FCP cDNA was cloned in PQE-UA vector (Qiagen) to facilitate its expression as an N-terminal His6-tagged protein. Site-directed mutagenesis was performed by using the QuikChange kit (Stratagene). Recombinant Protein Expression in E. coli and Anti-FCP Antiserum Production—BL21-RIL E. coli (Stratagene) was transformed with PQE-FCP plasmid or its variants. Cultures were grown in LB media containing 100 μg/ml ampicillin and 25 μg/ml chloramphenicol. Recombinant protein was induced at mid-logarithmic phase (A600 value of 0.6) by the addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside either at 37 °C for 4 h or at 18 °C for 14 h. Bacterial cells were harvested by centrifugation at 4000 × g for 30 min and suspended in buffer A (50 mm Tris, pH 7.4, 6 m urea, 500 mm NaCl) followed by sonication. Cell lysates were clarified by centrifugation at 20,000 × g for 30 min at 4 °C. Subsequently, supernatant was incubated with nickel-nitrilotriacetic acid-agarose (Qiagen) for 12 h at 4 °C. After washes in buffer A, recombinant His6-tagged proteins were eluted in 50 mm Tris, pH 8.0, containing 500 mm NaCl, 300 mm imidazole, 1 mm dithiothreitol, and 6 m urea. Eluted proteins were renatured by dialysis against buffer containing reduced amounts (4 to 0 m) of urea. Refolded recombinant proteins were analyzed by gel filtration and SDS-PAGE, and a protein band of the expected molecular mass of 37 kDa was detected by Coomassie staining. To confirm the identity of the recombinant protein, the 37-kDa protein band was excised and subjected to tryptic digest and mass spectrometric analysis by the Institute of Molecular Medicine, The Chatterjee Group, New Delhi (supplemental Fig. S1). To raise anti-sera against FCP, 100 μg of recombinant FCP was emulsified with complete Freund's adjuvant and used to raise antisera in mice and rabbits. Dot-blot Phosphoinositide Binding Assays—Dot-blot assays were performed as described previously (16Jensen R.B. La Cour T. Albrethsen J. Nielsen M. Skriver K. Biochem. J. 2001; 359: 165-173Crossref PubMed Scopus (40) Google Scholar). Briefly, various phosphoinositides were serially diluted and spotted on nitro-cellulose membrane and air-dried for 2 h. Subsequently, the membrane was blocked with 3% bovine serum albumin in buffer A (50 mm Tris-HCl, pH 7.4, 150 mm sodium chloride, 0.1% Tween 20) for 3 h. The membrane was incubated with recombinant His6-FCP (0.5 μg/ml) or its variants diluted in blocking buffer for 12 h at 4 °C. The membrane was washed 5 times with buffer A before3hof incubation with anti-His6 antibody (BD Biosciences). Subsequently, the membrane was incubated with horseradish peroxidase-labeled anti-mouse IgG, and phosphatidylinositol-bound protein was detected by chemiluminescence. Yeast Two-hybrid Growth Assays—Yeast two-hybrid liquid culture growth assays were performed as previously described (17Hoppe H.C. Ngo H.M. Yang M. Joiner K.A. Nat. Cell Biol. 2000; 2: 449-456Crossref PubMed Scopus (117) Google Scholar). Yeast were transformed with all possible paired plasmid combinations as well as with individual plasmids as controls, and protein-protein interactions were determined based on growth in His-dropout medium. Stringency was further assessed in increasing concentrations of 3-amino-1,2,4-trizol. Immunoblotting and Immunolocalization—For immunoblot experiments described in Fig. 5, parasite material was separated from erythrocytes and parasitophorous vacuole-derived material by lysis in 0.1% saponin analogous to a previously published procedure (33Klemba M. Gluzman I. Goldberg D.E. J. Biol. Chem. 2004; 279: 43000-43007Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). An equal number of parasites fractionated into a pellet or concentrated supernatant was denatured by boiling in 2% SDS, applied to SDS-PAGE, and subsequently transferred to nitrocellulose. Immunoblotting was performed using rabbit anti-FCP and horseradish peroxidase-labeled goat anti-rabbit IgG. Blots were developed using a WestDura ECL kit (Pierce). For immunofluorescence, parasitized red blood cells were spread on slides and fixed in methanol for 10 min. Subsequently, slides were blocked with 3% bovine serum albumin for 12 h at 4 °C and incubated with a 1:50 dilution of mouse anti-FCP antisera. After washing with phosphate-buffered saline, slides were stained with goat anti-mouse IgG conjugated to Texas Red. Cells were visualized on a Zeiss LSM510 confocal microscope equipped with a CCD camera. Control experiments were performed using preimmune bleeds. For immunoelectron microscopy, parasites were fixed in 4% paraformaldehyde (EM Polysciences Inc.), 0.1% glutaraldehyde (EM Polysciences Inc.), and 0.25 m HEPES, pH 7.4, embedded in 1× phosphate-buffered saline containing 10% bovine skin gelatin (Sigma) and incubated at 4 °C in cryo-protectant containing 10% polyvinylpyrolidone and 2.7 m sucrose. Subsequently, parasite blocks were applied to pins, snap-frozen in liquid nitrogen, and subjected to ultra thin sectioning in a liquid nitrogen-cooled cryo-ultramicrotome. Sections were applied to nickel-coated grids, incubated with a 1:1000 dilution of affinity-purified rabbit anti-FCP IgG at 25 °C, and stained with gold-conjugated goat anti-rabbit IgG. Electron micrographs were obtained digitally by a CCD camera using a Philips Techni Bio-Twin electron microscope. Parasite Transfection, Localization, and Dominant Negative Effects—Transient transfections of malaria parasites with EXP-GFP vectors (see above details) were performed by a modification of the previously described preloading technique (18Deitsch K. Driskill C. Wellems T. Nucleic Acids Res. 2001; 29: 850-853Crossref PubMed Scopus (243) Google Scholar). 200 μl of packed red blood cells were washed once in incomplete cytomix (19van den Hoff M.J. Moorman A.F. Lamers W.H. Nucleic Acids Res. 1992; 202902 Crossref PubMed Scopus (385) Google Scholar) (containing 120 mm KCl, 0.15 mm CaCl2, 2 mm EGTA, 5 mm MgCl2, 10 mm K2HPO4/KH2PO4, and 25 mm Hepes, pH 7.6) and then resuspended to 400 μl in ice-cold incomplete cytomix. Red blood cells were then preloaded with 50 μg of plasmid DNA by electroporation in a ECM630 Electro Cellular Manipulator™ (BTX) at settings of 310 V, 25 ohms, and 950 microfarads. To ensure synchronous transfections and facilitate morphological interpretation of potential dominant negative effects, cultures were synchronized as described above. Subsequently, late stage parasites were once again purified by Percoll sorbitol gradient as described above and then added to preloaded red blood cells and cultured for 40-46 h before microscopic analysis for GFP expression and morpho-logical detail. Microscopy was performed at 1000× magnification using a Nikon Optiphot II microscope equipped with epifluorescence and Nomarski optics or at 1000× using a Zeiss Axioscope with epifluorescence. FV localization was verified by photography of cells under simultaneous fluorescence and bright field phase contrast at low light levels. Images were collected by CCD camera, labeled in Adobe PhotoShop 6.0 (Adobe), and arranged in Adobe Illustrator 10 (Adobe). Plasmodium Encodes a Single FYVE Domain Protein—Sequence alignment of FYVE domains yielded the consensus sequence R(R/K)HHCR, which was used to search the P. falciparum genome data base. This revealed only a single FYVE domain-containing protein (Pf14_0574). Analysis by SOCKET (20Walshaw J. Woolfson D.N. J. Mol. Biol. 2001; 307: 1427-1450Crossref PubMed Scopus (308) Google Scholar) indicated the presence of a coiled-coil domain (residues 163-266) downstream of the FYVE zinc finger domain (residues 42-92). Thus, we named the protein FCP for FYVE and coiled-coil domain-containing protein. An additional gene (PF13_0055), annotated as encoding a potential FYVE domain, more closely resembled a RING domain and, hence, was not investigated further. Sequencing of FCP cDNA confirmed a lack of introns, and comparison of the amino acid sequence with other orthologous counterparts indicated that FCP was highly conserved with 70-80% amino acid identity within the malaria parasite genus Plasmodium (Fig. 1A). Additional searches indicated that at least two potential FYVE domain-containing proteins exist in the related parasite Toxoplasma gondii. TBLASTN searches excluding the FYVE domains of other family members including EEA1, Hrs, YOT1, Fab1, and Vac1 failed to identify genes encoding proteins of significant homology in P. falciparum. This provided the first indication that FCP might not function in the same fashion as the endosomal FYVE proteins of higher eukaryotes. X-ray crystal structures for Saccharomyces cerevisiae Vps27p, Drosophila melanogaster Hrs, and human EEA1 have further defined the secondary and tertiary structure of the FYVE domain as containing a core fold similar to that of the RING domain (21Misra S. Hurley J.H. Cell. 1999; 97: 657-666Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). FCP encodes all of the conserved residues and attributes known to be required for PI3P binding by other FYVE domain proteins. Among these are eight invariant zinc-coordinating cysteine residues arranged in four CXXC motifs, an upstream WXXD motif, and a downstream RVC motif, each intimately involved in PI3P binding (Fig. 1B). In addition, the glycine residue immediately adjacent to the second pair (CXXC) of zinc-coordinating cysteines is invariant among all known FYVE domain proteins and is believed to provide flexibility required for formation of the core fold of the domain (Fig. 1B). The R(R/K)HHCR consensus sequence is known to be the principal site of PI3P binding (Fig. 1B). FCP Specifically Interacts with PI3P—To better understand phosphoinositide interactions with FCP, we performed lipid dot-blot assays using recombinant wild type and site-directed mutants of FCP. Wild type FCP bound specifically to PI3P (Fig. 2B). Deletion of the FYVE domain (data not shown) resulted in a complete loss of phosphoinositide binding as did a site-directed substitution K55A (Fig. 2B), which resides within the conserved motif R(R/K)HHCR (Figs. 1B and 2A). The crystal structure of the EEA1 FYVE domain bound to inositol 1,3-diphosphate has revealed that amino acid residues form a coordination network with the 1′ and 3′ phosphates of PI3P, and it has been hypothesized that these residues may lend specificity to FYVE-PI3P interaction (22Dumas J.J. Merithew E. Sudharshan E. Rajamani D. Hayes S. Lawe D. Corvera S. Lambright D.G. Mol. Cell. 2001; 8: 947-958Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). To test this we replaced all flanking arginines (Arg-52, Arg-54, and Arg-59) with neutral alanine residues. This three-amino acid substitution resulted in a loss in specificity for PI3P as indicated by increased binding to phosphoinositides, PI 3,4,5-triphosphate and PI 4,5-diphosphate. This loss in PI3P binding specificity demonstrated that indeed a critical interaction does exist between these basic residues and the 1′ and 3′ phosphates of PI3P (Figs. 2, A and B). FCP Self-associates—Several FYVE proteins have been shown to dimerize by virtue of a coiled-coil domain, thereby increasing their avidity for PI3P (23Hayakawa A. Hayes S.J. Lawe D.C. Sudharshan E. Tuft R. Fogarty K. Lambright D. Corvera S. J. Biol. Chem. 2004; 279: 5958-5966Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 24Stenmark H. Aasland R. Driscoll P.C. FEBS Lett. 2002; 513: 77-84Crossref PubMed Scopus (158) Google Scholar). For example, EEA1 dimerizes when bound by Rab5 and then simultaneously binds two PI3P molecules in the early endosome (25Simonsen A. Lippe R. Christoforidis S. Gaullier J.M. Brech A. Callaghan J. Toh B.H. Murphy C. Zerial M. Stenmark H. Nature. 1998; 394: 494-498Crossref PubMed Scopus (922) Google Scholar). In this way EEA1 is believed to participate in tethering of opposing vesicular and target membranes during the homotypic fusion of early endosomes (26Woodman P.G. Traffic. 2000; 1: 695-701Crossref PubMed Scopus (88) Google Scholar). To test this hypothesis we employed a yeast two-hybrid growth assay (17Hoppe H.C. Ngo H.M. Yang M. Joiner K.A. Nat. Cell Biol. 2000; 2: 449-456Crossref PubMed Scopus (117) Google Scholar). Yeast expression plasmids were constructed by fusing the yeast GAL4 AD or GAL4 DNA BD to the N terminus of FCP. Persistent growth of yeast in HIS-medium in increasing concentrations of 3-amino-1,2,4-trizol indicates an interaction between bait and target proteins, each fused to AD or BD, respectively. A representative plot (Fig. 3A) shows inhibited growth of yeast expressing a negative cont
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