A Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.036772
ISSN1083-351X
AutoresHayley E. Bullen, Christopher J. Tonkin, Rebecca A. O’Donnell, Wai‐Hong Tham, Anthony T. Papenfuss, Sven B. Gould, Alan F. Cowman, Brendan S. Crabb, Paul R. Gilson,
Tópico(s)interferon and immune responses
ResumoThe phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton. The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton. Apicomplexan parasites cause a multitude of illnesses through infection of both human and livestock hosts. Members of this phylum include the opportunistic human parasites Toxoplasma gondii and Cryptosporidium parvum, pathogens of livestock, including Theileria annulata and Eimeria tenalla, and most notably the Plasmodium species, the causative agents of malaria in humans. Infection with P. falciparum results in ∼1–3 million deaths and a further 500 million infections annually (1Snow R.W. Guerra C.A. Noor A.M. Myint H.Y. Hay S.I. Nature. 2005; 434: 214-217Crossref PubMed Scopus (2138) Google Scholar). During various stages of the Apicomplexan lifecycle the parasites require motility to migrate through their insect and vertebrate hosts and to invade and internalize themselves within targeted host cells (2Baum J. Gilberger T.W. Frischknecht F. Meissner M. Trends Parasitol. 2008; 24: 557-563Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 3Cowman A.F. Crabb B.S. Cell. 2006; 124: 755-766Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar, 4Tardieux I. Ménard R. Traffic. 2008; 9: 627-635Crossref PubMed Scopus (33) Google Scholar). The parasite's unique mechanism of gliding motility is powered by an Apicomplexan-specific motor complex termed the actin-myosin motor (5Keeley A. Soldati D. Trends Cell Biol. 2004; 14: 528-532Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), which resides between the outer plasma membrane and inner membrane complex (IMC) 4The abbreviations used are:IMCinner membrane complexDRMdetergent-resistant membraneHAhemagglutininGFPgreen fluorescent proteinRIPAradioimmune precipitation assaybis-tris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolmAbmonoclonal antibodyChFPcherry fluorescent proteinDSPdithiobis succinimidyl propionateIMPintramembranous particleALValveolinGPIglycosylphosphatidylinositol. (6Soldati D. Meissner M. Curr. Opin Cell Biol. 2004; 16: 32-40Crossref PubMed Scopus (95) Google Scholar). The IMC is a continuous patchwork of flattened vesicular cisternae located directly beneath the plasma membrane and overlying the cytoskeletal network (7Bergman L.W. Kaiser K. Fujioka H. Coppens I. Daly T.M. Fox S. Matuschewski K. Nussenzweig V. Kappe S.H. J. Cell Sci. 2003; 116: 39-49Crossref PubMed Scopus (157) Google Scholar, 8Pinder J.C. Fowler R.E. Dluzewski A.R. Bannister L.H. Lavin F.M. Mitchell G.H. Wilson R.J. Gratzer W.B. J. Cell Sci. 1998; 111: 1831-1839Crossref PubMed Google Scholar). The IMC appears to arise from Golgi-associated vesicles flattened during parasite maturation to form large membranous sheets, which envelope the parasite and leave only a small gap at the extreme parasite apex (9Bannister L.H. Hopkins J.M. Fowler R.E. Krishna S. Mitchell G.H. Parasitology. 2000; 121: 273-287Crossref PubMed Scopus (91) Google Scholar). inner membrane complex detergent-resistant membrane hemagglutinin green fluorescent protein radioimmune precipitation assay 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol monoclonal antibody cherry fluorescent protein dithiobis succinimidyl propionate intramembranous particle alveolin glycosylphosphatidylinositol. The myosin component of the actin-myosin motor has previously been defined as a tetrameric complex consisting of a class XIV myosin termed Myo-A (10Pinder J. Fowler R. Bannister L. Dluzewski A. Mitchell G.H. Parasitol Today. 2000; 16: 240-245Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), a myosin tail interacting protein (also called myosin light chain) (7Bergman L.W. Kaiser K. Fujioka H. Coppens I. Daly T.M. Fox S. Matuschewski K. Nussenzweig V. Kappe S.H. J. Cell Sci. 2003; 116: 39-49Crossref PubMed Scopus (157) Google Scholar) and the two glideosome-associated proteins GAP45 and GAP50 (11Gaskins E. Gilk S. DeVore N. Mann T. Ward G. Beckers C. J. Cell Biol. 2004; 165: 383-393Crossref PubMed Scopus (206) Google Scholar). These motor components are linked to the outer IMC membrane via the membrane proteins GAP45/50 (11Gaskins E. Gilk S. DeVore N. Mann T. Ward G. Beckers C. J. Cell Biol. 2004; 165: 383-393Crossref PubMed Scopus (206) Google Scholar). Between the plasma membrane and the IMC are actin filaments held in place through aldolase-mediated contact with the C-terminal tails of plasma membrane-spanning adhesive proteins whose ectodomains bind substrate and host cells (2Baum J. Gilberger T.W. Frischknecht F. Meissner M. Trends Parasitol. 2008; 24: 557-563Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). To power the forward movement of apicomplexan zoite stages, myosin pulls the actin filaments and their attached adhesins rearward. For this to succeed the GAP-myosin complex must presumably be fixed to the IMC, possibly via interactions with unidentified proteins linking the motor to the underlying cytoskeleton. Studies of fluorescently tagged GAP50 confirm it is relatively immobile within the IMC, however attempts to identify potential anchoring proteins have not been successful and have instead indicated that GAP50 may be immobilized by the lipid-raft like properties of the IMC membranes (12Johnson T.M. Rajfur Z. Jacobson K. Beckers C.J. Mol. Biol. Cell. 2007; 18: 3039-3046Crossref PubMed Scopus (61) Google Scholar). The actin-myosin complex is confined to the outer IMC membrane while the opposing innermost IMC membrane is studded with 9 nm intramembranous particles, revealed by electron microscopy of freeze fractured Toxoplasma tachyzoites and Plasmodium ookinetes (13Morrissette N.S. Murray J.M. Roos D.S. J. Cell Sci. 1997; 110: 35-42Crossref PubMed Google Scholar, 14Raibaud A. Lupetti P. Paul R.E. Mercati D. Brey P.T. Sinden R.E. Heuser J.E. Dallai R. J. Struct. Biol. 2001; 135: 47-57Crossref PubMed Scopus (45) Google Scholar). The size of these particles suggests that the proteins involved are likely to form high molecular weight complexes that overlay the parasite's cytoskeletal network and possibly anchor the IMC to the cytoskeleton (12Johnson T.M. Rajfur Z. Jacobson K. Beckers C.J. Mol. Biol. Cell. 2007; 18: 3039-3046Crossref PubMed Scopus (61) Google Scholar, 13Morrissette N.S. Murray J.M. Roos D.S. J. Cell Sci. 1997; 110: 35-42Crossref PubMed Google Scholar, 14Raibaud A. Lupetti P. Paul R.E. Mercati D. Brey P.T. Sinden R.E. Heuser J.E. Dallai R. J. Struct. Biol. 2001; 135: 47-57Crossref PubMed Scopus (45) Google Scholar, 15Mann T. Beckers C. Mol. Biochem. Parasitol. 2001; 115: 257-268Crossref PubMed Scopus (184) Google Scholar). Due to the close apposition of the inner and outer IMC membranes (14Raibaud A. Lupetti P. Paul R.E. Mercati D. Brey P.T. Sinden R.E. Heuser J.E. Dallai R. J. Struct. Biol. 2001; 135: 47-57Crossref PubMed Scopus (45) Google Scholar, 16Pomel S. Luk F.C. Beckers C.J. PLoS Pathog. 2008; 4: e1000188Crossref PubMed Scopus (78) Google Scholar), it is possible that the intramembranous particles could bridge the IMC lumen and interact with the GAP-myosin complex contributing to its stabilization within the IMC. To identify putative proteins that might be components of the intramembranous particles, we examined data from the detergent-resistant membrane (DRM) proteome of schizont-stage P. falciparum parasites containing developing merozoites (17Sanders P.R. Gilson P.R. Cantin G.T. Greenbaum D.C. Nebl T. Carucci D.J. McConville M.J. Schofield L. Hodder A.N. Yates 3rd, J.R. Crabb B.S. J. Biol. Chem. 2005; 280: 40169-40176Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 18Sanders P.R. Cantin G.T. Greenbaum D.C. Gilson P.R. Nebl T. Moritz R.L. Yates 3rd, J.R. Hodder A.N. Crabb B.S. Mol. Biochem. Parasitol. 2007; 154: 148-157Crossref PubMed Scopus (71) Google Scholar). DRMs, or lipid-rafts, were of considerable interest, because they appeared to harbor proteins involved in host cell invasion such as glycosylphosphatidylinositol (GPI)-anchored merozoite surface proteins. Our data also indicated that P. falciparum schizont-stage DRMs contained the IMC proteins PfGAP45/50 (17Sanders P.R. Gilson P.R. Cantin G.T. Greenbaum D.C. Nebl T. Carucci D.J. McConville M.J. Schofield L. Hodder A.N. Yates 3rd, J.R. Crabb B.S. J. Biol. Chem. 2005; 280: 40169-40176Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), and recent studies in T. gondii have also suggested that the IMC is enriched in DRMs (12Johnson T.M. Rajfur Z. Jacobson K. Beckers C.J. Mol. Biol. Cell. 2007; 18: 3039-3046Crossref PubMed Scopus (61) Google Scholar). Another study indicated that when P. falciparum DRM protein complexes were separated by blue native gel electrophoresis, a band was produced containing PfGAP45/50 and PfMyo-A as well as a novel six-pass transmembrane protein (PlasmoDB: PFD1110w, GenBankTM: CAD49269) (18Sanders P.R. Cantin G.T. Greenbaum D.C. Gilson P.R. Nebl T. Moritz R.L. Yates 3rd, J.R. Hodder A.N. Crabb B.S. Mol. Biochem. Parasitol. 2007; 154: 148-157Crossref PubMed Scopus (71) Google Scholar). This protein was related to another six-pass transmembrane DRM protein (PlasmoDB: MAL13P1.130, GenBankTM: CAD52385) we had previously identified in P. falciparum schizont-stage DRMs (17Sanders P.R. Gilson P.R. Cantin G.T. Greenbaum D.C. Nebl T. Carucci D.J. McConville M.J. Schofield L. Hodder A.N. Yates 3rd, J.R. Crabb B.S. J. Biol. Chem. 2005; 280: 40169-40176Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). We show here that MAL13P1.130 and PFD1110w, termed PfGAPM1 and PfGAPM2 (glideosome-associated protein with multiple-membrane spans), respectively, belong to a family of proteins specific to the Apicomplexa and demonstrate that P. falciparum GAPM proteins, and their orthologues in T. gondii, localize to the parasite IMC. The GAPMs form high molecular weight complexes that are resistant to dissociation and solubilization by a variety of common detergents and could therefore be components of the intramembranous particles seen in electron microscopy. When isolated by immunoprecipitation, the GAPM complexes co-purify with components of the actin-myosin motor and particularly the parasite cytoskeletal network suggesting GAPMs could anchor the IMC to the cytoskeleton and perhaps even play a role in tethering the motor to cytoskeleton. Orthologues of PfGAPM1, -2, and -3 were identified by BLASTP searches (E-value <0.01) of the following databases; NCBI Non-Redundant data base, PlasmoDB, GeneDB, and OrthoMclDB and are listed in supplemental Table S1. The GAPM proteins were aligned with MUSCLE (19Edgar R.C. BMC Bioinformatics. 2004; 5: 113Crossref PubMed Scopus (5912) Google Scholar), evolutionary distances were estimated under the JTT model, and neighbor joining trees were constructed with Phylip (J. Felsenstein, Phylogeny Inference Package version 3.6, Dept. of Genome Sciences, University of Washington, Seattle). Bootstrap support was also calculated. gapm genes were PCR-amplified using cDNA extracted from either strain 3D7 P. falciparum parasites or strain RH T. gondii parasites (primers listed in supplemental Table S2). Products were ligated into the pGEM T-easy vector (Promega) and were verified by sequencing. The P. falciparum gapm genes were then excised with PstI and ligated into the PstI site of a modified version of pTGFP-GPI (20Gilson P.R. O'Donnell R.A. Nebl T. Sanders P.R. Wickham M.E. McElwain T.F. de Koning-Ward T.F. Crabb B.S. Mol. Microbiol. 2008; 68: 124-138Crossref PubMed Scopus (23) Google Scholar, 21Meissner M. Krejany E. Gilson P.R. de Koning-Ward T.F. Soldati D. Crabb B.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2980-2985Crossref PubMed Scopus (92) Google Scholar), called pTM2HA and pTM2GFP to create gene fusions with a double HA epitope and GFP, respectively, called pPfGAPM1/2/3-HA and pPfGAPM1/2/3-GFP (supplemental Fig. S2). Tggapm1a, Tggapm2b, and Tggapm3 were excised from pGEM T-easy with BglII and AvrII and ligated into pCTCh3H to create a gene fusion with mCherry and the triple HA epitope called pTggapm1/2/3-ChFP-HA (supplemental Fig. S2). pCTCh3H is a derivative of pCTG in which GFP has been replaced with the mCherry/HA fusion (22van Dooren G.G. Tomova C. Agrawal S. Humbel B.M. Striepen B. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 13574-13579Crossref PubMed Scopus (148) Google Scholar). P. falciparum strain 3D7 parasites were transfected with either 100 μg of pPfGAPM1/2/3-HA or pPfGAPM1/2/3-GFP and cultured continuously (23Trager W. Jensen J.B. Science. 1976; 193: 673-675Crossref PubMed Scopus (6158) Google Scholar). Transfectants were selected with 10 nm WR99210 (24Crabb B.S. Rug M. Gilberger T.W. Thompson J.K. Triglia T. Maier A.G. Cowman A.F. Methods Mol. Biol. 2004; 270: 263-276PubMed Google Scholar). T. gondii strain-RH parasites were transfected with pTgGAPM1/2/3-ChFP-HA plasmids using standard conditions (25Striepen B. He C.Y. Matrajt M. Soldati D. Roos D.S. Mol. Biochem. Parasitol. 1998; 92: 325-338Crossref PubMed Scopus (164) Google Scholar) and cultured in human foreskin fibroblasts and vero cells. Addition of 20 μm chloramphenicol to cultures permitted selection of stable transgenic parasites. Schizont stage P. falciparum parasites were treated with 0.15% saponin in RPMI media to release hemoglobin from the red blood cells. The hemoglobin was removed by washing the parasite pellet three times in cold phosphate-buffered saline. Parasites were solubilized at room temperature for 10 min in either 1% Triton X-100, RIPA (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 25 mm Tris-HCl, pH 8, in phosphate-buffered saline), 2% SDS, or two-dimensional sample buffer (7 m urea, 2 m thiourea, 2% ASB-14). Soluble proteins were separated from the insoluble by centrifugation in a microcentrifuge at 13,000 rpm for 10 min. A portion of the soluble fractions as well as the insoluble pellet fractions (washed in phosphate-buffered saline) were incubated in two-dimensional sample buffer overnight at room temperature to further extract the GAPMs. Prior to electrophoresis protein samples were mixed with reducing SDS-PAGE sample buffer at 1× concentration (0.05 m Tris-HCl, pH 6.8, 10% glycerol, 2 mm EDTA, 2% SDS, 0.05% bromphenol blue, 100 mm dithiothreitol). Samples not containing urea were heated at 80 °C for 10 min prior to SDS-PAGE. T. gondii tachyzoites were solubilized by sonication in RIPA buffer and soluble fractions were subsequently treated with either reducing SDS-PAGE sample buffer or reducing two-dimensional sample buffer. Samples were electrophoresed in pre-cast 4–12% acrylamide gradient bis-tris gels (Invitrogen) and blotted onto nitrocellulose or polyvinylidene difluoride prior to probing with specific primary antibodies; mouse anti-HA (mAb 12CA5) (1:500), rabbit anti-ALV repeat (ARILKPLIQEKIVEIMKPEIEEKIIEVPQVQYIEKLVEVPHVILQEKLIHIPKPVIHERIKKCSKTIFQEKIVEVPQIKVVDKIVEVPQYVYQEKIIEVPKIMVQERIIPVPKKIVKEKIVEIPQIELKNIDIEKVQEIPEYIPE, 1:100), rabbit anti-GFP (a gift from Emanuella Handman, 1:500), rabbit anti-PfGAP45 (1:200) (26Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), rabbit anti-PfGAP50 (1:100) (26Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), rabbit anti-aldolase (1:500) (26Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), rabbit anti-SAG1 (1:1000) (a gift from David Sibley), or rabbit anti-TgGAP45 (1:2000) (a gift from Con Beckers). 700 and 800 nm secondary antibodies were all from Rockland Immunochemicals. Bound antibody probes were detected with LiCor Odyssey infrared imager. Live cell microscopy was performed on P. falciparum transfectants expressing PfGAPM1/2-GFP and T. gondii RH-strain transfectants expressing TgGAPM1/2/3-ChFP-HA stained with 1 mm 4,6-diamidino-2-phenylindole. For immunofluorescence microscopy, PfGAPM1/3-HA parasites were fixed in either ice-cold acetone/methanol or 4% paraformaldehyde and probed with the following primary antibody combinations; mouse anti-HA (mAb 12CA5, 1:50) and either rabbit anti-GAP45 (1:50) (26Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar) or rabbit anti-MSP119 (1:50). Secondary antibodies were Alexa Fluor 568 nm goat anti-mouse IgG (1:2000, Molecular Probes) and Alexa Fluor 488 nm goat anti-rabbit IgG (1:2000, Molecular Probes). TgGAPM-ChFP-HA transfectants were treated with Clostridium septicum α-toxin as previously described (27Wichroski M.J. Melton J.A. Donahue C.G. Tweten R.K. Ward G.E. Infect. Immun. 2002; 70: 4353-4361Crossref PubMed Scopus (61) Google Scholar). Treated parasites were fixed in 4% paraformaldehyde and probed with mouse anti-HA mAb 12CA5 (1:200) and rabbit anti-SAG1 (1:200) (a gift from David Sibley). For co-immunoprecipitation assays, saponin-lysed PfGAPM1-HA, PfGAPM2-GFP, PfGAPM3-HA, and control 3D7 parasites as well as TgGAPM1/2/3-HA-ChFP and control RH strain parasites (100 μl pellets), were solubilized by sonication in RIPA buffer (2 ml) with Complete protease inhibitor mixture (Roche Applied Science). Insoluble material was pelleted, and either goat-polyclonal anti-HA agarose beads (AbCam, 50–100 μl) or rat anti-GFP-agarose beads (Medical and Biological Laboratories, 50–100 μl) were added to the supernatant, and the mixture was incubated at 4 °C for 3–4 h. For immunoprecipitation assays with parasite-specific antibodies, soluble 1% Triton X-100 lysates of the PfGAPM1-HA, PfGAPM2-GFP, and PfGAPM3-HA parasites were prepared as described above. Rabbit anti-PfGAP45 (10 μl) or rabbit anti-ALV repeat (10 μl) was added to the lysate for 16 h at 4 °C. Sheep anti-rabbit Dynal beads (Invitrogen) were added to the lysates, and the mixture was incubated at 4 °C for 3 h. Bound proteins were eluted, reduced in two-dimensional sample buffer or 2% SDS, and analyzed by Western blotting as described as above. Saponin-lysed P. falciparum PfGAPM1-HA, PfGAPM2-GFP, or PfGAPM3-HA schizont stage parasites were prepared as described above (∼20-μl pellets) and resuspended in 0.4 ml of phosphate-buffered saline containing 0.5 mm dithiobis succinimidyl propionate for 30 min. Cross-linkers were later quenched in 100 mm NaCl, 25 mm Tris, pH 7.5. Cross-linked pellets were resuspended in two-dimensional sample buffer, and soluble fractions were subsequently either reduced (addition of 100 mm dithiothreitol) or left non-reduced. Two hypothetical proteins PfGAPM1 (MAL13P1.130) and PfGAPM2 (PFD1110w), with six transmembrane domains were previously identified in DRMs extracted from developing P. falciparum schizont/merozoite lysates (17Sanders P.R. Gilson P.R. Cantin G.T. Greenbaum D.C. Nebl T. Carucci D.J. McConville M.J. Schofield L. Hodder A.N. Yates 3rd, J.R. Crabb B.S. J. Biol. Chem. 2005; 280: 40169-40176Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 18Sanders P.R. Cantin G.T. Greenbaum D.C. Gilson P.R. Nebl T. Moritz R.L. Yates 3rd, J.R. Hodder A.N. Crabb B.S. Mol. Biochem. Parasitol. 2007; 154: 148-157Crossref PubMed Scopus (71) Google Scholar). Alignment of the amino acid sequences of these proteins revealed a low to moderate degree of similarity (29.9% similar and 18.1% identical) (Fig. 1A). Sequence similarity searches of the Plasmodium genome data base (28Aurrecoechea C. Brestelli J. Brunk B.P. Dommer J. Fischer S. Gajria B. Gao X. Gingle A. Grant G. Harb O.S. Heiges M. Innamorato F. Iodice J. Kissinger J.C. Kraemer E. Li W. Miller J.A. Nayak V. Pennington C. Pinney D.F. Roos D.S. Ross C. Stoeckert Jr., C.J. Treatman C. Wang H. Nucleic Acids Res. 2009; 37: D539-D543Crossref PubMed Scopus (798) Google Scholar) indicated that P. falciparum encoded an additional member of the family termed PfGAPM3 (PlasmoDB: PF14_0065, GenBankTM: AAN36677). All three proteins were predicted by TMHMM (29Krogh A. Larsson B. von Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9080) Google Scholar) to have six transmembrane domains with the N- and C-terminal tails and loops 2 and 4 facing the cytoplasmic side of the membrane for the GAPM1 and -2 and the opposite orientation for GAPM3 (Fig. 1B). Subsequent searches of the Apicomplexan genome data base (30Aurrecoechea C. Heiges M. Wang H. Wang Z. Fischer S. Rhodes P. Miller J. Kraemer E. Stoeckert Jr., C.J. Roos D.S. Kissinger J.C. Nucleic Acids Res. 2007; 35: D427-D430Crossref PubMed Scopus (85) Google Scholar) revealed that most other Apicomplexan genomes also encode three of the GAPM proteins. Phylogenetic analysis of their sequences indicated that each of these proteins falls into one of three distinct orthologous groups, which we have called GAPM1, GAPM2, and GAPM3 (Fig. 1B). Interestingly, T. gondii encodes five GAPMs, two each in groups GAPM1 (TgGAPM1a and TgGAPM1b; GenBankTM: EEB00395 and EEB00396) and GAPM2 (TgGAPM2a and TgGAPM2b; GenBankTM: EEB03551 and EEB00710) and one GAPM3 protein (TgGAPM3; GenBankTM: EEA98700) (Fig. 1). Protein similarity searches of GenBankTM revealed no other obvious homologs of the GAPM family outside the Apicomplexan phylum. GAPM sequence alignment revealed interesting patterns of amino acid conservation across the entire GAPM family as well as within the three groups (Fig. 1 and supplemental Fig. S1). Inspection of the residues forming the five short loops separating the six transmembrane domains reveals that loops two and four are highly conserved across all Apicomplexa. Comparatively, loops 1, 3, and 5 are less conserved. Such a high level of conservation in loops 2 and 4 suggests that these residues may assist in maintaining overall GAPM integrity or facilitating interactions with other proteins. Interestingly, the N and C termini appear to be highly conserved within but not between each of the three GAPM groups and may potentially determine group specific functions. To facilitate fluorescent and immunodetection of GAPMs in the absence of antibodies to the endogenous proteins, members from each of the three groups in both P. falciparum and T. gondii were tagged at their C terminus with either green fluorescent protein (GFP), cherry fluorescent protein (ChFP) (31Giepmans B.N. Adams S.R. Ellisman M.H. Tsien R.Y. Science. 2006; 312: 217-224Crossref PubMed Scopus (2314) Google Scholar) and/or a hemagglutinin epitope tag (HA). For tagging the GAPMs in P. falciparum, cDNA sequences were ligated in front and in-frame with the sequences of GFP or a double HA tag. The gene fusions were inserted into a modified version of the pTGFP-GPI plasmid (21Meissner M. Krejany E. Gilson P.R. de Koning-Ward T.F. Soldati D. Crabb B.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2980-2985Crossref PubMed Scopus (92) Google Scholar) (supplemental Fig. S2). In this plasmid system the expression of gene fusions can be suppressed by the addition of the tetracycline analogue anhydrotetracycline and induced by removing the drug. Once induced, the gene fusions come under transcriptional control of a transactivator protein (TATi2) whose expression is in turn regulated by a schizont blood stage merozoite surface protein 2 promoter (20Gilson P.R. O'Donnell R.A. Nebl T. Sanders P.R. Wickham M.E. McElwain T.F. de Koning-Ward T.F. Crabb B.S. Mol. Microbiol. 2008; 68: 124-138Crossref PubMed Scopus (23) Google Scholar, 21Meissner M. Krejany E. Gilson P.R. de Koning-Ward T.F. Soldati D. Crabb B.S. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 2980-2985Crossref PubMed Scopus (92) Google Scholar). Examination of the microarray gene transcription profile of the Pfgapms confirmed the endogenous genes were also expressed during schizogony (32Bozdech Z. Llinás M. Pulliam B.L. Wong E.D. Zhu J. DeRisi J.L. PLoS Biol. 2003; 1: E5Crossref PubMed Scopus (1260) Google Scholar, 33Le Roch K.G. Johnson J.R. Florens L. Zhou Y. Santrosyan A. Grainger M. Yan S.F. Williamson K.C. Holder A.A. Carucci D.J. Yates 3rd, J.R. Winzeler E.A. Genome Res. 2004; 14: 2308-2318Crossref PubMed Scopus (357) Google Scholar). Once parasite lines transfected with the Pfgapm gene fusion plasmids were established, anhydrotetracycline was removed and those expressing the GFP fusion proteins were examined by microscopy. GFP fluorescence was only observed for the PfGAPM1-GFP and PfGAPM2-GFP fusions and initially only in a low percentage of schizont and merozoite stage parasites. Over several weeks however, this percentage increased greatly even in the presence of anhydrotetracycline, which should have silenced expression. This indicated that the plasmids containing the PfGAPM-GFP fusion may have recombined into the Pfgapm chromosomal locus tagging the endogenous gene with GFP. Southern blot analysis of PfGAMP2-GFP parasites confirmed this (supplemental Fig. S2). The PfGAPM1-GFP parasites grew poorly and no further work was done with this line. Our attention instead turned to the HA-tagged PfGAPM transfectants. These were examined by immunofluorescence microscopy with a HA monoclonal antibody (12CA5 mAb; used for all subsequent experiments) after several weeks in culture. Most schizont/merozoites expressed the PfGAPM1-HA fusion protein and Southern blot analysis also confirmed integration into the endogenous locus (supplemental Fig. S2). PfGAPM3-HA-expressing parasites were similarly produced, but integration was not confirmed by Southern blot analysis. Although transfected lines containing the PfGAPM2-HA and PfGAPM3-GFP plasmids were generated, the resultant lines did not express detectable levels of fusion proteins possibly due to some unforeseen deleterious effects. To determine in which cellular membrane the GAPMs resided, P. falciparum schizonts were examined by immunofluorescence microscopy. In Plasmodium asexual blood stages, cell replication occurs by schizogony where several rounds of nuclear division and associated organelle development occur within a common cytoplasm bound by a single plasma membrane. Daughter merozoites are formed late in schizogony when cytokinesis pinches off the individual merozoites. We microscopically examined immature PfGAPM1-HA and PfGAPM3-HA transgenic schizonts, because the multiple forming IMCs are distinct from the single bounding plasma membrane. Parasites were labeled with anti-HA and either the IMC-specific PfGAP45 rabbit antibody or parasite plasma membrane-specific rabbit antibody for the C-terminal domain of merozoite surface protein 1 (MSP119). The localization pattern of PfGAPM1-HA and PfGAPM3-HA was indistinguishable from that of PfGAP45 and markedly distinct from that of known plasma membrane protein MSP1 (Fig. 2A and supplemental Fig. S4). Additionally, live fluorescence microscopy of PfGAPM1-GFP and PfGAPM2-GFP transgenic schizonts demonstrated an identical IMC expression pattern to that of PfGAPM1-HA and PfGAPM3-HA throughout schizogony and a developmental series of images is shown in supplemental Fig. S3. To ascertain if the GAPMs localize to the
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