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• W2756164895 abstract "Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. Myosin A (MyoA) is a Class XIV myosin implicated in gliding motility and host cell and tissue invasion by malaria parasites. MyoA is part of a membrane-associated protein complex called the glideosome, which is essential for parasite motility and includes the MyoA light chain myosin tail domain–interacting protein (MTIP) and several glideosome-associated proteins (GAPs). However, most studies of MyoA have focused on single stages of the parasite life cycle. We examined MyoA expression throughout the Plasmodium berghei life cycle in both mammalian and insect hosts. In extracellular ookinetes, sporozoites, and merozoites, MyoA was located at the parasite periphery. In the sexual stages, zygote formation and initial ookinete differentiation precede MyoA synthesis and deposition, which occurred only in the developing protuberance. In developing intracellular asexual blood stages, MyoA was synthesized in mature schizonts and was located at the periphery of segmenting merozoites, where it remained throughout maturation, merozoite egress, and host cell invasion. Besides the known GAPs in the malaria parasite, the complex included GAP40, an additional myosin light chain designated essential light chain (ELC), and several other candidate components. This ELC bound the MyoA neck region adjacent to the MTIP-binding site, and both myosin light chains co-located to the glideosome. Co-expression of MyoA with its two light chains revealed that the presence of both light chains enhances MyoA-dependent actin motility. In conclusion, we have established a system to study the interplay and function of the three glideosome components, enabling the assessment of inhibitors that target this motor complex to block host cell invasion. Three stages of the malaria parasite, sporozoites, ookinetes, and merozoites, are invasive to host cells or tissue, and two stages, sporozoites and ookinetes, are motile. Motility and invasion are active processes, and in Plasmodium as in other apicomplexan parasites, such as Toxoplasma gondii, an actomyosin motor is central to these processes (reviewed in Refs. 1Keeley A. Soldati D. The glideosome: a molecular machine powering motility and host-cell invasion by Apicomplexa.Trends Cell Biol. 2004; 14: 528-532Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar and 2Boucher L.E. Bosch J. The apicomplexan glideosome and adhesins: structures and function.J. Struct. Biol. 2015; 190: 93-114Crossref PubMed Scopus (66) Google Scholar). Myosin proteins are typically composed of a conserved globular head domain, a neck domain comprising variable numbers of myosin light chain-binding sites, and a diverse tail domain that is often responsible for dimerization and/or cargo binding. The myosin head binds to actin and also contains the motor activity, where ATP hydrolysis is translated into molecular movement (3Krendel M. Mooseker M.S. Myosins: tails (and heads) of functional diversity.Physiology. 2005; 20: 239-251Crossref PubMed Scopus (282) Google Scholar4Syamaladevi D.P. Spudich J.A. Sowdhamini R. Structural and functional insights on the myosin superfamily.Bioinform. Biol. Insights. 2012; 6: 11-21Crossref PubMed Scopus (23) Google Scholar, 5Hartman M.A. Spudich J.A. The myosin superfamily at a glance.J. Cell Sci. 2012; 125: 1627-1632Crossref PubMed Scopus (181) Google Scholar6Foth B.J. Goedecke M.C. Soldati D. New insights into myosin evolution and classification.Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 3681-3686Crossref PubMed Scopus (372) Google Scholar). Myosin A (MyoA), 8The abbreviations used are: MyoAmyosin AACNacetonitrileELCessential light chainGAPglideosome-associated proteinIMCinner membrane complexMTIPmyosin tail domain interacting proteinTCEPtris(2-carboxyethyl)phosphinemant-ADP2′-/3′-O-(N′-methylanthraniloyl)-ADPMLCmyosin light chainRLCregulatory light chainBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolDICdifferential interference-contrast. an Apicomplexa-specific class XIV myosin composed of a head and neck region, but lacking a tail domain, is the motor component of the glideosome protein complex that is attached to the inner membrane complex (IMC), a network of large, flattened vesicles located just below the surface plasma membrane of the parasite (7Heintzelman M.B. Schwartzman J.D. A novel class of unconventional myosins from Toxoplasma gondii.J. Mol. Biol. 1997; 271: 139-146Crossref PubMed Scopus (93) Google Scholar, 8Dobrowolski J.M. Carruthers V.B. Sibley L.D. Participation of myosin in gliding motility and host cell invasion by Toxoplasma gondii.Mol. Microbiol. 1997; 26: 163-173Crossref PubMed Scopus (200) Google Scholar9Pinder J.C. Fowler R.E. Dluzewski A.R. Bannister L.H. Lavin F.M. Mitchell G.H. Wilson R.J. Gratzer W.B. Actomyosin motor in the merozoite of the malaria parasite, Plasmodium falciparum: implications for red cell invasion.J. Cell Sci. 1998; 111: 1831-1839Crossref PubMed Google Scholar). myosin A acetonitrile essential light chain glideosome-associated protein inner membrane complex myosin tail domain interacting protein tris(2-carboxyethyl)phosphine 2′-/3′-O-(N′-methylanthraniloyl)-ADP myosin light chain regulatory light chain 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol differential interference-contrast. Some components of the glideosome have been defined in T. gondii and also in Plasmodium species. In addition to MyoA, there is a myosin light chain (MLC) that binds to the last few amino acid residues of MyoA, corresponding to part of the myosin neck region and called T. gondii MLC1 (10Herm-Götz A. Weiss S. Stratmann R. Fujita-Becker S. Ruff C. Meyhöfer E. Soldati T. Manstein D.J. Geeves M.A. Soldati D. Toxoplasma gondii myosin A and its light chain: a fast, single-headed, plus-end-directed motor.EMBO J. 2002; 21: 2149-2158Crossref PubMed Scopus (194) Google Scholar), or myosin tail domain–interacting protein (MTIP) in Plasmodium sp. (11Bergman L.W. Kaiser K. Fujioka H. Coppens I. Daly T.M. Fox S. Matuschewski K. Nussenzweig V. Kappe S.H.I. Myosin A tail domain interacting protein (MTIP) localizes to the inner membrane complex of Plasmodium sporozoites.J. Cell Sci. 2003; 116: 39-49Crossref PubMed Scopus (157) Google Scholar). This MLC is involved in anchoring MyoA to the IMC via a palmitoyl modification (12Jones M.L. Collins M.O. Goulding D. Choudhary J.S. Rayner J.C. Analysis of protein palmitoylation reveals a pervasive role in Plasmodium development and pathogenesis.Cell Host Microbe. 2012; 12: 246-258Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) and thus appears to take on a role usually fulfilled by a myosin tail (i.e. anchoring myosin at a specific subcellular location). Recently, two more T. gondii MyoA MLCs have been described and named essential light chains 1 and 2 (ELC1 and -2). The terminology follows that used for mammalian muscle myosins with two light chains in which the most proximal is an ELC and the distal is a regulatory light chain (RLC). ELC1 and -2 have been shown to bind to the MyoA neck adjacent to MLC1 (which binds at the RLC site) in a mutually exclusive manner and are important for motor activity (13Nebl T. Prieto J.H. Kapp E. Smith B.J. Williams M.J. Yates 3rd, J.R. Cowman A.F. Tonkin C.J. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.PLoS Pathog. 2011; 7: e1002222Crossref PubMed Scopus (74) Google Scholar, 14Williams M.J. Alonso H. Enciso M. Egarter S. Sheiner L. Meissner M. Striepen B. Smith B.J. Tonkin C.J. Two essential light chains regulate the MyoA lever arm to promote toxoplasma gliding motility.mBio. 2015; 6: e00845-15Crossref PubMed Scopus (32) Google Scholar). Bookwalter et al. (15Bookwalter C.S. Kelsen A. Leung J.M. Ward G.E. Trybus K.M. A Toxoplasma gondii Class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.J. Biol. Chem. 2014; 289: 30832-30841Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) have demonstrated that a heterologously expressed TgMyoA requires both MLC1 and an ELC in order to move actin at its fastest speed. In addition to MyoA and its associated light chains, a number of glideosome-associated proteins (GAPs) have been described, including GAP45 (16Gaskins E. Gilk S. DeVore N. Mann T. Ward G. Beckers C. Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii.J. Cell Biol. 2004; 165: 383-393Crossref PubMed Scopus (206) Google Scholar, 17Baum J. Richard D. Healer J. Rug M. Krnajski Z. Gilberger T.W. Green J.L. Holder A.A. Cowman A.F. A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites.J. Biol. Chem. 2006; 281: 5197-5208Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar18Rees-Channer R.R. Martin S.R. Green J.L. Bowyer P.W. Grainger M. Molloy J.E. Holder A.A. Dual acylation of the 45kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites.Mol. Biochem. Parasitol. 2006; 149: 113-116Crossref PubMed Scopus (86) Google Scholar) (and its Coccidia-specific homolog GAP70 (19Frénal K. Polonais V. Marq J.-B. Stratmann R. Limenitakis J. Soldati-Favre D. Functional dissection of the apicomplexan glideosome molecular architecture.Cell Host Microbe. 2010; 8: 343-357Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar)), GAP50, and a family of three proteins with multiple membrane spans known as GAPMs (20Bullen H.E. Tonkin C.J. O'Donnell R.A. Tham W.H. Papenfuss A.T. Gould S. Cowman A.F. Crabb B.S. Gilson P.R. A novel family of apicomplexan glideosome-associated proteins with an inner membrane-anchoring role.J. Biol. Chem. 2009; 284: 25353-25363Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Another glideosome component, GAP40, has been described in T. gondii (19Frénal K. Polonais V. Marq J.-B. Stratmann R. Limenitakis J. Soldati-Favre D. Functional dissection of the apicomplexan glideosome molecular architecture.Cell Host Microbe. 2010; 8: 343-357Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar) and has a homologue in Plasmodium based on genomic (21Gardner M.J. Hall N. Fung E. White O. Berriman M. Hyman R.W. Carlton J.M. Pain A. Nelson K.E. Bowman S. Paulsen I.T. James K. Eisen J.A. Rutherford K. Salzberg S.L. et al.Genome sequence of the human malaria parasite Plasmodium falciparum.Nature. 2002; 419: 498-511Crossref PubMed Scopus (3443) Google Scholar) and proteomic studies (22Lasonder E. Janse C.J. van Gemert G.-J. Mair G.R. Vermunt A.M.W. Douradinha B.G. van Noort V. Huynen M.A. Luty A.J.F. Kroeze H. Khan S.M. Sauerwein R.W. Waters A.P. Mann M. Stunnenberg H.G. Proteomic profiling of Plasmodium sporozoite maturation identifies new proteins essential for parasite development and infectivity.PLoS Pathog. 2008; 4: e1000195Crossref PubMed Scopus (169) Google Scholar, 23Treeck M. Sanders J.L. Elias J.E. Boothroyd J.C. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries.Cell Host Microbe. 2011; 10: 410-419Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar24Bowyer P.W. Simon G.M. Cravatt B.F. Bogyo M. Global profiling of proteolysis during rupture of Plasmodium falciparum from the host erythrocyte.Mol. Cell. Proteomics. 2011; 10.1074/mcp.M110.001636Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar), but it has not been shown experimentally to be part of the glideosome in this genus. In Plasmodium, most studies on MyoA have been limited to the asexual blood stage parasites in the mammalian host, although its essential role in gliding motility in P. berghei ookinetes has been demonstrated in promoter swap experiments (25Siden-Kiamos I. Ganter M. Kunze A. Hliscs M. Steinbüchel M. Mendoza J. Sinden R.E. Louis C. Matuschewski K. Stage-specific depletion of myosin A supports an essential role in motility of malarial ookinetes.Cell Microbiol. 2011; 13: 1996-2006Crossref PubMed Scopus (33) Google Scholar). In this study, we have examined the expression and location of MyoA throughout the malaria parasite life cycle, including all invasive stages, and its temporal profile during merozoite and ookinete development. We have examined the composition of the glideosome in asexual blood stages by immunoprecipitation and mass spectrometry of proteins associated with GFP-tagged MyoA, confirming the presence of GAP40 and identifying a new myosin light chain. This essential-type light chain binds to the MyoA neck adjacent to, and in addition to, MTIP. Recombinant PfMyoA binds actin and with both light chains bound demonstrates increased sliding velocity of actin compared with MyoA with just MTIP bound. This is a first important step to reconstituting the Plasmodium glideosome in vitro, which will be an invaluable system with which to dissect the function of, and interplay between, different components of the complex. Expression of the myoA gene was detected by quantitative RT-PCR throughout the parasite life cycle, particularly in schizonts and sporozoites but also in non-activated gametocytes (Fig. 1A). To examine the synthesis and location of the protein throughout the parasite life cycle, we produced a C-terminal GFP-tagged MyoA expressed from the endogenous genomic locus using a strategy we described recently for Plasmodium falciparum MyoA and Plasmodium berghei MyoB (26Yusuf N.A. Green J.L. Wall R.J. Knuepfer E. Moon R.W. Schulte-Huxel C. Stanway R.R. Martin S.R. Howell S.A. Douse C.H. Cota E. Tate E.W. Tewari R. Holder A.A. The Plasmodium class XIV myosin, MyoB, has a distinct subcellular location in invasive and motile stages of the malaria parasite and an unusual light chain.J. Biol. Chem. 2015; 290: 12147-12164Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) (Fig. 1B). Integration of sequence coding for GFP into the endogenous MyoA locus and expression of the tagged protein was confirmed by PCR amplification from genomic DNA using primers that only produce a product following specific integration (Fig. 1C), by Western blotting (Fig. 1D), and by fluorescence microscopy (Fig. 1, F–H). Tagging MyoA with GFP resulted in no detectable phenotype; there was no effect on the rate of asexual parasite growth, on male gametogenesis (the number of exflagellation centers), the number of oocysts per mosquito gut, or the efficiency of infection via sporozoite inoculation (as judged by the number of days following infected mosquito bite that parasites were observed by microscopy in blood smears). In a subcellular fractionation of schizonts, the protein was largely associated with the peripheral membrane (carbonate-soluble) and membrane (carbonate-insoluble) fractions rather than in the soluble cell lysate (Fig. 1E), consistent with its association with the glideosome and the IMC membrane compartments. Using live parasite microscopy, the protein was detected uniformly at the periphery of segmenting schizonts and extracellular merozoites in asexual blood stages and at a similar location in extracellular ookinetes and salivary gland sporozoites (Fig. 1F), consistent with location at the IMC. Interestingly, in sporozoites, there was also a clear perinuclear localization. In early cytomere stages of liver-stage schizonts, the signal appeared to be cytosolic, rather than membrane-associated. In late cytomere stages, 55 h after invasion by sporozoites, a clear peripheral location associated with the hepatic merozoites was observed (Fig. 1G). The presence of myoA mRNA in ookinete stages, as well as in non-activated gametocytes as a translationally repressed transcript, prompted us to examine the temporal profile of protein expression during the 24 h of ookinete development divided into six stages (27Janse C.J. Rouwenhorst R.J. Van der Klooster P.F. Van der Kaay H.J. Overdulve J.P. Development of Plasmodium berghei ookinetes in the midgut of Anopheles atroparvus mosquitoes and in vitro.Parasitology. 1985; 91: 219-225Crossref PubMed Scopus (29) Google Scholar) (Fig. 1H). MyoA-GFP was not visible in either activated or non-activated gametocytes and was barely detectable in the zygote and stage I and II ookinetes. Following the establishment of morphological polarity at about 10 h after fertilization (stage III), the protein was detectable in the growing protuberance rather than the spherical body in the intermediate retort forms, so-called because of their shape (stages III–V). This location was retained in later stages, with the MyoA-GFP associated with the parasite periphery in fully formed motile ookinetes. There was a concentration of fluorescence detected at the apical tip of the mature ookinete. Immunofluorescence using an antibody against the surface marker P28 was used to visualize the ookinete at all stages of development. The use of P. falciparum allows a more detailed analysis of asexual blood stages than can be achieved with P. berghei. MyoA-GFP was largely undetectable by live fluorescence microscopy until ∼38 h after merozoite invasion of an erythrocyte but was present in later stages at the periphery of developing segmented schizonts and free merozoites (but not associated with the food vacuole/residual body), consistent with its proposed location at the IMC (Fig. 2A). No GFP signal was detected in ring or trophozoite stages, indicating that the protein is not synthesized at this time, and any MyoA carried through with the invading merozoite is degraded rapidly following erythrocyte invasion. Interestingly, the protein was also not detected in early schizonts when the first formation of the IMC can already be detected with, for example, GFP-tagged GAP50 (28Yeoman J.A. Hanssen E. Maier A.G. Klonis N. Maco B. Baum J. Turnbull L. Whitchurch C.B. Dixon M.W.A. Tilley L. Tracking glideosome-associated protein-50 reveals the development and organization of the inner membrane complex of P. falciparum.Eukaryotic Cell. 2011; 10: 556-564Crossref PubMed Scopus (40) Google Scholar) or GAP45 (29Ridzuan M.A.M. Moon R.W. Knuepfer E. Black S. Holder A.A. Green J.L. Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development.PLoS One. 2012; 7: e33845Crossref PubMed Scopus (45) Google Scholar) and seen as small ring-shaped structures at the tips of developing merozoites. Western blot analysis of parasite extracts at the corresponding time points (Fig. 2B (i)) confirmed that GAP50 is present in early schizonts (detectable from 24 h and strongly expressed 30 h postinvasion). Although a small amount of MyoA-GFP can be detected 27–36 h after merozoite invasion, the strongest expression is detected from 38 h postinvasion. A Western blot analysis of the MyoA-GFP immunoprecipitate obtained from these extracts (Fig. 2B (ii)) indicates that known components of the glideosome complex, MTIP, GAP45, and GAP50, are associated with MyoA from 38 h after erythrocyte invasion by a merozoite. Tagging MyoA with GFP had no effect on the rate of asexual blood stage parasite growth. The peripheral location of MyoA-GFP in mature schizonts and free merozoites was confirmed by an indirect immunofluorescence assay (Fig. 3A). Antibodies against GFP co-localized with antibodies against MTIP, GAP45, and GAP50, consistent with the presence of these proteins in a complex. During erythrocyte invasion, the peripheral location of MyoA did not change during the transition from initial attachment of the merozoite to the intracellular ring stage at the completion of invasion (Fig. 3B). However, thereafter, the signal disappeared until resynthesis during schizogony. A number of proteins associated with the glideosome have been identified in both Plasmodium and Toxoplasma species, and the presence of GAP45, GAP50, and MTIP, some of the known components in P. falciparum asexual blood stages, has been confirmed above. To examine what other proteins might be associated, we examined further the protein complex immunoprecipitated with GFP-specific antibodies. To do this, we first used parasites in which GAP45 was tagged internally with GFP (29Ridzuan M.A.M. Moon R.W. Knuepfer E. Black S. Holder A.A. Green J.L. Subcellular location, phosphorylation and assembly into the motor complex of GAP45 during Plasmodium falciparum schizont development.PLoS One. 2012; 7: e33845Crossref PubMed Scopus (45) Google Scholar) and then those in which MyoA was tagged at its C terminus with GFP. In the first instance, we were interested to see whether or not GAP40, which is a highly phosphorylated protein (23Treeck M. Sanders J.L. Elias J.E. Boothroyd J.C. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries.Cell Host Microbe. 2011; 10: 410-419Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar, 30Lasonder E. Green J.L. Grainger M. Langsley G. Holder A.A. Extensive differential protein phosphorylation as intraerythrocytic Plasmodium falciparum schizonts develop into extracellular invasive merozoites.Proteomics. 2015; 15: 2716-2729Crossref PubMed Scopus (46) Google Scholar, 31Pease B.N. Huttlin E.L. Jedrychowski M.P. Talevich E. Harmon J. Dillman T. Kannan N. Doerig C. Chakrabarti R. Gygi S.P. Chakrabarti D. Global analysis of protein expression and phosphorylation of three stages of Plasmodium falciparum intraerythrocytic development.J. Proteome Res. 2013; 12: 4028-4045Crossref PubMed Scopus (119) Google Scholar32Solyakov L. Halbert J. Alam M.M. Semblat J.-P. Dorin-Semblat D. Reininger L. Bottrill A.R. Mistry S. Abdi A. Fennell C. Holland Z. Demarta C. Bouza Y. Sicard A. Nivez M.-P. et al.Global kinomic and phospho-proteomic analyses of the human malaria parasite Plasmodium falciparum.Nat. Commun. 2011; 2: 565Crossref PubMed Scopus (260) Google Scholar) present in the T. gondii glideosome (19Frénal K. Polonais V. Marq J.-B. Stratmann R. Limenitakis J. Soldati-Favre D. Functional dissection of the apicomplexan glideosome molecular architecture.Cell Host Microbe. 2010; 8: 343-357Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), is present in the Plasmodium complex. To visualize phosphorylated proteins associated with GFP-GAP45, we metabolically labeled P. falciparum schizont phosphoproteins using [32P]phosphate (Fig. 4). As a control, 3D7 schizonts in which GAP45 was unmodified were used (Fig. 4, lane 1 in A and B). The glideosome was precipitated from schizont lysates using antibodies to GFP, and 32P-labeled phosphoproteins were visualized by autoradiography (Fig. 4A). In addition to GFP-GAP45 itself, there were five labeled proteins that were absent from the control samples. The identities of some were confirmed by immunoprecipitation from schizont lysates with GFP antibodies followed by Western blotting with antibodies against known glideosome components MyoA, GAP45, GAP50, and MTIP (Fig. 4A). The most prominent of the phosphoproteins, with an apparent molecular mass of just under 50 kDa, was distinct from these four proteins, so its identity was examined further by mass spectroscopy following fractionation of the protein precipitate by SDS-PAGE and tryptic digestion of specific bands (Fig. 4, B and C). This analysis clearly identified the protein as GAP40 and confirmed the presence of GAP45 and GAP50 in the immunoprecipitate. The high-molecular weight phosphoprotein, marked with an asterisk in Fig. 4A, was not detected in the SYPRO Ruby-stained immunoprecipitate. High-level phosphorylation of a low-abundance protein is one possible explanation for this discrepancy. An immunoprecipitate from parasites expressing GFP-tagged MyoA was also analyzed by tryptic digestion, with LC-MS/MS analysis of resultant peptides to identify associated proteins. Again, 3D7 schizonts in which MyoA was unmodified were used as a negative control. Table 1 shows the proteins identified only in the MyoA-GFP precipitate by the presence of two or more specific peptides. This approach confirmed the presence of MTIP, GAP50, GAP45, and GAP40. It also identified GAPM2 and GAPM3, which are two of the three GAPM proteins previously shown to associate with the glideosome and IMC (20Bullen H.E. Tonkin C.J. O'Donnell R.A. Tham W.H. Papenfuss A.T. Gould S. Cowman A.F. Crabb B.S. Gilson P.R. A novel family of apicomplexan glideosome-associated proteins with an inner membrane-anchoring role.J. Biol. Chem. 2009; 284: 25353-25363Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The absence of GAPM1 in the precipitate could be explained by the extremely detergent-resistant property of this protein (20Bullen H.E. Tonkin C.J. O'Donnell R.A. Tham W.H. Papenfuss A.T. Gould S. Cowman A.F. Crabb B.S. Gilson P.R. A novel family of apicomplexan glideosome-associated proteins with an inner membrane-anchoring role.J. Biol. Chem. 2009; 284: 25353-25363Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). In addition to these known or expected components of the glideosome, 13 additional proteins were identified; whereas it is likely that some of them represent co-purified contaminants, it cannot be excluded that some represent real components of the complex. For example, it is unlikely that signal peptide peptidase, MESA, and the hexose transporter are part of the glideosome because of their known or predicted subcellular location: the endoplasmic reticulum (33Marapana D.S. Wilson D.W. Zuccala E.S. Dekiwadia C.D. Beeson J.G. Ralph S.A. Baum J. Malaria parasite signal peptide peptidase is an ER-resident protease required for growth but not for invasion.Traffic. 2012; 13: 1457-1465Crossref PubMed Scopus (23) Google Scholar), the parasite-infected erythrocyte surface (34Coppel R.L. Lustigman S. Murray L. Anders R.F. MESA is a Plasmodium falciparum phosphoprotein associated with the erythrocyte membrane skeleton.Mol. Biochem. Parasitol. 1988; 31: 223-231Crossref PubMed Scopus (56) Google Scholar), and the parasite plasma membrane (35Woodrow C.J. Penny J.I. Krishna S. Intraerythrocytic Plasmodium falciparum expresses a high affinity facilitative hexose transporter.J. Biol. Chem. 1999; 274: 7272-7277Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), respectively. On the other hand, the protein encoded by PF3D7_1420200 is a tetratricopeptide repeat protein related to TgUNC, demonstrated to be a member of the UCS family of myosin-specific chaperones (15Bookwalter C.S. Kelsen A. Leung J.M. Ward G.E. Trybus K.M. A Toxoplasma gondii Class XIV myosin, expressed in Sf9 cells with a parasite co-chaperone, requires two light chains for fast motility.J. Biol. Chem. 2014; 289: 30832-30841Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In addition, of particular interest was the protein encoded by PF3D7_1017500, which is a 134-residue polypeptide with sequence homology to members of the EF-hand superfamily (36Gough J. Karplus K. Hughey R. Chothia C. Assignment of homology to genome sequences using a library of hidden Markov models that represent all proteins of known structure.J. Mol. Biol. 2001; 313: 903-919Crossref PubMed Scopus (927) Google Scholar) and reminiscent of myosin light chains and other calmodulin-like proteins. This protein is a candidate second light chain for MyoA, particularly because it has been proposed that there is “space” for a second light chain located adjacent to the MTIP-binding site (37Farrow R.E. Green J. Katsimitsoulia Z. Taylor W.R. Holder A.A. Molloy J.E. The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum.Semin. Cell Dev. Biol. 2011; 22: 953-960Crossref PubMed Scopus (26) Google Scholar), and additional light chains have been characterized for MyoA in T. gondii (13Nebl T. Prieto J.H. Kapp E. Smith B.J. Williams M.J. Yates 3rd, J.R. Cowman A.F. Tonkin C.J. Quantitative in vivo analyses reveal calcium-dependent phosphorylation sites and identifies a novel component of the Toxoplasma invasion motor complex.PLoS Pathog. 2011; 7: e1002222Crossref PubMed Scopus (74) Google Scholar, 1" @default.
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• W2756164895 date "2017-10-01" @default.
• W2756164895 modified "2023-10-17" @default.
• W2756164895 title "Compositional and expression analyses of the glideosome during the Plasmodium life cycle reveal an additional myosin light chain required for maximum motility" @default.
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• W2756164895 doi "https://doi.org/10.1074/jbc.m117.802769" @default.
• W2756164895 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/5663884" @default.
• W2756164895 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/28893907" @default.
• W2756164895 hasPublicationYear "2017" @default.

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