An actomyosin motor complex assembled below the parasite's plasma membrane drives erythrocyte invasion by Plasmodium falciparum merozoites. The complex is comprised of several proteins including myosin (MyoA), myosin tail domain interacting protein (MTIP) and glideosome associated proteins (GAP) 45 and 50, and is anchored on the inner membrane complex (IMC), which underlies the plasmalemma. A ternary complex of MyoA, MTIP and GAP45 is formed that then associates with GAP50. We show that full length GAP45 labelled internally with GFP is assembled into the motor complex and transported to the developing IMC in early schizogony, where it accumulates during intracellular development until merozoite release. We show that GAP45 is phosphorylated by calcium dependent protein kinase 1 (CDPK1), and identify the modified serine residues. Replacing these serine residues with alanine or aspartate has no apparent effect on GAP45 assembly into the motor protein complex or its subcellular location in the parasite. The early assembly of the motor complex suggests that it has functions in addition to its role in erythrocyte invasion.
References
[1]
World Health Organization (2011) 204 p. World Malaria Report 2010.
[2]
Bannister LH, Hopkins JM, Fowler RE, Krishna S, Mitchell GH (2000) A brief illustrated guide to the ultrastructure of Plasmodium falciparum asexual blood stages. Parasitol Today (Regul Ed) 16: 427–433.
[3]
Baum J, Gilberger T, Frischknecht F, Meissner M (2008) Host-cell invasion by malaria parasites: insights from Plasmodium and Toxoplasma. Trends Parasitol 24: 557–563. doi:10.1016/j.pt.2008.08.006.
[4]
Farrow RE, Green JL, Holder AA, Molloy JE (2011) The mechanism of erythrocyte invasion by the malarial parasite, Plasmodium falciparum. Seminars in cell & developmental biology. doi:10.1016/j.semcdb.2011.09.022.
[5]
Gaskins E, Gilk S, DeVore N, Mann T, Ward G, et al. (2004) Identification of the membrane receptor of a class XIV myosin in Toxoplasma gondii. J Cell Biol 165: 283–293.
[6]
Rees-Channer R, Martin SR, Green JL, Bowyer P, Grainger M, et al. (2006) Dual acylation of the 45 kDa gliding-associated protein (GAP45) in Plasmodium falciparum merozoites. Mol Biochem Parasitol 149: 113–116.
[7]
Yeoman JA, Hanssen E, Maier AG, Klonis N, Maco B, et al. (2011) Tracking glideosome-associated protein-50 reveals the development and organization of the inner membrane complex of P. falciparum. Eukaryotic Cell 10: 556–564. doi:10.1128/EC.00244-10.
[8]
Jones ML, Kitson EL, Rayner JC (2006) Plasmodium falciparum erythrocyte invasion: a conserved myosin associated complex. Mol Biochem Parasitol 147: 74–84. doi:10.1016/j.molbiopara.2006.01.009.
[9]
Green JL, Rees-Channer RR, Howell SA, Martin SR, Knuepfer E, et al. (2008) The Motor Complex of Plasmodium falciparum: phosphorylation by a calcium-dependent protein kinase. J Biol Chem 283: 30980–30989. doi:10.1074/jbc.M803129200.
[10]
Vaid A, Thomas DC, Sharma P (2008) Role of Ca2+/calmodulin-PfPKB signaling pathway in erythrocyte invasion by Plasmodium falciparum. J Biol Chem 283: 5589–5597. doi:10.1074/jbc.M708465200.
[11]
Billker O, Lourido S, Sibley LD (2009) Calcium-dependent signaling and kinases in apicomplexan parasites. Cell Host Microbe 5: 612–622. doi:10.1016/j.chom.2009.05.017.
[12]
Gilk SD, Gaskins E, Ward GE, Beckers CJM (2009) GAP45 phosphorylation controls assembly of the Toxoplasma myosin XIV complex. Eukaryotic Cell 8: 190–196. doi:10.1128/EC.00201-08.
[13]
Striepen B, Jordan CN, Reiff S, van Dooren GG (2007) Building the Perfect Parasite: Cell Division in Apicomplexa. PLoS Pathog 3: e78. doi:10.1371/journal.ppat.0030078.
[14]
Hu G, Cabrera A, Kono M, Mok S, Chaal BK, et al. (2010) Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum. Nature Biotechnology 28: 91–98. doi:10.1038/nbt.1597.
[15]
Bullen HE, Tonkin CJ, O'Donnell RA, Tham W-H, Papenfuss AT, et al. (2009) A Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role. J Biol Chem 284: 25353–25363. doi:10.1074/jbc.M109.036772.
[16]
Agop-Nersesian C, Naissant B, Ben Rached F, Rauch M, Kretzschmar A, et al. (2009) Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. PLoS Pathog 5: e1000270. doi:10.1371/journal.ppat.1000270.
[17]
Frénal K, Polonais V, Marq J-B, Stratmann R, Limenitakis J, et al. (2010) Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host Microbe 8: 343–357. doi:10.1016/j.chom.2010.09.002.
[18]
Johnson TM, Rajfur Z, Jacobson K, Beckers CJ (2007) Immobilization of the Type XIV Myosin Complex in Toxoplasma gondii. Mol Biol Cell. doi:10.1091/mbc.E07-01-0040.
[19]
Papakrivos J, Newbold CI, Lingelbach K (2004) A potential novel mechanism for the insertion of a membrane protein revealed by a biochemical analysis of the Plasmodium falciparum cytoadherence molecule PfEMP-1. Mol Microbiol 55: 1272–1284. doi:10.1111/j.1365-2958.2004.04468.x.
[20]
Treeck M, Sanders JL, Elias JE, Boothroyd JC (2011) The Phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii Reveal Unusual Adaptations Within and Beyond the Parasites' Boundaries. Cell Host Microbe 10: 410–419. doi:10.1016/j.chom.2011.09.004.
[21]
Matsumura F (2005) Regulation of myosin II during cytokinesis in higher eukaryotes. Trends Cell Biol 15: 371–377. doi:10.1016/j.tcb.2005.05.004.
[22]
Burgess DR (2005) Cytokinesis: new roles for myosin. Curr Biol 15: R310–1. doi:10.1016/j.cub.2005.04.008.
[23]
Field C, Li R, Oegema K (1999) Cytokinesis in eukaryotes: a mechanistic comparison. Curr Opin Cell Biol 11: 68–80.
[24]
Delbac F, Sanger A, Neuhaus E, Stratmann R, Ajioka J, et al. (2001) Toxoplasma gondii myosins B/C: one gene, two tails, two localizations, and a role in parasite division. J Cell Biol 155: 613–623.
[25]
Walliker D, Quakyi IA, Wellems TE, McCutchan TF, Szarfman A, et al. (1987) Genetic analysis of the human malaria parasite Plasmodium falciparum. Science 236: 1661–1666.
[26]
Lambros C, Vanderberg J (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65: 418–420.
[27]
Wu Y, Sifri CD, Lei HH, Su XZ, Wellems TE (1995) Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci USA 92: 973–977.
[28]
Fidock DA, Wellems TE (1997) Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil. Proc Natl Acad Sci USA 94: 10931–10936.
[29]
Hastie CJ, McLauchlan HJ, Cohen P (2006) Assay of protein kinases using radiolabeled ATP: a protocol. Nature protocols 1: 968–971. doi:10.1038/nprot.2006.149.
