Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa.
Toxoplasma gondii is an obligate intracellular parasite, and its sub-cellular organization shows clear adaptations to this life-style. In addition to organelles shared among all eukaryotes, the organism possesses a number of specialized compartments with important roles in host cell invasion and intra-cellular survival. These unique aspects of the parasite's biology are also reflected in its genome. The ongoing genome sequencing efforts for T. gondii and related apicomplexans predict a high proportion of genes unique to the phylum, which lack homologs in other model organisms. Knowing the sub-cellular localization of these gene products will be an important first step towards their functional characterization. We used a library approach wherein parasite genomic DNA was fused to the yellow fluorescent protein (YFP) gene. Parasites transformed with this library were screened by flow cytometry and fluorescence microscopy. Clones tagged in a wide variety of sub-cellular compartments (nucleus, mitochondria, ER, dense granules (secreted), spliceosome, plasma membrane, apicoplast, inner membrane complex) were isolated and confirmed using compartment specific markers. Clones with tags in parasite-specific localizations were subjected to insert rescue and phenotypic verification using an in vitro recombination system. Among the genes identified is a novel inner membrane complex gene (IMC3) conserved among Apicomplexa.
Center for Tropical and Emerging Global Diseases, University of Georgia, 724 Biological Sciences Building, Athens 30602, USA.
Marc-Jan
MJ
Gubbels
Marnix
M
Wieffer
Boris
B
Striepen
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry; D005434:Flow Cytometry; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D015388:Organelles / Q000737:chemistry; D010641:Phenotype; D021381:Protein Transport; D015800:Protozoan Proteins / Q000032:analysis* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D014162:Transfection
D001426:Bacterial Proteins; D016054:DNA, Protozoan; D008164:Luminescent Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria