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Eukaryotic Cell, October 2006, p. 1622-1634, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00114-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of PhIL1, a Novel Cytoskeletal Protein of the Toxoplasma gondii Pellicle, through Photosensitized Labeling with 5-[125I]Iodonaphthalene-1-Azide

Stacey D. Gilk,1 Yossef Raviv,2 Ke Hu,3,{dagger} John M. Murray,3 Con J. M. Beckers,4 and Gary E. Ward1*

Department of Microbiology and Molecular Genetics, 316 Stafford Hall, University of Vermont, Burlington, Vermont 05404,1 Intramural Research Support Program, SAIC, Center for Cancer Research Nanobiology Program, Bldg. 469, Room 213, National Cancer Institute, Frederick, Frederick, Maryland 21702,2 Department of Cell and Developmental Biology, 421 Curie Boulevard, University of Pennsylvania, Philadelphia, Pennsylvania 19104,3 Department of Cell and Developmental Biology, 109 Taylor Hall, University of North Carolina, Chapel Hill, Chapel Hill, North Carolina 275994

Received 19 April 2006/ Accepted 20 July 2006

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[125I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [125I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, 316 Stafford Hall, University of Vermont, Burlington, VT 05405. Phone: (802) 656-4868. Fax: (802) 656-8749. E-mail: Gary.Ward{at}uvm.edu.

{dagger} Present address: Department of Cell Biology, Scripps Research Institute CB-163, 10550 N. Torrey Pines Rd., La Jolla, CA 92037.

Eukaryotic Cell, October 2006, p. 1622-1634, Vol. 5, No. 10
1535-9778/06/$08.00+0     doi:10.1128/EC.00114-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gilk, S. D., Gaskins, E., Ward, G. E., Beckers, C. J. M. (2009). GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex. Eukaryot Cell 8: 190-196 [Abstract] [Full Text]  


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