Selective labelling of proteins synthesized by intracellular parasites using ricin and host cells lacking mitochondrial DNA.
Studies focused on the synthesis of developmentally regulated proteins by intracellular parasites have been limited due to the lack of a simple method for selectively labelling proteins produced by the parasite. A method has now been developed in which ricin, the toxin, is employed to selectively inhibit host cell protein synthesis while protein synthesis by the intracellular parasite is unaffected. Ricin is composed of two subunits, one of which binds to cell surface receptors containing terminal galactose residues while the other subunit enters the cell, inactivates ribosomes and, as a consequence, cytoplasmic protein synthesis. Due to the loss of the receptor-binding subunit, ricin cannot permeate the host cell mitochondria or the intracellular parasite, and therefore protein synthesis within these compartments continues uninterrupted. This system was explored using Eimeria tenella- and Toxoplasma gondii-infected avian rho0 cells. This host cell type was selected because it lacks mitochondrial DNA and supports the intracellular development of E. tenella sporozoites through first-generation merogony. Host mitochondrial proteins are not synthesized when labelling in the presence of ricin because these cells lack mitochondrial DNA. Therefore, those proteins which are radiolabelled with 35S methionine in ricin-treated infected monolayers are exclusively those of the intracellular parasite. Alternatively cells with intact mitochondria can be utilized, and in this case the host mitochondrial protein synthesis can be inhibited by chloramphenicol.
Gurnett
A M
AM
Department of Parasite Biochemistry and Molecular Biology, Merck Research Laboratories, Rahway, NJ 07065.
Dulski
P M
PM
Rattray
S
S
Carrington
M
M
Schmatz
D M
DM
D000818:Animals; D016786:Eimeria tenella / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D008715:Methionine; D008928:Mitochondria / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism*; D012276:Ricin / Q000494:pharmacology*; D014122:Toxoplasma / Q000378:metabolism*
D015800:Protozoan Proteins; D012276:Ricin; D008715:Methionine