Selective labeling of intracellular parasite proteins by using ricin.
Studies focused on the synthesis by intracellular parasites of developmentally regulated proteins have been limited due to the lack of a simple method for selectively labeling proteins produced by the parasite. A method has now been developed in which ricin is employed to selectively inhibit host-cell protein synthesis. Ricin is a heterodimer composed of two subunits, a lectin and a glycosidase, and it binds to terminal galactose residues on the cell surface via the lectin. Following endocytosis of the intact molecule, a disulfide bond linking the two subunits is cleaved, and only the glycosidase subunit enters the cytoplasm, where it inhibits cytoplasmic protein synthesis by catalyzing the cleavage of the 28S rRNA. Due to the loss of the receptor-binding lectin subunit, ricin cannot permeate host-cell mitochondria or intracellular parasites, and, therefore, protein synthesis within these compartments continues uninterrupted. This system has been used to selectively label parasite proteins from Eimeria tenella and Toxoplasma gondii by using the avian cell line DU-24. In these cells, mitochondrial protein synthesis was inhibited by using chloramphenicol. The use of the avian rho0 cell line DUS-3 provided an additional advantage, because these cells lack mitochondrial DNA. Therefore, those proteins radiolabeled with [35S]methionine/cysteine in ricin-treated, parasite-infected rho0 cells are exclusively those of the intracellular parasite. This technique should be applicable for studying protein synthesis by other intracellular parasites.
Gurnett
A M
AM
Department of Parasite Biochemistry and Molecular Biology, Merck Research Laboratories, Rahway, NJ 07065.
Dulski
P M
PM
Darkin-Rattray
S J
SJ
Carrington
M J
MJ
Schmatz
D M
DM
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002645:Chickens; D003048:Coccidiosis / Q000469:parasitology; D003545:Cysteine / Q000378:metabolism; D016786:Eimeria tenella / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts; D006801:Humans; D008297:Male; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D012276:Ricin; D012867:Skin; D013462:Sulfur Radioisotopes; D014122:Toxoplasma / Q000378:metabolism*
D015800:Protozoan Proteins; D013462:Sulfur Radioisotopes; D012276:Ricin; D008715:Methionine; D003545:Cysteine