The micronutrient selenium is found in proteins as selenocysteine (Sec), the 
21st amino acid cotranslationally inserted in response to a UGA codon. In vitro 
studies in archaea and mouse showed that Sec-tRNA(Sec) formation is a 3-step 
process starting with serylation of tRNA(Sec) by seryl-tRNA synthetase (SerRS), 
phosphorylation of serine to form phosphoserine (Sep)-tRNA(Sec) by 
phosphoseryl-tRNA(Sec) kinase (PSTK), and conversion to Sec-tRNA(Sec) by 
Sep-tRNA:Sec-tRNA synthase (SepSecS). However, a complete study of eukaryotic 
selenoprotein synthesis has been lacking. Here, we present an analysis of 
Sec-tRNA(Sec) formation in the parasitic protozoon Trypanosoma brucei in vivo. 
Null mutants of either PSTK or SepSecS abolished selenoprotein synthesis, 
demonstrating the essentiality of both enzymes for Sec-tRNA(Sec) formation. 
Growth of the 2 knockout strains was not impaired; thus, unlike mammals, 
trypanosomes do not require selenoproteins for viability. Analysis of 
conditional RNAi strains showed that SerRS, selenophosphate synthase, and the 
Sec-specific elongation factor, EFSec, are also essential for selenoprotein 
synthesis. These results with T. brucei imply that eukaryotes have a single 
pathway of Sec-tRNA(Sec) synthesis that requires Sep-tRNA(Sec) as an 
intermediate.