Nucleic Acids Research, 2006, Vol. 34, No. 2 503 Figure 3. Amino acid sequence alignments of four Plasmodium selenoproteins. Conserved residues are highlighted. Sec (indicated by U) is shown in boldface. Only partial sequences are available for P.chabaudi selenoprotein 1 and P.vivax selenoprotein 3. Figure 4. Plasmodium SECIS elements support Sec insertion in mammalian cells. (A) Schematic illustration of GFP fusion expression constructs. 1, GFP-SelH fusion containing a mouse SelH SECIS element (control); 2, GFP-SelH fusion containing a P.falciparum Sel4 SECIS element; 3, GFP-Sel1 fusion containing a P.falciparum Sel1 SECIS element; and 4, GFP alone (control). (B) Selenoprotein expression in NIH 3T3 cells. Designation of constructs in A corresponds to the designation of lanes in B. The constructs were transfected into NIH3T3 cells, cells were labeled with 75Se, protein resolved by SDS–PAGE gels and analyzed by PhosphorImager. The location of GFP-SelH and GFP-Sel1 fusion selenoproteins is indicated by arrows on the left. Migration of major endogenous selenoproteins thioredoxin reductase 1 (TR1) and glutathione peroxidase 1 (GPx1) is shown on the right. the cells were labeled with 75Se. PhosphorImager analyses of 75Se-labeled proteins revealed the presence of a 40 kDa GFP- SelH selenoprotein band if either mouse SelH or Plasmodium Sel4 SECIS elements were used (Figure 4B, lanes 1 and 2). In addition, a 36 kDa GFP-Sel1 selenoprotein band was observed (Figure 4B, lane 3), whereas transfection with the GFP con- struct showed only endogenous mouse proteins (Figure 4B, lane 4). These data show that both Sel1 and Sel4 SECIS ele- ments are functional in NIH 3T3 cells and that Plasmodium SECIS elements can support Sec insertion into either Plasmodium or mouse selenoproteins in mammalian cells. Thus, Sec insertion systems are functionally similar in the Apicomplexa parasites and mammals. Components of the Sec insertion machinery in Plasmodium All known components of the Sec insertion machinery are present in Plasmodia, including tRNASec (Figure 5A), EFSec/eSelB and SPS (21). Plasmodium EFSec/eSelB (32,33) was distinct from EF-Tu, which is the elongation factor that inserts all other amino acids. SBP2 was previously described only in animals (11), but hypothetical or putative proteins with accession numbers NP_473214, EAA22418 and Pv_6635.phat_70 for P.falciparum, P.yoelii and P.vivax, respectively, represent good candidates for SBP2 in Plas- modia. Identiﬁcation of both EFSec/eSelB and SBP2 homo- logs suggests that the general strategy for Sec insertion (separation of SECIS-binding Sec insertion functions) is con- served across eukaryotes. Consistent with the recent study (21), the gene sequences of Plasmodium tRNASec (Figure 5B) revealed that they are 90 nt in length compared with 87 [reviewed in (10)] or 88 nt (34) found in other eukaryotic tRNASec genes. The structure of P.falciparum tRNASec folded into a cloverleaf model includ- ing the CCA 30-terminus that is added post-transcriptionally is 93 nt long as shown in Figure 5A. This tRNA has similar features to those of tRNASecs found in other eukaryotes. The acceptor and TyC stems consist of 9/4 base pairs (i.e. 9 bp in the acceptor stem and 4 in the TyC stem) and