Domain organization and sequence of SelL. (A) Schematic representation of SelL and the location of the Prx 2-like domain. (B) Multiple sequence alignment of SelL ORFs corresponding to the boxed segment in A. GenBank accession nos. for SelL sequences are given in SI Table 1. Conserved residues are highlighted. Sec residues are shaded in red and marked with asterisks. Predicted secondary structure elements common to all aligned sequences are shown at the top.

SelL proteins and SECIS elements. (A) Secondary structures of different SECIS elements tested for expression of the GST-fused SelL fragment. The original bacterial SelL SECIS element (pGEX-SelL-SECIS construct) is designated as SECIS; SECISdel and SECISins are mutated SECIS elements with a bulged U (pGEX-SelL-SECISdel and pGEX-SelL-SECISins constructs); and FDH H SECIS is a minimal SelB-binding region of E. coli FDH H SECIS element (the pGEX-SelL-FDH H SECIS construct). SelB-binding elements are shown in bold type, and Sec codons are shown in red. Optimal location of Sec UGA codons relative to SECIS, SECISdel, and FDH H SECIS is shown by a larger open box and relative to SECISins by a smaller gray box. (B) GST-fused bacterial SelL expression constructs used in this study. The green box corresponds to GST, the orange box corresponds to the SelL fragment containing two Sec codons (red vertical lines) and a SECIS element (stem-loop structure), and the gray box corresponds to a (His)₆-tag. (C) A construct coding for His-tagged zebrafish SelL. The brown box indicates SelL ORF and an embedded orange box shows Prx 2-like domain. The gray box depicts a (His)₆-tag. The 3′ UTRs with a SECIS element are shown in black, and Sec UGA codons are in red. (D) Structures of eukaryotic SelL SECIS elements. Conserved nucleotides in the SECIS core and in the apical loop are shown in bold. (E) Expression of GST-SelL fragment in E. coli. (Upper) The selenoprotein pattern visualized with a PhosphorImager. (Lower) Coomassie blue staining of the same membrane (protein loading control): lane 1, GEX-4T-1 (control expressing GST); lane 2, pGEX-SelL-FDH H SECIS; lane 3, pGEX-SelL-SECISins; lane 4, pGEX-SelL-SECISdel; lane 5, pGEX-SelL-SECIS; lane 6, pGEX-SelL(CLPU)-SECISins; lane 7, pGEX-SelL(CLPU)-SECIS; and lane 8, pGEX-SelL(ULPC)-SECIS. Migration of full-length GST-SelL and truncated GST-SelL proteins is shown on the right. (F) Efficiency of Sec insertion into indicated Sec and Cys GST-SelL forms with the original SelL and E. coli FDH H SECIS elements. Control corresponds to pGEX-4T-1 vector expressing GST. (Upper) Western blotting with anti-His-tag antibodies. (Lower) The selenoprotein pattern of cells labeled with ⁷⁵Se. Mutations made in the ULPU redox motif of SelL to generate mutant forms are indicated at the top. Positions of full-length and truncated GST-SelL forms are indicated on the right. (G and H) The ⁷⁵Se-labeled GST-SelL and its Sec-to-Cys mutants were expressed from constructs containing E. coli FDH H SECIS, purified on Talon resin and analyzed by SDS/PAGE after Coomassie blue staining (G) and Western blotting with anti-GST (H Top) and anti-His-tag (H Middle) antibodies and by exposure of the membrane to a PhosphorImager (H Bottom): lane 1, ULPU form of GST-SelL; lane 2, ULPC form; lane 3, CLPU form; and lane 4, CLPC form. GST that was copurified with GST-SelL is shown; migration of the 28-kDa marker is pointed out by arrows on the right in H.

SelL has a diselenide bond. (A) Identification of the diselenide bond in GST-fused SelL by mass spectrometry. Isotopic pattern of triple-charged peptide [HFAULPUR + 3H]³⁺, with chemical formula C₄₁H₆₃N₁₃O₉Se₂, is indicated by blue solid lines. Dashed vertical lines correspond to the calculated pattern. (B) An MS/MS spectrum of the double-charged parent ion [HFAULPUR + 2H]²⁺. Amino acid sequences of the corresponding daughter ions containing two Sec are shown above the peaks. (C) Expression of D. rerio SelL in mammalian cells. HEK 293 cells were cotransfected in a 1:1 ratio with a construct coding for a C-terminal domain of SBP2 and the following His-tagged pCI-neo-based constructs: lane 1, D. rerio SelL; lane 2, SelL(ULPC); lane 3, SelL(CLPU); lane 4, SelL(CLPC); and lane 5, GFP (control). Metabolically ⁷⁵Se-labeled cells were subjected to SDS/PAGE, proteins were transferred onto a PVDF membrane, and the radioactivity pattern was visualized by using a PhosphorImager (Upper); the same membrane was used in Western blotting with anti-His-tag antibodies (Lower). (D) Comparison of HEK 293 cells transfected with pCI-SelL construct (lane 1) and cotransfected with pCI-SelL construct and SBP2-expressing construct (lane 2). Migration of His-tagged SelL and major endogenous proteins, thioredoxin reductase 1 (TR1) and glutathione peroxidase (GPx1), is indicated.