Figure 6

The SID-like region in SBP2L does not promote Sec incorporation. (A) Schematic of the SBP2L/SBP2 domain swap. (B) 2 fmol of the indicated [³⁵S]-Met labeled in vitro translated proteins (top gel) were added to an in vitro translation reaction containing a luciferase Sec incorporation reporter. Luciferase activity (Sec incorporation) is expressed as a percent of that obtained with wild-type CT-SBP2 (bottom graph). (C) 8 fmol of [³⁵S]-Met labeled in vitro translated proteins were incubated with [³²P]-labeled wild-type (wt) or mutant (mt) GPX4 SECIS elements and resolved on a 4% non-denaturing polyacrylamide gel. Arrow 1 marks CT-SBP2L and SBP2-SBP2L complexes, arrow 2 marks CT-SPB2 and SBP2L-SBP2 complexes. The asterisk marks a probe shift resulting from an unidentified component in rabbit reticulocyte lysate. (D) Graphical representation of EMSA data shown in (D) expressed as the percent of probe shifted relative to that obtained with wild-type CT-SBP2. (E) 2.4 fmol of the indicated [³⁵S]-Met labeled in vitro translated proteins were added to an in vitro translation reaction containing a luciferase Sec incorporation reporter. Mock contains no added in vitrotranslated proteins. 'CT-SBP2L subs.' and 'SBP2L-SBP2 subs.' indicate proteins bearing the following substitutions: D494G, SKA556PLM, EK563QR and A567P (human SBP2L numbering). Sec incorporation is expressed as a percentage of CT-SBP2. All data are mean ± SEM of three independent experiments.