A Split-Ubiquitin Yeast Two-Hybrid Screen to Examine the Substrate Specificity of atToc159 and atToc132, Two Arabidopsis Chloroplast Preprotein Import Receptors
(A) Schematic representation of the LHCA4(TP)-DHFRHis and FNR1(TP)-DHFRHis constructs with C-terminal histidine tags (His-Tag) used as baits in the binding reactions. The numbers refer to the amino acid numbers of the TPs from LHC4 and FNR. (B and C) Equal amounts of in vitro translated [35S]atToc132AG, or [35S]atToc159AG were incubated in the presence of GTP with the indicated amounts of immobilized hexahistidine-tagged LHCA4(TP)-DHFRHis (A) or FNR1(TP)-DHFRHis (B). Bound proteins were eluted and separated by SDS-PAGE, and detected in dried gels using a phosphorimager. Top panels of B and C show representative experiments of triplicates. Lane, IVT in each panel contains 10% of the in vitro translation product added to each reaction. The graphs show quantitative analysis of the triplicate binding experiments with SE bars. atToc159AG binds to LHCA4(TP)-DHFRHis and FNR1(TP)-DHFRHis at levels that are two-fold and five-fold higher than atToc132AG, respectively. The atToc132AG and atToc159AG do not significantly bind to 800 pmol of a negative control immobilized hexahistidine-tagged DHFRHis protein (D).