Figure 2

Sequential expression and characterization of HRT1/HRBP-nanodisc complexes. (a) Schematic representation of protein expression protocols. The mRNAs for HRT1 and HRBP were translated together in the presence of nanodiscs (Co-expression); purified HRT1-nanodisc and HRBP-nanodisc complexes were mixed (Mixture); or HRBP or HRT1 was synthesized in the presence of HRT1-nanodisc or HRBP-nanodisc complexes, respectively (Sequential expression 1 and 2, respectively). It is also possible that HRT1 and HRBP exist as homodimers on nanodiscs (see Discussion). (b) Purified protein-nanodisc complexes (P) as well as a portion of the translation reaction mixture corresponding to 1% of the input for purification (T) were subjected to SDS-PAGE and stained with CBB. 1st and 2nd indicate the first and second expression, respectively. Lanes 1 to 4 correspond to expression of HRT1 in the presence of empty nanodiscs, expression of HRBP in the presence of empty nanodiscs, expression of HRBP in the presence of HRT1-nanodisc complexes, and expression of HRT1 in the presence of HRBP-nanodisc complexes, respectively. Arrowheads indicate HRT1 (blue), HRBP (red), and MSP (gray) as well as a co-purified protein from the wheat-germ extract (black). (c) Polyisoprene synthesis activity assay for the purified protein-nanodisc complexes. Co, co-expression; Mix, mixture; Seq 1, sequential expression 1; Seq 2, sequential expression 2. The incorporation of [¹⁴C]IPP into macromolecules extracted with 1-butanol was measured. Data are means ± s.d. from three independent experiments. (d) Analysis of chain length for the ¹⁴C-labeled polyisoprenes extracted by 1-butanol was performed by TLC and autoradiography. The positions of the origin (Ori), solvent front (S.F.), and authentic standards are indicated on the right side.