Reconstitution of prenyltransferase activity on nanodiscs by components of the rubber synthesis machinery of the Para rubber tree and guayule

Abstract

Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. The proteins HRT1, HRT2, and HRBP have been identified as candidate components of the rubber biosynthetic machinery. To clarify the contribution of these proteins to prenyltransferase activity, we established a cell-free translation system for nanodisc-based protein reconstitution and measured the enzyme activity of the protein-nanodisc complexes. Co-expression of HRT1 and HRBP in the presence of nanodiscs yielded marked polyisoprene synthesis activity. By contrast, neither HRT1, HRT2, or HRBP alone nor a complex of HRT2 and HRBP manifested such activity. Similar analysis of guayule (Parthenium argentatum) proteins revealed that three HRT1 homologs (PaCPT1-3) manifested prenyltransferase activity only when co-expressed with PaCBP, the homolog of HRBP. Our results thus indicate that two heterologous subunits form the core prenyltransferase of the rubber biosynthetic machinery. A recently developed structure modeling program predicted the structure of such heterodimer complexes including HRT1/HRBP and PaCPT2/PaCBP. HRT and PaCPT proteins were also found to possess affinity for a lipid membrane in the absence of HRBP or PaCBP, and structure modeling implicated an amphipathic α-helical domain of HRT1 and PaCPT2 in membrane binding of these proteins.

Figure 1

Preparation and characterization of protein-nanodisc complexes. (a) HRT1, HRT2, HRBP, HRT1 plus HRBP, HRT2 plus HRBP, and β2AR (lanes 1–6, respectively) were synthesized with a wheat-germ cell-free system in the presence of nanodiscs. The protein-nanodisc complexes were purified by Ni–NTA column chromatography and, together with a portion of the translation reaction mixture corresponding to 1% of the input for purification (Total), were subjected to SDS-PAGE and staining with CBB. Arrowheads indicate HRT1 or HRT2 (blue), HRBP (red), β2AR (black), and MSP (gray). The positions of molecular size markers are indicated on the left. (b) Polyisoprene synthesis activity assay for the purified protein-nanodisc complexes in (a). The incorporation of [¹⁴C]IPP into macromolecules extracted with 1-butanol was measured. Data are means ± s.d. from three independent experiments.

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.

Figure 3

Characterization of guayule homologs of HRT1 and HRBP. (a) The guayule proteins PaCPT1, PaCPT2, PaCPT3, and PaCBP were synthesized with a wheat-germ cell-free system in the presence of nanodiscs either separately (lanes 1–4, respectively) or together in the combinations PaCPT1/PaCBP, PaCPT2/PaCBP, or PaCPT3/PaCBP (lanes 5–7, respectively). The protein-nanodisc complexes were then purified and analyzed by SDS-PAGE and CBB staining. β2AR was similarly synthesized and purified as a control (lane 8). A portion of each translation reaction mixture corresponding to 1% of the input for purification (Total) was also analyzed. Arrowheads indicate PaCPT1, PaCPT2, or PaCPT3 (blue); PaCBP (red); β2AR (large black); MSP (gray); or a wheat-germ protein co-purified by Ni–NTA column chromatography (small black). Single and double asterisks indicate a truncated form of PaCPT3 translated from an internal initiation codon (Supplementary Fig. S4) and an uncharacterized translation product for PaCPT1 mRNA, respectively. (b) Polyisoprene synthesis activity assay for the purified protein-nanodisc complexes in (a). The incorporation of [¹⁴C]IPP into macromolecules extracted with 1-butanol was measured. Data are means ± s.d. from three independent experiments. (c) Analysis of chain length for the ¹⁴C-labeled polyisoprenes extracted by 1-butanol was performed by TLC and autoradiography.

Figure 4

Prenyltransferase subunit compatibility between H. brasiliensis and P. argentatum. (a) Protein-nanodisc complexes were prepared by co-translation of HRT1 and HRBP (lane 1), HRT1 and PaCBP (lane 2), PaCPT2 and HRBP (lane 3), or PaCPT2 and PaCBP (lane 4). The purified complexes were analyzed by SDS-PAGE and CBB staining. Arrowheads indicate HRT1 or PaCPT2 (blue), HRBP or PaCBP (red), or MSP (gray). (b) Polyisoprene synthesis activity assay for the purified complexes in (a). The incorporation of [¹⁴C]IPP into macromolecules extracted with 1-butanol was measured. Data are means ± s.d. from three independent experiments. (c) Analysis of chain length for the ¹⁴C-labeled polyisoprenes extracted by 1-butanol was performed by TLC and autoradiography.

Figure 5

Structure models for plant cPT/cPTL complexes. (a) Structure of the human prenyltransferase complex DHDDS/NgBR (PDB code 6W2L). (b–f) Structural models for HRT1/HRBP, HRT2/HRBP, PaCPT2/PaCBP, HRT1/PaCBP, and PaCPT2/HRBP, respectively. Plant cPT and NH₂-terminally truncated cPTL proteins are colored cyan and orange, respectively. Each pair of images represents views before and after vertical rotation through 180 degrees.

Figure 6

Amino acid residues at the putative interface of plant cPT/cPTL complexes. (a) Interface of the human prenyltransferase complex DHDDS/NgBR (PDB code 6W2L). (b–d) Putative interface regions of structural models for HRT1/HRBP, PaCPT2/PaCBP, and PaCPT2/HRBP, respectively. cPT and NH₂-terminally truncated cPTL proteins are colored cyan and orange, respectively.

Figure 7

Putative membrane binding domain of plant cPT proteins. (a, b) Structural models for HRT1/HRBP (a) and PaCPT2/PaCBP (b) built from the full-length amino acid sequences (Supplementary Fig. S5). HRT1 and PaCPT2 are shown in cyan, and HRBP and PaCBP in gray, with the exception that NH₂-terminal α-helices of HRT1 and PaCPT2 that are predicted to interact with lipid membranes are colored according to a scale from white to red depending on the hydrophobicity of the amino acid residues (with dark red indicating the greatest hydrophobicity). (c, d) Helical wheel diagrams of the NH₂-terminal α-helices of HRT1 (c) and PaCPT2 (d). Hydrophobic, positively charged, and uncharged polar residues are indicated in red, blue, and green, respectively. (e) Protein-nanodisc complexes were prepared by co-translation of HRT1 and HRBP, HRT1 and HRBP(55–257), HRT1(30–290) and HRBP, or HRT1(30–290) and HRBP(55–257) (lanes 1–4, respectively). The purified complexes were subjected to SDS-PAGE followed by CBB staining or immunoblot analysis with antibodies to HRT1, to HRBP, or to His₆ (for detection of MSP). A portion of each translation reaction mixture corresponding to 1% of the input for purification (Total) was also analyzed. Arrowheads indicate HRT1 or HRT1(30–290) (blue), HRBP or HRBP(55–257) (red), or MSP (gray). (f) Relative prenyltransferase activity of HRT1/HRBP-nanodisc and HRT1(30–290)/HRBP-nanodisc complexes. The raw data used to calculate relative activity are 12,220 ± 678 dpm for HRT1/HRBP and 160 ± 78 dpm for HRT1(30–290)/HRBP (means ± s.d. from three independent experiments).