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.

Reviving Natural Rubber Synthesis via Native/Large Nanodiscs

“…The proteoliposomes successfully incorporated the RTase complex but did not exhibit any RTase activity in vitro, while the WRPs carrying the reconstituted proteins demonstrated activity, as shown in Figure 3 A [ 13 ]. The researchers propose that proteoliposomes could be explored further, involving additional candidate proteins and specific lipid molecules, to gain a deeper understanding of rubber particle structure and function [ 2 , 13 , 21 , 22 ].…”

“…Nanodiscs, which are disc-shaped lipid bilayers stabilized by membrane scaffold proteins (MSPs), have been widely used to investigate protein and membrane lipid structure, function, and interactions [ 21 , 23 ]. The size of nanodiscs is determined by the length of MSPs and the stoichiometry of lipids used in the self-assembly process, resulting in monodispersed particles with similar sizes [ 29 , 30 , 31 ].…”

“…The successful reconstitution of the HRT1-HRBP complex on MSP nanodiscs in a CFPSS has led to the demonstration of CPT activity, marking a significant milestone, as shown in Figure 3 B [ 21 ]. However, in the case of combining the purified HRT1–nanodisc complex with the HRBP–nanodisc complex, no CPT activity was observed.…”

“…Hence, the researchers co-expressed them on nanodiscs with the HRT1-HRBP complex. However, due to the low affinity of REF and SRPPs to the nanodiscs’ membranes or flat lipid bilayer structures, they were lost during the purification step [ 21 ].…”

Transfer mechanism of cell-free synthesized membrane proteins into mammalian cells

“…The cotranslational insertion of MPs into nanodisc (ND) membranes by cell-free (CF) expression is an efficient method to produce a variety of functional MPs including G-protein coupled receptors (GPCRs), transporters, channels, or enzymes (Henrich et al, 2016;Henrich et al, 2017b;He et al, 2017;Rues et al, 2018;Keller et al, 2019;Kuroiwa et al, 2022). The NDs are first preassembled with the lipid or lipid mixture that is best suited for the stability and folding of the selected MP.…”