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.
Highlights
Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity
The cDNAs for HRT1, HRT2, HRT1-REF bridging protein (HRBP), and β2-adrenergic receptor (β2AR) were cloned into the cell-free expression plasmid pYT08, and the resultant plasmids were subjected to transcription and subsequent cell-free protein synthesis in the presence of preassembled asolectin nanodiscs (Supplementary Fig. S1)
The resulting protein-nanodisc complexes were isolated by immobilized metal affinity chromatography (IMAC) and analyzed by SDS–polyacrylamide gel electrophoresis (PAGE) with Coomassie brilliant blue (CBB) staining (Fig. 1a)
Summary
Natural rubber of the Para rubber tree (Hevea brasiliensis) is synthesized as a result of prenyltransferase activity. CPTL proteins associated with rubber particles have been identified in other NR-producing plants including lettuce (Lactuca sativa) and Russian dandelion, and knockdown of the genes encoding these proteins was found to reduce rubber c ontent[4,11,12] These in vivo findings implicated cPTL as a key player in NR biosynthesis. Introduction of the lettuce cPT protein LsCPT3 to washed rubber particles from H. brasiliensis reconstituted synthetic activity for NR-size polyisoprene even without co-expression of LsCPTL28. These observations have questioned the notion that cPTL is essential for cPT activity
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