RESUMEN
Methods for functional analysis of proteins specifically localizing to lipid monolayers such as rubber particles and lipid droplets are limited. We have succeeded in establishing a system in which artificially prepared lipid monolayer particles are added to a cell-free translation system to confirm the properties of proteins that specifically bind to lipid monolayers in a translation-coupled manner.
Asunto(s)
Sistema Libre de Células , Lípidos , Biosíntesis de Proteínas , Lípidos/química , Unión Proteica , Proteínas/química , Proteínas/metabolismoRESUMEN
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.
