Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis

Abstract

Natural rubber (NR) is stored in latex as rubber particles (RPs), rubber molecules surrounded by a lipid monolayer. Rubber transferase (RTase), the enzyme responsible for NR biosynthesis, is believed to be a member of the cis-prenyltransferase (cPT) family. However, none of the recombinant cPTs have shown RTase activity independently. We show that HRT1, a cPT from Heveabrasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed HeveaRPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactucasativa also exhibited RTase activity, indicating proper introduction of cPT on RP is the key to reconstitute active RTase. RP proteomics and interaction network analyses revealed the formation of the protein complex consisting of HRT1, rubber elongation factor (REF) and HRT1-REF BRIDGING PROTEIN. The RTase activity enhancement observed for the complex assembled on WRPs indicates the HRT1-containing complex functions as the NR biosynthetic machinery.DOI: http://dx.doi.org/10.7554/eLife.19022.001

Lipid droplet functions beyond energy storage

“…It is stored in specialized LDs called rubber particles, which lack structural proteins of the oleosin family common on other types of plant LDs [42]. Rubber is synthesized by the sequential condensation of isopentenyl diphosphate subunits [48]. The enzyme responsible, rubber transferase, likely corresponds to one or more cis -prenyltransferases localized to the ER and/or to the phospholipid monolayer of the rubber particle [42, 48].…”

“…Rubber is synthesized by the sequential condensation of isopentenyl diphosphate subunits [48]. The enzyme responsible, rubber transferase, likely corresponds to one or more cis -prenyltransferases localized to the ER and/or to the phospholipid monolayer of the rubber particle [42, 48]. The polyisoprene latex cargo of rubber particle LDs plays important roles in the protection and defense of plants against wounding, insect herbivory, and environmental stresses [49].…”

Membrane Dynamics and Multiple Functions of Oil Bodies in Seeds and Leaves

“…SRPPs are localized on the rubber particle membrane in rubber-accumulating plants (Berthelot et al, 2014). Rubber particles are surrounded by a phospholipid monolayer and store polyisoprene (Yamashita et al, 2016). The structure of rubber particles is similar to that of oil bodies.…”

“…These combined results suggest that LDAPs enhance oil body formation, lipid biosynthesis, plant growth, and drought resistance. Yeast two-hybrid analysis indicates that H. brasiliensis LDAP/SRPP binds rubber elongation factor (REF; Yamashita et al, 2016). H. brasiliensis cis-prenyltransferases (HRTs) interact with HRT1-REF bridging protein, which binds REF and HRT for efficient rubber biosynthesis (Yamashita et al, 2016).…”

“…Yeast two-hybrid analysis indicates that H. brasiliensis LDAP/SRPP binds rubber elongation factor (REF; Yamashita et al, 2016). H. brasiliensis cis-prenyltransferases (HRTs) interact with HRT1-REF bridging protein, which binds REF and HRT for efficient rubber biosynthesis (Yamashita et al, 2016). LDAPs may recruit a complex for lipid biosynthesis, although they do not interact with seed oil bodies containing oleosins (Gidda et al, 2016), suggesting that LDAPs are antagonistic to oleosins in oil body targeting.…”