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

Satoshi Yamashita, Haruhiko Yamaguchi, Toshiyuki Waki, Yuichi Aoki, Makie Mizuno, Fumihiro Yanbe, Tomoki Ishii, Ayuta Funaki, Yuzuru Tozawa, Yukino Miyagi-Inoue, Kazuhisa Fushihara, Toru Nakayama, Seiji Takahashi

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50 Citations (Scopus)

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 detergentwashed 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.

Original languageEnglish
Article numbere19022
JournaleLife
Volume5
Issue numberOCTOBER2016
DOIs
Publication statusPublished - 2016 Oct 28

ASJC Scopus subject areas

  • Neuroscience(all)
  • Immunology and Microbiology(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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    Yamashita, S., Yamaguchi, H., Waki, T., Aoki, Y., Mizuno, M., Yanbe, F., Ishii, T., Funaki, A., Tozawa, Y., Miyagi-Inoue, Y., Fushihara, K., Nakayama, T., & Takahashi, S. (2016). Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis. eLife, 5(OCTOBER2016), [e19022]. https://doi.org/10.7554/eLife.19022.001