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 Hevea brasiliensis, exhibits distinct RTase activity in vitro only when it is introduced on detergent-washed Hevea RPs (WRPs) by a cell-free translation-coupled system. Using this system, a heterologous cPT from Lactuca sativa 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.

Data availability

The following data sets were generated
    1. Takahashi S
    2. Nakayama T
    (2015) Hevea brasiliensis HRTBP mRNA for transferase binding protein,
    Publicly available at the DNA Data Bank of Japan (Accession no: LC057267).
The following previously published data sets were used

Article and author information

Author details

  1. Satoshi Yamashita

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  2. Haruhiko Yamaguchi

    Sumitomo Rubber Industries, Ltd, Kobe, Japan
    Competing interests
    The authors declare that no competing interests exist.
  3. Toshiyuki Waki

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  4. Yuichi Aoki

    Graduate School of Information Sciences, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  5. Makie Mizuno

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  6. Fumihiro Yanbe

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  7. Tomoki Ishii

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  8. Ayuta Funaki

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  9. Yuzuru Tozawa

    Graduate School of Science and Engineering, Saitama University, Saitama, Japan
    Competing interests
    The authors declare that no competing interests exist.
  10. Yukino Miyagi-Inoue

    Sumitomo Rubber Industries, Ltd, Kobe, Japan
    Competing interests
    The authors declare that no competing interests exist.
  11. Kazuhisa Fushihara

    Sumitomo Rubber Industries, Ltd, Kobe, Japan
    Competing interests
    The authors declare that no competing interests exist.
  12. Toru Nakayama

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    Competing interests
    The authors declare that no competing interests exist.
  13. Seiji Takahashi

    Graduate School of Engineering, Tohoku University, Sendai, Japan
    For correspondence
    takahasi@seika.che.tohoku.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2288-4340

Funding

No external funding was received for this work.

Reviewing Editor

  1. Kazunori Okada, University of Tokyo, Japan

Version history

  1. Received: June 21, 2016
  2. Accepted: October 25, 2016
  3. Accepted Manuscript published: October 28, 2016 (version 1)
  4. Accepted Manuscript updated: November 3, 2016 (version 2)
  5. Version of Record published: November 15, 2016 (version 3)

Copyright

© 2016, Yamashita et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Satoshi Yamashita
  2. Haruhiko Yamaguchi
  3. Toshiyuki Waki
  4. Yuichi Aoki
  5. Makie Mizuno
  6. Fumihiro Yanbe
  7. Tomoki Ishii
  8. Ayuta Funaki
  9. Yuzuru Tozawa
  10. Yukino Miyagi-Inoue
  11. Kazuhisa Fushihara
  12. Toru Nakayama
  13. Seiji Takahashi
(2016)
Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis
eLife 5:e19022.
https://doi.org/10.7554/eLife.19022

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https://doi.org/10.7554/eLife.19022

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    Funding:

    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.