Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis
- Tohoku University, Japan
- Sumitomo Rubber Industries, Ltd, Japan
- Saitama University, Japan
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
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Hevea brasiliensis HRTBP mRNA for transferase binding protein,Publicly available at the DNA Data Bank of Japan (Accession no: LC057267).
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Lactuca sativa cis-prenyltransferase 3 mRNA, complete cds.Publicly available at the NCBI Nucleotide (Accession no: F752488).
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TSA: Hevea brasiliensis contigxxxxx, mRNA sequence.Publicly available at the NCBI BioProject (Accession no: PRJNA828950).
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Lactuca sativa cis-prenyltransferase-like 2 mRNA, complete cdsPublicly available at the NCBI Nucleotide (Accession no: KF752485).
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Funding
No external funding was received for this work.
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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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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