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
-
Hevea brasiliensis HRTBP mRNA for transferase binding protein,Publicly available at the DNA Data Bank of Japan (Accession no: LC057267).
-
Lactuca sativa cis-prenyltransferase 3 mRNA, complete cds.Publicly available at the NCBI Nucleotide (Accession no: F752488).
-
TSA: Hevea brasiliensis contigxxxxx, mRNA sequence.Publicly available at the NCBI BioProject (Accession no: PRJNA828950).
-
Lactuca sativa cis-prenyltransferase-like 2 mRNA, complete cdsPublicly available at the NCBI Nucleotide (Accession no: KF752485).
Article and author information
Author details
Funding
No external funding was received for this work.
Reviewing Editor
- Kazunori Okada, University of Tokyo, Japan
Version history
- Received: June 21, 2016
- Accepted: October 25, 2016
- Accepted Manuscript published: October 28, 2016 (version 1)
- Accepted Manuscript updated: November 3, 2016 (version 2)
- 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.
Metrics
-
- 6,272
- views
-
- 1,437
- downloads
-
- 105
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Identification and reconstitution of the rubber biosynthetic machinery on rubber particles from Hevea brasiliensis
eLife 5:e19022.
https://doi.org/10.7554/eLife.19022
Further reading
-
- Biochemistry and Chemical Biology
- Cell Biology
Mediator of ERBB2-driven Cell Motility 1 (MEMO1) is an evolutionary conserved protein implicated in many biological processes; however, its primary molecular function remains unknown. Importantly, MEMO1 is overexpressed in many types of cancer and was shown to modulate breast cancer metastasis through altered cell motility. To better understand the function of MEMO1 in cancer cells, we analyzed genetic interactions of MEMO1 using gene essentiality data from 1028 cancer cell lines and found multiple iron-related genes exhibiting genetic relationships with MEMO1. We experimentally confirmed several interactions between MEMO1 and iron-related proteins in living cells, most notably, transferrin receptor 2 (TFR2), mitoferrin-2 (SLC25A28), and the global iron response regulator IRP1 (ACO1). These interactions indicate that cells with high MEMO1 expression levels are hypersensitive to the disruptions in iron distribution. Our data also indicate that MEMO1 is involved in ferroptosis and is linked to iron supply to mitochondria. We have found that purified MEMO1 binds iron with high affinity under redox conditions mimicking intracellular environment and solved MEMO1 structures in complex with iron and copper. Our work reveals that the iron coordination mode in MEMO1 is very similar to that of iron-containing extradiol dioxygenases, which also display a similar structural fold. We conclude that MEMO1 is an iron-binding protein that modulates iron homeostasis in cancer cells.
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.
