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
Figures
Figure 1
Biosynthetic pathways of backbone structures of cis,trans-mixed polyisoprenoids and natural rubber, catalyzed by trans- and cis-prenyltransferases.
https://doi.org/10.7554/eLife.19022.002
Figure 2 with 2 supplements
Identification of a Hevea NgBR homologue.
(A) Solubilization of proteins from RPs. Proteins on RPs were solubilized by stepwise treatment with buffers containing 4 mM CHAPS (lane 1), 16 mM CHAPS (lane 2), and 7 M urea, 4 M thiourea, and 6.5 …
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Figure 2—source data 1
Proteins related to the natural rubber biosynthesis (upper list) and vesicular trafficking (lower list) identified in the RP proteomics.
RP, rubber particle.
- https://doi.org/10.7554/eLife.19022.004
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Figure 2—source data 2
Proteins identified in the proteomics of the detergent-washed RPs.
RPs, rubber particles.
- https://doi.org/10.7554/eLife.19022.005
Figure 2—figure supplement 1
Treatments of RPs with various detergents.
(A) Solubilization of proteins on RPs by the treatments with various detergents. RPs (50kRP) was incubated overnight with the TD buffer (see Materials and methods) containing various concentrations …
Figure 2—figure supplement 2
Multiple alignments of amino acid sequences.
(A) Comparison of the deduced amino acid sequences of HRT1 (BAB71776) and HRT2 (Q8W3U4). Underlined HRT2/HRT1 sequences indicate peptides detected by LC-MS/MS in the RP proteomics. (B) Comparison of …
Figure 3 with 1 supplement
Physical interaction analyses of the proteins by a split-ubiquitin-based Y2H assay.
Growth of yeast strains, harbouring genes fused with N-terminal (Nub) or C-terminal (Cub) halves of split ubiquitin fragments (listed on left side), on SD(-WL) and SD(-WLHAde). In each panel, …
Figure 3—figure supplement 1
Split-ubiquitin-based Y2H assays.
Growth of yeast strains, harbouring genes fused with N-terminal (Nub) or C-terminal (Cub) halves of split ubiquitin fragments (indicated on left side), on SD media lacking Trp and Leu [SD(–WL)], …
Figure 4 with 2 supplements
In planta interaction analyses of the proteins by monitoring BiFC.
(A) Fluorescent images of N. benthamiana leaf epidermal cells infected with Agrobacterium harbouring a binary vector constructed with pDOE-07 (HRT1–HRBP, HRT2–HRBP HRBP–REF and HRBP–HRBP) or pDOE-05 …
Figure 4—figure supplement 1
BiFC assays for in planta interactions with split mVenus protein fragments.
(A) Binary vectors pDOE-05 and pDOE-07 utilised for BiFC analyses, developed by Gookin and Assmann (Metcalfe, 1967). NmVen210: N-terminal fragment (1–210) of mVenus, which contains the A206K …
Figure 4—figure supplement 2
BiFC assays for in planta interactions with split mVenus protein fragments using the pDOE-05 binary vector.
Fluorescent images of N. benthamiana epidermal cells infected with Agrobacterium harbouring binary vector constructed with pDOE-05 were observed using confocal laser microscope. Fluorescence signals …
Figure 5 with 3 supplements
Subcellular localizations of HRT1, HRT2, HRBP and REF.
(A) Fluorescent images of N. benthamiana leaf epidermal cells expressing target proteins (indicated in each panel) fused with a fluorescent protein, mTq2, mVenus or mChe, at their C-terminus. …
Figure 5—figure supplement 1
Subcellular localizations of HRBP.
Fluorescent images of N. benthamiana leaf epidermal cells expressing target proteins (indicated in each panel) fused with a fluorescent protein, mTq2, mVenus or mChe, at their C-terminus. …
Figure 5—figure supplement 2
Subcellular localizations of HRBP and REF.
Fluorescent images of N. benthamiana leaf epidermal cells co-expressing REF:mTq2 and HRBP:mVenus were obtained using a confocal laser microscope and superimposed to create merged images (Merged1), …
Figure 5—figure supplement 3
BiFC assays for interaction between HRT1 and REF in cells expressing HRBP.
N. benthamiana epidermal cells were infected with an Agrobacterium strain harbouring a BiFC binary vector constructed with pDOE-07 to assay HRT1-REF interaction (upper panels) or with mixture of two …
Figure 6
Detection of the ternary complex formation on RP by co-immunoprecipitations.
CHAPS-solubilized proteins from RPs were applied for immunoprecipitation using anti-HRBP antibody (A) or anti-HRT1/HRT2 antibody (B). The immunoprecipitation experiment without an antibody or the …
Figure 7 with 3 supplements
Cell-free translation-coupled protein introduction onto WRPs.
Immunodetection of HRT1, His6-tagged HRBP and REF with anti-HRT1/HRT2 antibody, anti-His6 antibody and anti-REF antibody, respectively. After in vitro translation to express the protein(s) indicated …
Figure 7—figure supplement 1
In vitro translation of HRT1, HRT2, HRBP and REF.
(A) SDS-PAGE of proteins derived from the cell-free protein expression system supplemented with liposomes. After the in vitro protein expression reaction (see Materials and methods), total crude …
Figure 7—figure supplement 2
Sustained proteins and RTase activity on WRP applied for the cell-free translation.
(A) Analyses of proteins sustained on 50kRP and the WRP applied for the cell-free translation-coupled protein introduction. Proteins were solubilized from each sample with a SDS-PAGE sample buffer, …
Figure 7—figure supplement 3
Workflow of protein expression on washed rubber particles with the wheat germ cell-free system.
https://doi.org/10.7554/eLife.19022.021
Figure 8
Size distributions of RPs after the in vitro translation reaction.
Typical results obtained by measurement of scattering light distributions from more than three measurements by dynamic light scattering are shown. The number in parenthesis indicates the apparent …
Figure 9
Enzymatic characterization of HRT1, HRBP and REF introduced on WRPs.
(A) In vitro RTase activities of HRT1, HRBP and REF, introduced onto 1 µg of WRPs, in the reaction with FPP and 14C-IPP as substrates. The activity is expressed as the net IPP incorporation into the …
Figure 10
Enzymatic characterizations of LsCPT3 and LsCPT2 introduced on the Hevea WRPs.
(A) In vitro RTase activities of LsCPT3 and LsCPT2, introduced onto 1 µg of WRPs, in the reaction with FPP and 14C-IPP as substrates. The activity is expressed as the net IPP incorporation into …
Figure 11
Schematic models of the rubber biosynthetic machinery on RPs (A) and RP formation correlated with the interactions of proteins (B) in the latex of H. brasiliensis. RPs, rubber particles.
https://doi.org/10.7554/eLife.19022.025Additional files
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Supplementary file 1
List of primers used in this study.
- https://doi.org/10.7554/eLife.19022.026
