Probing the function of protein farnesyltransferase in Tripterygium wilfordii
We found two subunits FTase/GGTaseI-α and FTase-β formed a heterodimer to transfer a farnesyl group from FPP to protein N-dansyl-GCVLS, confirming they are responsible for protein farnesylation in planta.
Tripterygium wilfordii is a medicinal plant with a broad spectrum of anti-inflammatory, immunosuppressive and anti-cancer activities. Recently, a number of studies have focused on investigating the biosynthetic pathways of its bioactive compounds, whereas little attention has been paid to the enzymes which play important roles in regulating diverse developmental processes of T. wilfordii. In this study, we report for the first time the identification and characterization of two subunits of farnesyltransferase (FTase), farnesyltransferase/geranylgeranyltransferase I-α (TwFTase/GGTase I-α) and farnesyltransferase-β (TwFTase-β), in this important medicinal plant. Cell-free in vivo assays, yeast two-hybrid (Y2H) and pull-down assays showed that the two subunits interact with each other to form a heterodimer to perform the role of specifically transferring a farnesyl group from FPP to the CAAX-box protein N-dansyl-GCVLS. Furthermore, we discovered that the two subunits had the same cytoplasmic localization pattern and displayed the same tissue expression pattern. These results indicated that we identified a functional TwFTase enzyme which contains two functionally complementary subunits TwFTase/GGTase I-α and TwFTase-β, which provides us promising genetic targets to construct transgenic plants or screen for more adaptable T. wilfordii mutants, which are able to survive in changing environments.
KeywordsTripterygium wilfordii Protein farnesyltransferase Y2H assay Functional analysis Subcellular localization Gene expression
This research was supported by the National Natural Science Foundation of China (81773830 to W.G.), High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (CIT&TCD20170324 to W.G.) and the Key project at central government level: the ability establishment of sustainable use for valuable Chinese medicine resources (2060302 to L.H.).
PS, LH and WG designed the project; LG, SL, HG, JW, YZ (Zhang), YZ(Zhao) and TH performed the experiments and database analysis; PS, LG and LT wrote the manuscript; JZ, BM and XL interpreted the data; All authors read and approved the final manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Bartel P, Chien CT, Sternglanz R, Fields S (1993) Elimination of false positives that arise in using the two-hybrid system. Biotechniques 14:920–924Google Scholar
- Del SG, Manfioletti G, Schneider C (1989) The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques 7:514–520Google Scholar
- Iwabuchi K, Li B, Bartel P, Fields S (1993) Use of the two-hybrid system to identify the domain of p53 involved in oligomerization. Oncogene 8:1693–1696Google Scholar
- Su P, Guan H, Zhang Y, Wang X, Gao L, Zhao Y, Hu T, Zhou J, Ma B, Tu L et al (2017) Probing the single key amino acid responsible for the novel catalytic function of ent-kaurene oxidase supported by NADPH-cytochrome P450 reductases in Tripterygium wilfordii. Front Plant Sci 8:1756CrossRefGoogle Scholar
- Zhai Q, Zhang X, Wu F, Feng H, Deng L, Xu L, Zhang M, Wang Q, Li C (2015) Transcriptional mechanism of jasmonate receptor COI1-mediated delay of flowering time in Arabidopsis. Plant Cell 27:2814–2828Google Scholar