NDLI: A plant protein farnesylation system in prokaryotic cells reveals Arabidopsis AtJ3 produced and farnesylated in E. coli maintains its function of protecting proteins from heat inactivation

Content Provider	Springer Nature : BioMed Central
Author	Wu, Jia-Rong Zohra, Rida Duong, Ngoc Kieu Thi Yeh, Ching-Hui Lu, Chung-An Wu, Shaw-Jye
Abstract	Background Protein farnesylation involves the addition of a 15-carbon polyunsaturated farnesyl group to proteins whose C-terminus ends with a CaaX motif. This post-translational protein modification is catalyzed by a heterodimeric protein, i.e., farnesyltransferase (PFT), which is composed of an α and a β subunit. Protein farnesylation in plants is of great interest because of its important roles in the regulation of plant development, responses to environmental stresses, and defense against pathogens. The methods traditionally used to verify whether a protein is farnesylated often require a specific antibody and involve isotope labeling, a tedious and time-consuming process that poses hazardous risks. Results Since protein farnesylation does not occur in prokaryotic cells, we co-expressed a known PFT substrate (i.e., AtJ3) and both the α and β subunits of Arabidopsis PFT in E. coli in this study. Farnesylation of AtJ3 was detected using electrophoretic mobility using SDS-PAGE and confirmed using mass spectrometry. AtJ3 is a member of the heat shock protein 40 family and interacts with Arabidopsis HSP70 to protect plant proteins from heat-stress-induced denaturation. A luciferase-based protein denaturation assay demonstrated that farnesylated AtJ3 isolated from E. coli maintained this ability. Interestingly, farnesylated AtJ3 interacted with E. coli HSP70 as well and enhanced the thermotolerance of E. coli. Meanwhile, AtFP3, another known PFT substrate, was farnesylated when co-expressed with AtPFTα and AtPFTβ in E. coli. Moreover, using the same strategy to co-express rice PFT α and β subunit and a potential PFT target, it was confirmed that OsDjA4, a homolog of AtJ3, was farnesylated. Conclusion We developed a protein farnesylation system for E. coli and demonstrated its applicability and practicality in producing functional farnesylated proteins from both mono- and dicotyledonous plants.
Related Links	https://plantmethods.biomedcentral.com/counter/pdf/10.1186/s13007-023-01087-x.pdf
Ending Page	12
Page Count	12
Starting Page	1
File Format	HTM / HTML
ISSN	17464811
DOI	10.1186/s13007-023-01087-x
Journal	Plant Methods
Issue Number	1
Volume Number	19
Language	English
Publisher	BioMed Central
Publisher Date	2023-10-26
Access Restriction	Open
Subject Keyword	Plant Sciences Biological Techniques Arabidopsis AtJ3 HSP40 OsDjA4 Post-translational modification Protein farnesylation Protein farnesyltransferase Heat stress Thermotolerance
Content Type	Text
Resource Type	Article
Subject	Plant Science Biotechnology Genetics
Journal Impact Factor	4.7/2023
5-Year Journal Impact Factor	5.6/2023

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