| dc.description.abstract | Farnesylation of proteins involves the covalent attachment of a 15-carbon unsaturated farnesyl lipid group to a protein ending with a CaaX sequence at the C-terminus. This post-translational modification is catalyzed by the protein farnesyltransferase (PFT), which comprises α and β subunits. Protein farnesylation is pivotal in plant growth, regulating plant development, combating environmental stresses, and defending against pathogen infections. Traditionally, assessing whether a candidate protein is farnesylated involves laborious and potentially hazardous processes, such as preparing specific antibodies for the candidate protein and utilizing isotopically labeled farnesol, a precursor of farnesyl pyrophosphate in the farnesylation reaction. Given that protein farnesylation does not occur in prokaryotic cells, this study aims to establish a system in E. coli for rapidly identifying whether plant candidate proteins are farnesylated. This system involves introducing into E. coli a plasmid carrying the ColE1 replication origin, the α and β subunit genes of Arabidopsis PFT, and a plasmid carrying the candidate protein gene labeled with a 6xHis sequence, along with the RSF1031 replication origin. This study selected a known substrate of Arabidopsis PFT, AtJ3, as the candidate protein. The results demonstrate that all three Arabidopsis genes can be expressed simultaneously in E. coli cells. The produced AtJ3, after simple affinity purification, can be identified as post-translationally modified by its mobility on SDS-PAGE, with mass spectrometry confirming the modification as farnesylation at the C-terminus. AtJ3 belongs to the heat shock protein 40 family, interacts with HSP70, and protects plant proteins from denaturation damage caused by high temperatures. Based on luciferase activity analysis experiments, this study also demonstrates that farnesylated AtJ3 isolated from E. coli retains its molecular chaperone function. Interestingly, farnesylated AtJ3 produced in E. coli can also interact with E. coli HSP70 and enhance the thermotolerance of E. coli. Furthermore, using the same strategy to co-express rice PFTα and β subunits along with a potential rice PFT substrate, OsDjA4, in E. coli cells also showed OsDjA4 to be farnesylated. In summary, this study successfully developed a system using E. coli to identify protein farnesylation modifications, applicable to both monocot and dicot plants. The farnesylated proteins produced in E. coli retain their original functions in eukaryotic cells and can be utilized to study the molecular and biochemical functions of farnesylated proteins. | en_US |