Functional Analysis of the Arabidopsis Mitochondrial Chaperones mtHSC70 and MGE
Date Issued
2015
Date
2015
Author(s)
Hung, Meng-Ju
Abstract
In prokaryotes, DnaK, DnaJ, and GrpE form a set of molecular chaperone machine to facilitate protein folding. The DnaK protein consists of an ATPase domain and a substrate binding domain. With the help of the cochaperone DnaJ, DnaK binds to the protein substrate and change the conformation by hydrolyzing ATP to ADP. The ADP-bound DnaK has increased affinity for the substrate, which promotes the folding of its protein substrate. GrpE serves as a nucleotide exchange factor that replaces ADP with ATP on DnaK and decreases its affinity to the substrate, leading to release of the folded protein. The DnaK-DnaJ-GrpE chaperone (or KJE chaperone in short) also exists in the chloroplasts and mitochondria in eukaryotes, involving in protein import as well as protein folding. In Arabidopsis nuclear genome, there are two homologous genes encoding mitochondrial DnaK, named mtHSC70-1 and mtHSC70-2. Similarly, mitochondrial GrpE is also encoded by two different genes, named MGE1 and MGE2. Previous studies showed that mtHSC70-2 and MGE2 are heat-inducible and that the Arabidopsis mutants without mtHSC70-1, mtHSC70-2, or MGE2 could not tolerate certain heat stress conditions. It is possible that mtHSC70 or MGE have evolved to cope with different heat stress conditions. However, the molecular functions of these components are not clear. In this study, I investigated whether mtHSC70-1/MGE1 and mtHSC70-2/MGE2 are differentially responsible for mitochondrial protein import. The levels of certain mitochondria matrix proteins in the mitochondrial enriched fraction were analyzed by Western blot. The results show that the defect in mtHSC70-2 or MGE2 do not obviously affect the import of the tested proteins at high temperature, suggesting that mtHSC70-1 and MGE1 also function under high temperature. The defect in mtHSC70-1 also does not affect the protein import at high temperature, suggesting functional redundancy of the two mtHSC70 chaperones encoding genes. Another interesting aspect concerns the structure of MGE2. In many higher plants structural variant of MGE2 can be produced due to pre-mRNA alternative splicing and the retention of intron 2, which encodes a conserved serine and arginine/lysine (SR/K)-rich sequence. To understand whether the SR/K-rich sequence is important for heat tolerance in plants, I constructed pMGE2::MGE2 plasmid containing the full length MGE2 genomic DNA and pMGE2::MGE2D that encodes MGE2 without the SR/K-rich sequence for transforming the MGE2 knockout mutant. The results show that the MGE2 or MGE2D transcripts in the transgenic lines were heat inducible and down regulated when the heat treatment prolonged. The MGE2 or MGE2D proteins were similarly accumulated during the heat stress treatment. Moreover, both pMGE2::MGE2 and pMGE2::MGE2D rescued the heat sensitivity of the MGE2 knockout mutant from prolonged exposure to moderately high temperature. In conclusion, the results suggest that the SR/K-rich sequence is not required for the stability of MGE2 and is also not required for the thermotolerance to moderately high temperature.
Subjects
mitochondrial chaperones
heat stress
protein import
Type
thesis
File(s)![Thumbnail Image]()
Loading...
Name
ntu-104-R02b22023-1.pdf
Size
23.32 KB
Format
Adobe PDF
Checksum
(MD5):b9cc4f10b25daaef7b1590b60a06efc6