Here, we explored heat dependent thylakoid FtsH protease substrates and investigated proteotoxicity induced by thermal damage and processive protease dysfunction on the thylakoid membrane. Through our thylakoid enriched proteome analysis and biochemical experiments, carbonylated stromal proteins were suggested as possible FtsH targets. Furthermore, we observed in the thylakoid fractions in the absence of FtsH stromal reactive oxygen species-detoxifying enzymes, as well as heat shock proteins and chaperones, which are known to be upregulated at the transcriptional level when this protease is absent, which is called the damaged protein response, resembling unfolded protein response in eukaryotic cells. Interestingly, the thylakoid-enriched high-density fractions included stromal translation factors and RNA-binding proteins, along with aminoacyl-tRNA synthetase, reminiscent of the formation of stress granules. Unexpectedly, extraplastid proteins such as mitochondrial chaperones, peroxidase, tricarboxylic acid cycle and respiratory chain enzymes, as well as cytosolic ribosomes, translation factors, heat shock proteins, antioxidants and metabolic enzymes, were also found deposited in the high-density fractions depending on the loss of thylakoid FtsH, with more prominent effects of thermal stress on the cytosolic proteins. This may reflect intracellular adaptation to the proteotoxic influences from the organelle.
Keywords: chloroplast; heat stress; processive protease; proteostasis; stress granule; thylakoid membrane.