Abstract
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.
Highlights
IntroductionChloroplast proteins are mostly nucleus-encoded, and synthesized by 80S ribosomes in the cytosol as preproteins having an N-terminal cleavable transit peptide facilitating their import into the organelle, while the remaining ~100 proteins are chloroplast-encoded and synthesized by 70S ribosomes within the plastid
Wild-type and var2-7 mutant [10] Arabidopsis plants grown for 4 weeks under normal growth conditions were either harvested immediately or exposed to heat stress at 45 ◦ C for 3 h and harvested, each followed by biochemical enrichment of thylakoid membrane fractions by simple low-speed centrifugation
A conserved Filamentation temperature-sensitive H (FtsH) processive protease plays a central role in thylakoid proteostasis; loss of its function could cause proteotoxic stress
Summary
Chloroplast proteins are mostly nucleus-encoded, and synthesized by 80S ribosomes in the cytosol as preproteins having an N-terminal cleavable transit peptide facilitating their import into the organelle, while the remaining ~100 proteins are chloroplast-encoded and synthesized by 70S ribosomes within the plastid. These proteins are destined to the intraorganellar locations, i.e., stroma, envelopes and thylakoid membranes. The thylakoid membrane provides the site for photosynthesis, and their membrane structure is highly organized with photosynthetic protein machineries such as photosystems [2]. A protein quality control (PQC) mechanism repairing a primary target of photodamage, namely the reaction center D1 protein in the photosystem II (PSII), is present on the thylakoid membrane, called the PSII repair cycle [3]
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