Accumulation of a Misfolded Protein in Chloroplasts Triggers an Organelle-Specific Response

CANTOIA, ALEJO; BLANCO, NICOLÁS E; CECCARELLI, EDUARDO A.; ROSANO, GERMÁN L.

Congreso; LVI SAIB Meeting ?XV SAMIGE Meeting; 2020

Sociedad Argentina de Investigaciones en Bioquímica y Biología Molecular

Proteostasis maintenance plays a key role in all living systems. It is especially important in organelles, where protein turnoverand replacement are highly elevated. Chaperones and proteases are responsible for carrying out this protein quality control(PQC). They recognize and remove unnecessary, unfolded, or aggregated proteins. However, in certain situations the PQC isoverloaded and cells trigger a response to deal with the accumulation of unwanted proteins. This process is called the unfoldedprotein response (UPR), which is a mechanism that activates signaling pathways leading to increased expression of specificchaperones and proteases, with the aim of restoring proteostasis. We propose that the accumulation of misfolded proteins inchloroplasts can trigger a chloroplastic UPR in plants. To test this, we have used a mutant version of ferredoxin NADP+reductase (FNR) bearing a deletion in amino acids Asp289, Trp290 and Ile291 (Taq3-FNR). These mutations resulted in a markeddecrease in solubility of the protein. Taq3-FNR and a wild-type version (WT-FNR) were inserted in a binary plasmid in framewith a transit peptide allowing for import into chloroplasts. Each version of FNR was also fused in-frame with cyan fluorescentprotein (CFP) for confocal microscopy assays. All variants of FNR (with or without CFP fusion) were transiently expressedin Nicotiana benthamiana leaves. The expression of the FNR proteins and chloroplast chaperones - like ClpB - was assessedby western blot. Proteome disturbances caused by treatments were assessed by a label-free quantitation approach usingLC/MS/MS. By measuring fluorescence intensity in leaves by confocal microscopy and band intensity in protein extracts byWestern blot, we confirmed that Taq3-FNR accumulates in chloroplasts at a lower concentration than WT-FNR, suggestingincreased turnover. Overexpression of some chloroplastic PQC proteins was found in leaves infiltrated with Taq3-FNR. Ourresults suggest that Taq3-FNR generates a specific response in chloroplasts, due to its presence in the stroma. As ClpB is anuclear encoded protein, the response should involve a chloroplasts-to-nucleus communication, a common feature in other UPRs. Finally, we are also studying the effect of Taq3-FNR and CFP-Taq3-FNR stable expression in Arabidopsis thaliana.Early results indicate that the phenotype of these stable lines resembles that of ClpB-overexpressing lines, which furtherindicate that accumulation of Taq3-FNR in the chloroplast causes an increment in the levels of chloroplastic PQC chaperones