ACCUMULATION OF A MISFOLDED PROTEIN IN CHLOROPLASTS TRIGGERS AN ORGANELLE-SPECIFIC RESPONSE

CANTOIA, ALEJO; BLANCO, NICOLAS; ROSANO, GERMÁN L; CECCARELLI, EDUARDO A

Online

Congreso; CONGRESO ONLINE LVI SAIB ?XV SAMIGE; 2020

Sociedad Arg. de investigación en Bioquímica y Biología Molecular - Sociedad Argentina de Microbiología

Proteostasis maintenance plays a key role in all living systems. It is especially important in organelles, where protein turnover and 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 is overloaded and cells trigger a response to deal with the accumulation of unwanted proteins. This process is called the unfolded protein response (UPR), which is a mechanism that activates signaling pathways leading to increased expression of specific chaperones and proteases, with the aim of restoring proteostasis. We propose that the accumulation of misfolded proteins in chloroplasts 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 marked decrease in solubility of the protein. Taq3-FNR and a wild-type version (WT-FNR) were inserted in a binary plasmid in frame with a transit peptide allowing for import into chloroplasts. Each version of FNR was also fused in-frame with cyan fluorescent protein (CFP) for confocal microscopy assays. All variants of FNR (with or without CFP fusion) were transiently expressed in Nicotiana benthamiana leaves. The expression of the FNR proteins and chloroplast chaperones - like ClpB - was assessed by western blot. Proteome disturbances caused by treatments were assessed by a label-free quantitation approach using LC/MS/MS. By measuring fluorescence intensity in leaves by confocal microscopy and band intensity in protein extracts by Western blot, we confirmed that Taq3-FNR accumulates in chloroplasts at a lower concentration than WT-FNR, suggesting increased turnover. Overexpression of some chloroplastic PQC proteins was found in leaves infiltrated with Taq3-FNR. Our results suggest that Taq3-FNR generates a specific response in chloroplasts, due to its presence in the stroma. As ClpB is a nuclear 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 further indicate that accumulation of Taq3-FNR in the chloroplast causes an increment in the levels of chloroplastic PQC chaperones.