TheArabidopsisNOT4A E3 ligase coordinates PGR3 expression to regulate chloroplast protein translation

Abstract

31Chloroplast function requires the coordinated action of nuclear-and chloroplast-derived 32 proteins, including several hundred nuclear-encoded pentatricopeptide repeat (PPR) 33 proteins that regulate plastid mRNA metabolism. Despite their large number and importance, 34 regulatory mechanisms controlling PPR expression are poorly understood. Here we show 35 that the Arabidopsis NOT4A ubiquitin-ligase positively regulates PROTON GRADIENT 3 36 (PGR3), a PPR protein required for translating 30S ribosome subunits and several thylakoid-37 localised photosynthetic components within chloroplasts. Loss of NOT4A function leads to a 38 strong depletion of plastid ribosomes, which reduces mRNA translation and negatively 39 impacts photosynthetic capacity, causing pale-yellow and slow-growth phenotypes. 40Quantitative transcriptome and proteome analyses reveal that these defects are due to a 41 lack of PGR3 expression in not4a, and we show that normal plastid function is restored 42 through transgenic PGR3 expression. Our work identifies NOT4A as crucial for ensuring 43 robust photosynthetic function during development and stress-response, through modulating 44 PGR3 levels to coordinate chloroplast protein synthesis.The synthesis of energy from the sun, photosynthesis, supports organic life on earth. Light 68 harvesting in green plants takes place within the specialized chloroplast organelle, believed 69 to have arisen from engulfment of a photosynthetic prokaryote by an ancestral eukaryotic 70 cell (Archibald, 2015). Coevolution and merging of these organisms has resulted in nuclear 71 and chloroplast genomes separated within cellular compartments. In land plants, the 72 chloroplast genome comprises of ~130 genes, yet chloroplasts contain around 3000 different 73 proteins (Zoschke and Bock, 2018). Consequently, chloroplast function requires expression 74 not only of chloroplast encoded proteins, but a multitude of nuclear encoded genes, which 75 are imported into chloroplasts post-translationally. One such group of nuclear derived factors 76 is the pentatricopeptide repeat domain (PPR) containing proteins. The PPR protein family 77 has significantly expanded in plants (~450 in Arabidopsis, vs <10 in humans and yeast; 78 (Schmitz-Linneweber and Small, 2008)), and members are characterized by a 35-amino acid 79 repeat sequence that facilitates RNA binding and enables them to provide critical gene 80 expression control within chloroplasts and mitochondria (Barkan and Small, 2014). Through 81 binding to organellar RNAs, PPR proteins stabilize gene transcripts, facilitate post-82 transcriptional processing and promote translation of the encoded proteins (Barkan and 83 Small, 2014; Manna, 2015). Whilst their function in the regulation of gene expression control 84 within organelles has been described, including many of the RNA species to which they 85 bind, little is known about how their expression is regulated prior to import. 86Precise, selective removal of proteins is essential to cellular development and response. In 87...

Cellular Control of Protein Turnover via the Modification of the Amino Terminus

“…In plants as in yeast, amino-terminal acetylation does not destabilize the bulk of cytoplasmic proteins [ 70 ]. However, this does not rule out the existence of specific plant proteins with Ac/N-degrons because both Doa10 and Not4 [ 71 ], in particular, have homologs in plants.…”

The Secret Life of Chloroplast Precursor Proteins in the Cytosol

“…Mutations in the plant homolog of the yeast E3 ligase NOT4, which recognizes N-acetylated proteins as degron, showed a chloroplast-related phenotype but revealed no indication for systematic changes of precursor stability. However, this cannot be excluded for individual proteins (Bailey et al, 2020).…”