Phosphorylation at serine-260 of Toc33 is essential for chloroplast biogenesis.

Chloroplast biogenesis, essential for photosynthesis, depends on the import of nuclear-encoded proteins through the translocon at the outer envelope of chloroplasts (TOC) complexes. Despite its importance, the mechanisms regulating this process remain largely elusive. We identify serine-260 (S260) as a critical phosphorylation site in Toc33, a core TOC component. This phosphorylation stabilizes Toc33 by preventing its ubiquitination and degradation. Constitutive triple response 1 (CTR1), a negative regulator of ethylene signaling, and its paralog RAF-like kinase are involved in phosphorylating Toc33. Disruption of Toc33 phosphorylation impairs its stability and photosynthetic protein import, consequently affecting chloroplast structural integrity and biogenesis. Our findings underscore the essential role of TOC phosphorylation in chloroplast biogenesis and reveal an unexpected regulatory network involving RAF-like kinases in organelle development.