STIC2 selectively binds ribosome-nascent chain complexes in the cotranslational sorting of Arabidopsis thylakoid proteins

Chloroplast-encoded multi-span thylakoid membrane proteins are crucial for photosynthetic complexes, yet the coordination of their biogenesis remains poorly understood. To identify factors that specifically support the cotranslational biogenesis of the reaction center protein D1 of photosystem (PS) II, we generated and affinity-purified stalled ribosome-nascent chain complexes (RNCs) bearing D1 nascent chains. Stalled RNCs translating the soluble ribosomal subunit uS2c were used for comparison. Quantitative tandem-mass spectrometry of the purified RNCs identified around 140 proteins specifically associated with D1 RNCs, mainly involved in protein and cofactor biogenesis, including chlorophyll biosynthesis, and other metabolic pathways. Functional analysis of STIC2, a newly identified D1 RNC interactor, revealed its cooperation with chloroplast protein SRP54 in the de novo biogenesis and repair of D1, and potentially other cotranslationally-targeted reaction center subunits of PSII and PSI. The primary binding interface between STIC2 and the thylakoid insertase Alb3 and its homolog Alb4 was mapped to STIC2’s β-sheet region, and the conserved Motif III in the C-terminal regions of Alb3/4. Synopsis The biogenesis and maintenance of photosynthetic complexes requires precise coordination of cotranslational maturation, folding, targeting and insertion of their plastid-encoded subunits. This work offers insight into the interactome of photosystem II reaction center-specific ribosome-nascent chain complexes, and identifies a role for STIC2 in their cotranslational protein sorting. Proteins from metabolic, biogenesis, and protein processing pathways associate with D1-translating ribosomes. Knock-out of STIC2 and the targeting factor cpSRP54 in Arabidopsis causes sensitivity to high light and low accumulation of photosystem subunits. STIC2 partially associates with ribosomes and affects chloroplast translation. The C-terminal motif III of the thylakoid membrane proteins Alb3 and Alb4 mediates their interaction within the β-sheet region of STIC2. The chloroplast protein STIC2 associates with ribosomes translating the photosystem II reaction center protein D1 and supports insertion into the thylakoid membrane.