Single-molecule fluorescence and cross-linking reveal ligand-gated Toc34 oligomerization dynamics.

Researchers have characterized the structural framework of the chloroplast TOC (translocon at the outer envelope membrane of chloroplasts) complex, but how this machinery orchestrates preprotein recognition and translocation at the molecular level remains unclear. Toc34, a membrane-anchored GTPase receptor within the TOC complex, plays a central regulatory role through its ability to oligomerize in a nucleotide-reversible, substrate-dependent manner. To investigate the dynamic behavior of Toc34, we employed single-molecule photobleaching, Förster resonance energy transfer (FRET), and chemical cross-linking to examine its oligomeric states under various conditions. Photobleaching revealed that Toc34 predominantly forms dimers in the basal state, but this dimerization is disrupted in the presence of the RuBisCO small subunit transit peptide and nonhydrolyzable GTP analogs (GMP-PNP, GTPγS), suggesting a conformational shift linked to precursor engagement and GTPase cycling. FRET analysis of GDP-bound Toc34 identified three conformational populations with distinct energy transfer efficiencies; no FRET signal was observed upon binding transit peptides/GTP analogs, indicative of increased conformational mobility or complete monomerization. Cross-linking data further support this oligomeric transition. These results demonstrate that Toc34 undergoes regulated conformational changes driven by nucleotide state and precursor binding, acting as a molecular switch that modulates TOC complex activity during chloroplast protein import. This study provides mechanistic insight into Toc34 ligand-gated conformational switching, revealing how dynamic GTPase interactions contribute to the fidelity and efficiency of chloroplast biogenesis and have broader implications for protein trafficking in plant organelles.