AtTOC159 Receptors are Targeted to the Chloroplast Outer Membrane by a β-Signal and Galactolipid-Specific Transit Peptide-Like Sequence at the C-Terminus

Chloroplasts, the essential organelles responsible for photosynthesis, rely on the coordinated import of nuclear-encoded proteins for their biogenesis and function. TOC159 receptors are critical components of the translocon at the outer membrane of chloroplasts (TOC complex), responsible for recognizing and importing preproteins. However, the mechanisms by which TOC159 itself is targeted and integrated into the chloroplast outer envelope are only partially understood. In this study, we explore the structural aspects of the C-terminal membrane (M) domain of Arabidopsis thaliana TOC159, with a focus on its role in chloroplast outer envelope targeting and TOC complex integration. Structural predictions using AlphaFold and far-UV circular dichroism (CD) spectroscopy experiments suggested that the M domain forms an integral β-barrel that fuses with the β-barrel of TOC75, forming a hybrid channel at the chloroplast outer envelope. We identified a novel bi-partite targeting signal within the M domain, composed of a C-terminal β-signal with the consensus sequence G-Q-Φ-[ST]-Φ-[RK]-X-[SN]-[ST] and a transit peptide (TP)-like sequence. Using fluorescence microscopy and mutational analyses, we demonstrated that the β-signal is essential for chloroplast targeting, while the TP-like sequence enhances targeting ejiciency. Furthermore, CD spectroscopy and Langmuir-Blodgett trough experiments revealed that the TP-like sequence preferentially interacts with galactolipids unique to the chloroplast outer membrane, particularly sulfoquinovosyl diacylglycerol (SQDG), facilitating chloroplast outer envelope targeting specificity. Overall, our findings provide a deeper understanding of TOC159 receptor targeting mechanisms, TOC complex biogenesis and highlight the broader significance of lipid-mediated targeting in intracellular protein trajicking. These results pave the way for future studies on the role of TP-like sequences in chloroplast outer membrane protein targeting and other novel targeting pathways more generally.