A conserved sequence insert of fibrillins is involved in plastoglobule association and lipid binding.

Plastoglobule lipid droplets of chloroplasts serve complex roles affecting plant development, stress tolerance, and photosynthesis. They harbor a set of ~40 proteins that collectively dictate plastoglobule functions. Due to the monolayer structure of plastoglobules, which encompasses a neutral lipid core, these proteins must associate monotopically on the plastoglobule surface. However, targeting determinants have not been identified for plastoglobule proteins, and the protein-membrane interaction mechanisms that establish the plastoglobule proteome remain unclear. Here, we demonstrate that most plastoglobule-localized fibrillins (FBNs) harbor an extra sequence insert with a predicted amphipathic helical structure placed at the lip of the β-barrel. Deletions of the insert in two plastoglobule-targeted FBNs disrupts their localization, indicating that the inserts are necessary for effective plastoglobule association. Molecular dynamics simulations support the specific interaction of the amphipathic helical insert of AtFBN1a with membranes rich in lipid-packing defects, which are expected to be especially prevalent on the tightly curved surface of plastoglobules. Proteomic analyses indicate that AtFBN1a influences the plastoglobule proteome through outcompeting and recruiting specific proteins. We also demonstrate that the plastoglobule-localized FBNs, AtFBN1a and AtFBN7a, bind unsaturated fatty acids (FAs), particularly C18:1, and that elimination of the amphipathic helical inserts suppresses FA binding in AtFBN1a but promotes FA binding in AtFBN7a. Predicted amphipathic helices can be identified on two-thirds of plastoglobule proteins, which indicates that the use of amphipathic helices may be a general mechanism by which proteins selectively associate with plastoglobules.