Lipid metabolism and contact site homologs are present at the chloroplast envelope-thylakoid interface.

Biogenesis and maintenance of the photosynthetic thylakoid membrane requires transport of lipids from their site of synthesis in the chloroplast envelopes to their destination in the thylakoid. While vesicle trafficking is likely involved, we hypothesized a complementary mechanism involving direct membrane interactions. Using domain homology and proteomic profiling of chloroplast membrane fractions, we identified candidate lipid transport proteins present in a distinct, intermediate-density membrane population. This fraction contained an overrepresentation of lipid metabolic enzymes and proteins homologous to known membrane organization factors. Several candidates, including TVP38 FAMILY PROTEIN (TVPFP), PLASMA MEMBRANE FUSION PROTEIN (PMFP), and LETM1-LIKE, localized to discrete subdomains within chloroplasts. Loss-of-function tvpfp or pmfp mutants exhibited altered chloroplast ultrastructure, including changes in thylakoid-envelope proximity, supporting their roles in maintaining membrane architecture. These findings, which identify a chloroplast membrane subdomain enriched in proteins with specialized functions, offer a foundation for elucidating the molecular architecture of these regions.