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The reticulons and receptor expression-enhancing proteins (REEPs) in the endoplasmic reticulum (ER) are necessary and sufficient for generating ER tubules. However, the mechanism of curvature generation remains elusive. Here, we systematically analyze components of the REEP family based on AI-predicted structures. In yeast REEP Yop1p, TM1/2 and TM3/4 form hairpins and TM2-4 exist as a bundle. Site-directed cross-linking reveals that TM2 and TM4 individually mediate homotypic dimerization, allowing further assembly into a curved shape. Truncated Yop1p lacking TM1 (equivalent to REEP1) retains the curvature-generating capability, undermining the role of the intrinsic wedge. Unexpectedly, both REEP1 and REEP5 fail to replace Yop1p in the maintenance of ER morphology, mostly due to a subtle difference in oligomerization tendency, which involves not only the TM domains, but also the TM-connecting cytosolic loop and previously neglected C-terminal helix. Several hereditary spastic paraplegia-causing mutations in REEP1 appear at the oligomeric interfaces identified here, suggesting compromised self-association of REEP as a pathogenic mechanism. These results indicate that membrane curvature stabilization by integral membrane proteins is dominantly achieved by curved, oligomeric scaffolding., (© 2023. The Author(s).)

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage-inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins., (© 2021 Chen et al.)