Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum (ER) is particularly intriguing, as it is comprised of morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation1–5, but how exactly they mediate network formation and whether they are all required is unclear. Here, we have reconstituted a dynamic tubular membrane network with purified ER proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p6,7 and the curvature-stabilizing protein Yop1p8,9 from Saccharomyces cerevisiae form a tubular network upon GTP addition. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favors the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon8 and REEP10 proteins from different species. Atlastin, the vertebrate ortholog of Sey1p6,11, forms a GTP-hydrolysis dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly
