Reconstitution of the tubular endoplasmic reticulum network with purified components

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Abstract

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation1,2,3,4,5, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs6,7) and the curvature-stabilizing protein Yop1p (refs8,9) fromSaccharomyces cerevisiaeform a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon8and receptor expression-enhancing proteins (REEPs)10from different species. Atlastin, the vertebrate orthologue 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.

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