Reconstitution of the tubular endoplasmic reticulum network with purified components.

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 formation, 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 (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form 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 reticulon and receptor expression-enhancing proteins (REEPs) from different species. Atlastin, the vertebrate orthologue of Sey1p, 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.

The ER morphology-regulating lunapark protein induces the formation of stacked bilayer discs.

Lunapark (Lnp) is a conserved membrane protein that localizes to and stabilizes three-way junctions of the tubular ER network. In higher eukaryotes, phosphorylation of Lnp may contribute to the conversion of the ER from tubules to sheets during mitosis. Here, we report on the reconstitution of purified Lnp with phospholipids. Surprisingly, Lnp induces the formation of stacked membrane discs. Each disc is a bicelle, with Lnp sitting in the bilayer facing both directions. The interaction between bicelles is mediated by the cytosolic domains of Lnp, resulting in a constant distance between the discs. A phosphomimetic Lnp mutant shows reduced bicelle stacking. Based on these results, we propose that Lnp tethers ER membranes in vivo in a cell cycle-dependent manner. Lnp appears to be the first membrane protein that induces the formation of stacked bicelles.

A Partial Calcium-Free Linker Confers Flexibility to Inner-Ear Protocadherin-15.

Tip links of the inner ear are protein filaments essential for hearing and balance. Two atypical cadherins, cadherin-23 and protocadherin-15, interact in a Ca2+-dependent manner to form tip links. The largely unknown structure and mechanics of these proteins are integral to understanding how tip links pull on ion channels to initiate sensory perception. Protocadherin-15 has 11 extracellular cadherin (EC) repeats. Its EC3-4 linker lacks several of the canonical Ca2+-binding residues, and contains an aspartate-to-alanine polymorphism (D414A) under positive selection in East Asian populations. We present structures of protocadherin-15 EC3-5 featuring two Ca2+-binding linker regions: canonical EC4-5 linker binding three Ca2+ ions, and non-canonical EC3-4 linker binding only two Ca2+ ions. Our structures and biochemical assays reveal little difference between the D414 and D414A variants. Simulations predict that the partial Ca2+-free EC3-4 linker exhibits increased flexural flexibility without compromised mechanical strength, providing insight into the dynamics of tip links and other atypical cadherins.