The tubular structure of the endoplasmic reticulum (ER) appears to be generated by integral membrane proteins, the reticulons and a protein family consisting of DP1 in mammals and Yop1p in yeast. Here, individual members of these families were found to be sufficient to generate membrane tubules. When we purified yeast Yop1p and incorporated it into proteoliposomes, narrow tubules (approximately 15 to 17 nanometers in diameter) were generated. Tubule formation occurred with different lipids; required essentially only the central portion of the protein, including its two long hydrophobic segments; and was prevented by mutations that affected tubule formation in vivo. Tubules were also formed by reconstituted purified yeast Rtn1p. Tubules made in vitro were narrower than normal ER tubules, due to a higher concentration of tubule-inducing proteins. The shape and oligomerization of the "morphogenic" proteins could explain the formation of the tubular ER.

Graphical Abstract Highlights d Super-resolution live-cell imaging up to 266 fps at 97-nm resolution d Hitchhiking interactions among organelles remodel ER and mitochondrial networks d ER-mitochondrion contacts promote coalescence of mitochondrial membranes d Collision of late endosomes or lysosomes carried along microtubules split ER tubules

The endoplasmic reticulum (ER) consists of tubules that are shaped by the reticulons and DP1/Yop1p, but how the tubules form an interconnected network is unknown. Here, we show that mammalian atlastins, which are dynamin-like, integral membrane GTPases, interact with the tubule-shaping proteins. The atlastins localize to the tubular ER and are required for proper network formation in vivo and in vitro. Depletion of the atlastins or overexpression of dominant-negative forms inhibits tubule interconnections. The Sey1p GTPase in S. cerevisiae is likely a functional ortholog of the atlastins; it shares the same signature motifs and membrane topology and interacts genetically and physically with the tubule-shaping proteins. Cells simultaneously lacking Sey1p and a tubule-shaping protein have ER morphology defects. These results indicate that formation of the tubular ER network depends on conserved dynamin-like GTPases. Since atlastin-1 mutations cause a common form of hereditary spastic paraplegia, we suggest ER shaping defects as a novel neuropathogenic mechanism.

Cellular organelles have characteristic morphologies that arise as a result of different local membrane curvatures. A striking example is the endoplasmic reticulum (ER), which consists of ER tubules with high curvature in cross-section, peripheral ER sheets with little curvature except at their edges and the nuclear envelope with low curvature except where the nuclear pores are inserted. The ER may be shaped by several mechanisms. ER tubules are often generated through their association with the cytoskeleton and stabilized by two families of integral membrane proteins, the reticulons and DP1/Yop1p. Similar to how curvature is generated in budding vesicles, these proteins may use scaffolding and hydrophobic insertion mechanisms to shape the lipid bilayer into tubules. In addition, proteins of the dynamin family may deform the ER membrane to generate a tubular network. Mechanisms affecting local membrane curvature may also shape peripheral ER sheets and the nuclear envelope as well as mitochondria and caveolae.

We recently identified a class of membrane proteins, the reticulons and DP1/Yop1p, which shape the tubular endoplasmic reticulum (ER) in yeast and mammalian cells. These proteins are highly enriched in the tubular portions of the ER and virtually excluded from other regions. To understand how they promote tubule formation, we characterized their behavior in cellular membranes and addressed how their localization in the ER is determined. Using fluorescence recovery after photobleaching, we found that yeast Rtn1p and Yop1p are less mobile in the membrane than normal ER proteins. Sucrose gradient centrifugation and cross-linking analyses show that they form oligomers. Mutants of yeast Rtn1p, which no longer localize exclusively to the tubular ER or are even totally inactive in inducing ER tubules, are more mobile and oligomerize less extensively. The mammalian reticulons and DP1 are also relatively immobile and can form oligomers. The conserved reticulon homology domain that includes the two membrane-embedded segments is sufficient for the localization of the reticulons to the tubular ER, as well as for their diffusional immobility and oligomerization. Finally, ATP depletion in both yeast and mammalian cells further decreases the mobilities of the reticulons and DP1. We propose that oligomerization of the reticulons and DP1/Yop1p is important for both their localization to the tubular domains of the ER and for their ability to form tubules.Most organelles have defined shapes that are evolutionarily conserved, but little is known about how the particular architecture of an organelle is formed and maintained. The structure of the endoplasmic reticulum (ER) 6 is one such example. The ER consists of the nuclear envelope, a double membrane surrounding the nucleus, and the peripheral ER. The peripheral ER is further morphologically divided into flat sheets that extend two-dimensionally, and tubules that have high curvature in their cross-section. In yeast, the bulk of the peripheral ER is maintained at the cell cortex with a small number of tubules connecting it to the nuclear envelope, whereas in higher eukaryotes the peripheral ER is found throughout the entire cytoplasm. Regardless of the spatial arrangement, however, these domains form a continuous membrane and luminal system (1-5).Recently, we identified a class of integral membrane proteins, consisting of two distinct protein families, that structurally shape ER tubules (6). The first family is the reticulons, including four reticulon genes in mammals (RTN1-4), and two in yeast (RTN1 and RTN2). The other family consists of the DP1/Yop1p proteins, which includes mammalian DP1 and its yeast homolog, Yop1p. Both families are found in all eukaryotes and are ubiquitously expressed. These proteins localize predominantly to the tubular ER and avoid the low curvature domains of the nuclear envelope and the peripheral sheets in a variety of eukaryotic species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and in...

The generation of the tubular network of the endoplasmic reticulum (ER) requires homotypic membrane fusion that is mediated by the dynamin-like, membrane-bound GTPase atlastin (ATL). Here, we have determined crystal structures of the cytosolic segment of human ATL1, which give insight into the mechanism of membrane fusion. The structures reveal a GTPase domain and athree-helix bundle, connected by a linker region. One structure corresponds to a prefusion state, in which ATL molecules in apposing membranes interact through their GTPase domains to form a dimer with the nucleotides bound at the interface. The other structure corresponds to a postfusion state generated after GTP hydrolysis and phosphate release. Compared with the prefusion structure, the three-helix bundles of the two ATL molecules undergo a major conformational change relative to the GTPase domains, which could pull the membranes together. The proposed fusion mechanism is supported by biochemical experiments and fusion assays with wild-type and mutant full-length Drosophila ATL. These experiments also show that membrane fusion is facilitated by the C-terminal cytosolic tails following the two transmembrane segments. Finally, our results show that mutations in ATL1 causing hereditary spastic paraplegia compromise homotypic ER fusion.protein structure | membrane remodeling | organelle shaping | spastic paraplegia gene 3A | endoplasmic reticulum network formation

Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome.

Mitofusin 1 (MFN1) mediates mitochondrial fusion, but the mechanisms involved are unclear. Qi et al. present the crystal structures of a minimal GTPase domain of human MFN1, which suggest that MFN1 tethers apposing membranes through nucleotide-dependent dimerization.