presented his group's efforts to understand how organelle shapes are formed and maintained. The fi ndings identify two protein families that create tubules out of ER membrane. A sphere is the most stable membrane shape; deformations that increase membrane curvature cost energy and must be actively stabilized. Rapoport and colleagues, including Gia Voeltz, used an in vitro system for ER network formation to identify factors that create curved membrane shapes. With just membranes, salt, and GTP, their system produces a network of ER tubules.< I D > J C B 1 7 2 4 M R _ F i g. 1. e p s < / I D > This simple, self-contained system was not ideal for identifying the shape-creating components. "When we fi rst saw [that] everything was already in the membrane," said Rapoport in his talk, "we thought, 'what a bummer.'" But the group found a way around this diffi culty by using small molecule inhibitors to block in vitro ER formation, and then identifying the inhibitors' targets. One such target was an integral membrane protein called Reticulon4a (Rtn4a), previously named for its localization to ER membranes. All eukaryotes express at least one homologue of Rtn4a, and the proteins are the fi rst known markers specifi cally localized to the tubular ER and absent from sheets. Cells overexpressing Rtn proteins formed more tubules, but loss of the two yeast members did not prevent tubule formation under normal conditions. Only when mutant cells were subjected to osmotic stress were their tubules lost. Rtn proteins form homo-and hetero-oligomers, so the group fi gured that another Rtn-interacting protein might be required for tubule formation. Indeed, they found that Rtn pulled down another ubiquitous integral membrane protein called DP-1. Loss of both the yeast DP-1 and the more abundant of its two Rtns now blocked tubule formation. The group has proposed that Rtn and DP-1 might be wedge-shaped, with their wider sides in the outer membrane leafl et. The presence of these proteins would thus favor a highly curved membrane. They now plan to test whether purifi ed Rtn and DP-1 can turn liposomes into tubules. NL

The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by the curvature-stabilizing proteins reticulons and DP1/Yop1p, but how the sheets are formed is unclear. Here we identify several sheet-enriched membrane proteins in the mammalian ER, including proteins that translocate and modify newly synthesized polypeptides, as well as coiled-coil membrane proteins that are highly upregulated in cells with proliferated ER sheets, all of which are localized by membrane-bound polysomes. These results indicate that sheets and tubules correspond to rough and smooth ER, respectively. One of the coiled-coil proteins, Climp63, serves as a “luminal ER spacer” and forms sheets when overexpressed. More universally, however, sheet-formation appears to involve the reticulons and DP1/Yop1p, which localize to sheet edges and whose abundance determines the ratio of sheets to tubules. These proteins may generate sheets by stabilizing the high curvature of edges.

The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man 1, 2 and are implicated in regulation of sterol pathways 3,4 and in signal transduction 5 . The structure of the full-length yeast ORP Osh4 was determined at 1.5-1.9 Å resolution in complexes with ergosterol, cholesterol, and 7-, 20-, and 25-hydroxycholesterol. A single sterol molecule binds in a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible N-terminal lid and surrounded by functionally critical basic residues. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 Å resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favoring transport across aqueous barriers and transmitting signals. The unliganded structure exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. Based on these observations we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.OSBP was first discovered 6,7 as a cytosolic receptor for oxysterols that downregulate cholesterol synthesis 8 . The cloning of OSBP 1 led to the identification of a large family of OSBP-related proteins, the ORPs, with 7 members in S. cerevisiase and 12 in H. sapiens 2 . ORPs are essential for life in eukaryotes. The deletion of all 7 ORPs leads to severe defects in sterol and lipid distribution and endocytosis in yeast 3,4 , and OSBP is essential for embryonic development in mice (M. Brown, personal communication). All ORPs contain a core OSBPrelated domain (ORD), and many also contain pleckstrin homology (PH) domains, transmembrane regions, endoplasmic reticulum (ER)-targeting FFAT motifs, GOLD domains, and/or ankyrin repeats 2 . These additional domains localize ORPs by binding to phosphoinositides 9 , the ER protein VAP 10 , and other targeting signals. The localization of ORPs is dynamic. Oxysterol binding changes the subcellular localization of certain ORPs from the cytosol to the Golgi or ER 9,11 . ORPs can bind lipids other than oxysterols, including phosphoinositides and phosphatidic acid 12,13 . OSBP is a cholesterol sensing regulator of two protein phosphatases, a PTPPBS family member, and Ser/Thr phosphatase PP2A 5 .The structure of full-length Osh4 was determined by multiple isomorphous replacement and refined to a free R-factor of 23 % at 1.5 Å resolution (Supplementary Fig. 1 Table 1). Osh4 is built around a central antiparallel β-sheet of 19 strands (residues 115-293) which form a nearly complete β-barrel (Fig. 1a, Supplementary Fig. 2). The strands of the barrel are pitched at ~45 degrees to its axis. The barrel has structural similarity to the large β-barrels of various bacterial outer membrane transporters 14 as scored by Dali 15 ( Supplementary Fig. 3). There is no PH domain within the ORD 13 . A tunnel with largely hydrophobic walls...

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.

Increasing the size of the ER by lipid synthesis helps the cell deal with ER stress.

In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each other in vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.

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.

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...