Growing evidence suggests that close appositions between the endoplasmic reticulum (ER) and other membranes, including appositions with the plasma membrane (PM), mediate exchange of lipids between the two bilayers. The mechanisms of such exchange, which allows lipid transfer independently of vesicular transport, remain poorly understood. The presence of an SMP (synaptotagmin-like-mitochondrial-lipid binding protein) domain, a proposed lipid binding module, in several proteins localized at membrane contact sites raised the possibility that such domains may be implicated in lipid transport1,2. SMP-containing proteins include components of the ERMES complex, an ER-mitochondrial tether3, and the Extended-Synaptotagmins/tricalbins, which are ER-PM tethers4-6. Here we present at 2.44 Å resolution the crystal structure of a fragment of Extended-Synaptotagmin 2 (E-Syt2), including an SMP domain and two adjacent C2 domains. The SMP domain has a beta-barrel structure like protein modules in the TULIP superfamily. It dimerizes to form a ~90 Å long cylinder traversed by a channel lined entirely with hydrophobic residues, with the two C2A-C2B fragments forming arched structures flexibly linked to the SMP domain. Importantly, structural analysis complemented by mass spectrometry revealed the presence of glycerophospholipids in the E-Syt2 SMP channel, indicating a direct role for E-Syts in lipid transport. These findings provide strong evidence for a role of SMP domain containing proteins in the control of lipid transfer at membrane contact sites and have broad implication beyond the field of ER to PM appositions.

Acute metabolic changes of plasma membrane (PM) lipids, such as those mediating signaling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the Extended-Synaptotagmins (E-Syts), ER proteins which function as PI(4,5)P2 and Ca2+-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro and such transfer requires Ca2+ and their SMP domain, a lipid-harboring module. Genome edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but display enhanced and sustained accumulation of PM diacylglycerol (DAG) upon PI(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syts-dependent ER-PM tethers in response to stimuli that cleave PI(4,5)P2 and elevate Ca2+ may help reverse accumulation of DAG in the PM by transferring it to the ER for metabolic recycling.

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Influenza is a contagious respiratory illness in humans caused by influenza A and B viruses that results in an annual average of 36,000 deaths in the United States alone (www.cdc. gov/Flu). Influenza A viruses, infecting birds and mammals, periodically cause widespread pandemics, the most deadly of which was the 1918 "Spanish Flu" pandemic that claimed an estimated 50 million lives (1). In recent years, the appearance of new H5N1 and H1N1 influenza A viruses, known as "avian flu" and "swine flu," respectively, has fueled fears of an impending deadly influenza pandemic in the 21st century. Moreover, the rapid emergence of influenza virus strains resistant to current antiviral drugs directed against influenza A neuraminidase and M2 ion channel accentuate the need for the development of new classes of influenza antivirals. Toward this end, several recent structural and functional studies of influenza proteins have illuminated new potential drug targets in influenza (2, 3).One such target is the non-structural protein 1 of influenza A, NS1A, a key multifunctional virulence factor produced in the host infected cell that plays a critical role in evading the host antiviral response (4). This highly conserved protein is composed of two domains, the 73-residue N-terminal doublestranded RNA-binding domain (RBD) 4 and the C-terminal (residues 86-end) effector domain (ED), joined by a flexible linker (Fig. 1). By binding non-specifically to dsRNA, the N-terminal RBD functions primarily to inhibit the interferon-induced 2Ј-5Ј oligonucleotide A synthetase/RNase L pathway (5). The C-terminal ED binds a plethora of host cellular proteins, including the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) (6, 7), the p85␤ subunit of phosphatidylinositol 3-kinase (PI3K) (8, 9), protein kinase R (PKR) (10, 11), and human tripartite motif 25, TRIM25, the E3 ubiquitin ligase of the retinoic acid-inducible gene I (RIG-I) (12, 13). All of these activities of NS1A ultimately contribute to the ability of influenza virus to suppress host responses to infection, including interferon production and apoptosis, critical to the life cycle of the virus. The recent developments of novel NS1 antagonists (14) and attenuated influenza viruses containing * This work was supported, in whole or in part, by National Institutes of Health Grant U54-GM094597 through the NIGMS Protein Structure Initiative (to G. T. M.) and National Institutes of Health Grants U01 AI074497 (to G. T. M. and R. M. K.) and R01 AI11772 (to R. M. K. 3 To whom correspondence may be addressed. E-mail: guy@cabm.rutgers.edu. 4 The abbreviations used are: RBD, RNA-binding domain; ED, effector domain; HSQC, heteronuclear single quantum coherence; TROSY, transverse relaxation optimized spectroscopy; SUMO, small ubiquitin-like modifier.

Plasma membrane (PM) phosphoinositides play essential roles in cell physiology, serving as both markers of membrane identity and signaling molecules central to the cell's interaction with its environment. The first step in PM phosphoinositide synthesis is the conversion of phosphatidylinositol (PI) to PI4P, the precursor of PI(4,5)P and PI(3,4,5)P This conversion is catalyzed by the PI4KIIIα complex, comprising a lipid kinase, PI4KIIIα, and two regulatory subunits, TTC7 and FAM126. We here report the structure of this complex at 3.6-Å resolution, determined by cryo-electron microscopy. The proteins form an obligate ∼700-kDa superassembly with a broad surface suitable for membrane interaction, toward which the kinase active sites are oriented. The structural complexity of the assembly highlights PI4P synthesis as a major regulatory junction in PM phosphoinositide homeostasis. Our studies provide a framework for further exploring the mechanisms underlying PM phosphoinositide regulation.

Tethering proteins play a key role in vesicular transport, ensuring that cargo arrives at a specific destination. The bacterial effector protein SidC and its paralog SdcA have been described as tethering factors encoded by the intracellular pathogen Legionella pneumophila. Here, we demonstrate that SidC proteins are important for early events unique to maturation of vacuoles containing Legionella and discover mono-ubiquitination of Rab1 as a new SidC-dependent activity. The crystal structure of the SidC N-terminus revealed a novel fold that is important for function and could be involved in Legionella adaptations to evolutionarily divergent host cells it encounters in natural environments.

We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.DOI: http://dx.doi.org/10.7554/eLife.04463.001

Fragile-X syndrome is the most commonly inherited cause of autism and mental disabilities. The Fmr1 (Fragile-X Mental Retardation 1) gene is essential in humans and Drosophila for the maintenance of neural stem cells, and Fmr1 loss results in neurological and reproductive developmental defects in humans and flies. FMRP (Fragile-X Mental Retardation Protein) is a nucleo-cytoplasmic shuttling protein, involved in mRNA silencing and translational repression. Both Zfrp8 and Fmr1 have essential functions in the Drosophila ovary. In this study, we identified FMRP, Nufip (Nuclear Fragile-X Mental Retardation Protein-interacting Protein) and Tral (Trailer Hitch) as components of a Zfrp8 protein complex. We show that Zfrp8 is required in the nucleus, and controls localization of FMRP in the cytoplasm. In addition, we demonstrate that Zfrp8 genetically interacts with Fmr1 and tral in an antagonistic manner. Zfrp8 and FMRP both control heterochromatin packaging, also in opposite ways. We propose that Zfrp8 functions as a chaperone, controlling protein complexes involved in RNA processing in the nucleus.