Membrane Trafficking: Licensing a Cargo Receptor for ER Export

SummaryQuality control in the endoplasmic reticulum prevents packaging of immature and misfolded proteins into vesicles, but the actual mechanisms involved in this process have not been defined for most cargos. A recent study demonstrates that the engagement of mature cargo with its receptor triggers the recruitment of a vesicle cargo adaptor

Four GTPases Differentially Regulate the Sec7 Arf-GEF to Direct Traffic at the trans-Golgi Network

SummaryTraffic through the Golgi complex is controlled by small GTPases of the Arf and Rab families. Guanine nucleotide exchange factor (GEF) proteins activate these GTPases to control Golgi function, yet the full assortment of signals regulating these GEFs is unknown. The Golgi Arf-GEF Sec7 and the homologous BIG1/2 proteins are effectors of the Arf1 and Arl1 GTPases. We demonstrate that Sec7 is also an effector of two Rab GTPases, Ypt1 (Rab1) and Ypt31/32 (Rab11), signifying unprecedented signaling crosstalk between GTPase pathways. The molecular basis for the role of Ypt31/32 and Rab11 in vesicle formation has remained elusive. We find that Arf1, Arl1, and Ypt1 primarily affect the membrane localization of Sec7, whereas Ypt31/32 exerts a dramatic stimulatory effect on the nucleotide exchange activity of Sec7. The convergence of multiple signaling pathways on a master regulator reveals a mechanism for balancing incoming and outgoing traffic at the Golgi

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The g-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergen

Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Data Publication with the Structural Biology Data Grid Supports Live Analysis

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

15 pags., 6 figs., 1 tab.With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.Financial support from the STC Program of the National Science Foundation through BioXFEL under agreement no. 1231306, NSF ABI Innovations award no. 1565180, and the National Institutes of Health award no. R01GM095583 is gratefully acknowledged. The use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is generously supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The HERA system for in-helium experiments at MFX was developed by Bruce Doak and funded by the Max Planck Institute for Medical Research. This work was also supported by The Center for Structural Dynamics in Biology, NIH grant P41GM139687.Peer reviewe

The neutron and its role in cosmology and particle physics

Experiments with cold and ultracold neutrons have reached a level of precision such that problems far beyond the scale of the present Standard Model of particle physics become accessible to experimental investigation. Due to the close links between particle physics and cosmology, these studies also permit a deep look into the very first instances of our universe. First addressed in this article, both in theory and experiment, is the problem of baryogenesis ... The question how baryogenesis could have happened is open to experimental tests, and it turns out that this problem can be curbed by the very stringent limits on an electric dipole moment of the neutron, a quantity that also has deep implications for particle physics. Then we discuss the recent spectacular observation of neutron quantization in the earth's gravitational field and of resonance transitions between such gravitational energy states. These measurements, together with new evaluations of neutron scattering data, set new constraints on deviations from Newton's gravitational law at the picometer scale. Such deviations are predicted in modern theories with extra-dimensions that propose unification of the Planck scale with the scale of the Standard Model ... Another main topic is the weak-interaction parameters in various fields of physics and astrophysics that must all be derived from measured neutron decay data. Up to now, about 10 different neutron decay observables have been measured, much more than needed in the electroweak Standard Model. This allows various precise tests for new physics beyond the Standard Model, competing with or surpassing similar tests at high-energy. The review ends with a discussion of neutron and nuclear data required in the synthesis of the elements during the "first three minutes" and later on in stellar nucleosynthesis.Comment: 91 pages, 30 figures, accepted by Reviews of Modern Physic

Developing Digital Literacy Modules for Women in Cuenca, Ecuador

Our project worked with the María Amor Foundation in Cuenca, Ecuador to build a self-sustaining curriculum to teach digital literacy for women who are escaping domestic violence. To properly tailor our curriculum and gauge interest, we interviewed and surveyed the future participants and talked to senior employees. We learned that it needed to be as engaging as possible and start very basic as some participants had never used a computer before. The project resulted in the creation of nine independent bilingual modules designed for future workshops