Precision gamma-ray polarimetry applied to studies of bremsstrahlung produced by polarized electrons

The thesis reports on the measurement of bremsstrahlung linear polarization produced in collisions of longitudinally and transversely polarized electrons with gold atoms. The experiment was performed at the Mainzer Microtron MAMI in the Institut für Kernphysik of Johannes Gutenberg-Universität Mainz, Germany. Spin-oriented electrons with 2.15 MeV kinetic energy collided with a thin golden target and produced bremsstrahlung. Linear polarization of the emitted photons was measured by means of Compton polarimetry applied to a segmented high-purity germanium detector. Experimental results reveal a strong correlation between the electron spin orientation and bremsstrahlung linear polarization. This indicates a dominant role of the electron spin in atomic-field bremsstrahlung and Coulomb scattering

Architecture of the AP2:clathrin coat on the membranes of clathrin-coated vesicles

AbstractClathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. AP2 is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures, interactions and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2-appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.</jats:p

Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed

Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes.

The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signalling, immunity, neuroprotection and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non-BAR sorting nexin SNX3 using cryo-electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors allowing cargo recognition at subunit interfaces. Thus membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes

Species-independent translational leaders facilitate cell-free expression

Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems differ substantially from each other with respect to efficiency, scalability and the ability to produce functional eukaryotic proteins. Here we describe species-independent translational sequences (SITS) that mediate efficient cell-free protein synthesis in multiple prokaryotic and eukaryotic systems, presumably through bypassing the early translation initiation factors. We use these leaders in combination with targeted suppression of the endogenous Leishmania tarentolae mRNAs to create a cell-free system based on this protozoan. The system can be directly programmed with unpurified PCR products, enabling rapid generation of large protein libraries and protein variants. L. tarentolae extract can produce up to 300 μg/ml of recombinant protein in 2 h. We further demonstrate that protein-protein and protein–small molecule interactions can be quantitatively analyzed directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy

Leishmania cell-free protein expression system

Cell-free protein expression is an important tool for a rapid production, engineering and labeling of recombinant proteins. However the complex protocols for preparation of eukaryotic cell-free protein expression systems result in high manufacturing costs and limit their utility. Recently we reported a novel cell-free expression system based on the lysate of a fermentable protozoan Leishmania tarentolae. Herein we describe a protocol for high throughput protein expression using Leishmania cell-free lysate. The protocol combines PCR-based synthesis and engineering of translation templates with a combined transcription–translation system. The protocol is adapted to multiwell plate format and allows translation of large protein libraries. In the presented example we translate in vitro and isolate a nearly complete complement of mammalian Rab GTPases. Further applications and developments of the system are discussed