Thomas Wollert

When internalized receptors and other cargo are destined for lysosomal degradation, they are ubiquitinated and sorted by the endosomal sorting complex required for transport (ESCRT) complexes 0, I, II and III into multivesicular bodies. Multivesicular bodies are formed when cargo-rich patches of the limiting membrane of endosomes bud inwards by an unknown mechanism and are then cleaved to yield cargo-bearing intralumenal vesicles. The biogenesis of multivesicular bodies was reconstituted and visualized using giant unilamellar vesicles, fluorescent ESCRT-0, -I, -II and -III complexes, and a membrane-tethered fluorescent ubiquitin fusion as a model cargo. Here we show that ESCRT-0 forms domains of clustered cargo but does not deform membranes. ESCRT-I and ESCRT-II in combination deform the membrane into buds, in which cargo is confined. ESCRT-I and ESCRT-II localize to the bud necks, and recruit ESCRT-0-ubiquitin domains to the buds. ESCRT-III subunits localize to the bud neck and efficiently cleave the buds to form intralumenal vesicles. Intralumenal vesicles produced in this reaction contain the model cargo but are devoid of ESCRTs. The observations explain how the ESCRTs direct membrane budding and scission from the cytoplasmic side of the bud without being consumed in the reaction.

The endosomal sorting complex required for transport (ESCRT) system is essential for multivesicular body biogenesis, in which cargo sorting is coupled to the invagination and scission of intralumenal vesicles. The ESCRTs are also needed for budding of enveloped viruses including human immunodeficiency virus 1, and for membrane abscission in cytokinesis. In Saccharomyces cerevisiae, ESCRT-III consists of Vps20, Snf7, Vps24 and Vps2 (also known as Did4), which assemble in that order and require the ATPase Vps4 for their disassembly. In this study, the ESCRT-III-dependent budding and scission of intralumenal vesicles into giant unilamellar vesicles was reconstituted and visualized by fluorescence microscopy. Here we show that three subunits of ESCRT-III, Vps20, Snf7 and Vps24, are sufficient to detach intralumenal vesicles. Vps2, the ESCRT-III subunit responsible for recruiting Vps4, and the ATPase activity of Vps4 were required for ESCRT-III recycling and supported additional rounds of budding. The minimum set of ESCRT-III and Vps4 proteins capable of multiple cycles of vesicle detachment corresponds to the ancient set of ESCRT proteins conserved from archaea to animals.

Autophagy is a catabolic pathway that sequesters undesired cellular material into autophagosomes for delivery to lysosomes for degradation. A key step in the pathway is the covalent conjugation of the ubiquitin-related protein Atg8 to phosphatidylethanolamine (Atg8–PE) in autophagic membranes by a complex consisting of Atg16 and the Atg12–Atg5 conjugate. Atg8 controls the expansion of autophagic precursor membranes, but the underlying mechanism remains unclear. Here, we reconstitute Atg8 conjugation on giant unilamellar vesicles and supported lipid bilayers. We found that Atg8–PE associates with Atg12–Atg5-Atg16 into a membrane scaffold. By contrast, scaffold formation is counteracted by the mitochondrial cargo adaptor Atg32 through competition with Atg12–Atg5 for Atg8 binding. Atg4, previously known to recycle Atg8 from membranes, disassembles the scaffold. Importantly, mutants of Atg12 and Atg16 deficient in scaffold formation in vitro impair autophagy in vivo. This suggests that autophagic scaffolds are critical for phagophore biogenesis and thus autophagy.

<h2>Summary</h2> In causing disease, pathogens outmaneuver host defenses through a dedicated arsenal of virulence determinants that specifically bind or modify individual host molecules. This dedication limits the intruder to a defined range of hosts. Newly emerging diseases mostly involve existing pathogens whose arsenal has been altered to allow them to infect previously inaccessible hosts. We have emulated this chance occurrence by extending the host range accessible to the human pathogen <i>Listeria monocytogenes</i> by the intestinal route to include the mouse. Analyzing the recognition complex of the listerial invasion protein InlA and its human receptor E-cadherin, we postulated and verified amino acid substitutions in InlA to increase its affinity for E-cadherin. Two single substitutions increase binding affinity by four orders of magnitude and extend binding specificity to include formerly incompatible murine E-cadherin. By rationally adapting a single protein, we thus create a versatile murine model of human listeriosis.

Abstract Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1–kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro . We find that on assembly from its cytoplasmic subcomplexes, the Atg1–kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1–kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1–Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.

<h2>Summary</h2> The ESCRT-II-ESCRT-III interaction coordinates the sorting of ubiquitinated cargo with the budding and scission of intralumenal vesicles into multivesicular bodies. The interacting regions of these complexes were mapped to the second winged helix domain of human ESCRT-II subunit VPS25 and the first helix of ESCRT-III subunit VPS20. The crystal structure of this complex was determined at 2.0 Å resolution. Residues involved in structural interactions explain the specificity of ESCRT-II for Vps20, and are critical for cargo sorting in vivo. ESCRT-II directly activates ESCRT-III-driven vesicle budding and scission in vitro via these structural interactions. VPS20 and ESCRT-II bind membranes with nanomolar affinity, explaining why binding to ESCRT-II is dispensable for the recruitment of Vps20 to membranes. Docking of the ESCRT-II-VPS20₂ supercomplex reveals a convex membrane-binding surface, suggesting a hypothesis for negative membrane curvature induction in the nascent intralumenal vesicle.

This article is part of a Minifocus on the ESCRT machinery. For further reading, please see related articles: `No strings attached: the ESCRT machinery in viral budding and cytokinesis' by Bethan McDonald and Juan Martin-Serrano (J. Cell Sci. 122 , [2167-2177][1]) and `How do ESCRT proteins control

Lys-48-linked polyubiquitination regulates a variety of cellular processes by targeting ubiquitinated proteins to the proteasome for degradation. Although polyubiquitination had been presumed to occur by transferring ubiquitin molecules, one at a time, from an E2 active site to a substrate, we recently showed that the endoplasmic reticulum-associated RING finger ubiquitin ligase gp78 can mediate the preassembly of Lys-48-linked polyubiquitin chains on the catalytic cysteine of its cognate E2 Ube2g2 and subsequent transfer to a substrate. Active site-linked polyubiquitin chains are detected in cells on Ube2g2 and its yeast homolog Ubc7p, but how these chains are assembled is unclear. Here, we show that gp78 forms an oligomer via 2 oligomerization sites, one of which is a hydrophobic segment located in the gp78 cytosolic domain. We further demonstrate that a gp78 oligomer can simultaneously associate with multiple Ube2g2 molecules. This interaction is mediated by a novel Ube2g2 surface distinct from the predicted RING binding site. Our data suggest that a large gp78-Ube2g2 heterooligomer brings multiple Ube2g2 molecules into close proximity, allowing ubiquitin moieties to be transferred between neighboring Ube2g2s to form active site-linked polyubiquitin chains.

<h2>Summary</h2> In 1955, the biologist and Nobel Prize laureate Christian de Duve discovered that cells possess specialized organelles filled with hydrolytic enzymes and he called these organelles lysosomes. At the same time, electron microscopy studies by Novikoff and colleagues showed that intracellular dense bodies, which later turned out to be lysosomes, contain cytoplasmic components. Together, these groundbreaking observations revealed that cells can deliver cytoplasmic components to lysosomes for degradation. The hallmark of this degradative process, which de Duve called autophagy, is the formation of double-membrane-limited vesicles. Further morphological characterization of these vesicles (autophagosomes) revealed that they mainly contain bulk cytoplasm. Although this suggested that autophagy leads to a non-selective degradation of cytoplasmic material, de Duve anticipated that a regulated and selective type of this pathway must also exist. Today we know that, under normal conditions, macroautophagy is a highly selective pathway that sequesters damaged or superfluous material from the cytoplasm through the formation of double-membrane-limited autophagosomes. Upon fusion with lysosomes, the content of autophagosomes is degraded and the resulting building blocks are released into the cytoplasm. However, in response to cytotoxic stress or starvation, cells start to produce autophagosomes that capture bulk cytoplasm non-selectively. This stress response is essential for cells to survive adverse environmental conditions, whereas the selective sequestration of cargo is important to maintain cellular homeostasis.

Autophagy recycles cytoplasmic components by sequestering them in double membrane-surrounded autophagosomes. The two proteins Atg11 and Atg17 are scaffolding components of the Atg1 kinase complex. Atg17 recruits and tethers Atg9-donor vesicles, and the corresponding Atg1 kinase complex induces the formation of nonselective autophagosomes. Atg11 initiates selective autophagy and coordinates the switch to nonselective autophagy by recruiting Atg17. The molecular function of Atg11 remained, however, less well understood. Here, we demonstrate that Atg11 is activated by cargo through a direct interaction with autophagy receptors. Activated Atg11 dimerizes and tethers Atg9 vesicles, which leads to the nucleation of phagophores in direct vicinity of cargo. Starvation reciprocally regulates the activity of both tethering factors by initiating the degradation of Atg11 while Atg17 is activated. This allows Atg17 to sequester and tether Atg9 vesicles independent of cargo to nucleate nonselective phagophores. Our data reveal insights into the molecular mechanisms governing cargo selection and specificity in autophagy.

Abstract The accumulation of protein aggregates is involved in the onset of many neurodegenerative diseases. Aggrephagy is a selective type of autophagy that counteracts neurodegeneration by degrading such aggregates. In this study, we found that LC3C cooperates with lysosomal TECPR1 to promote the degradation of disease-related protein aggregates in neural stem cells. The N-terminal WD-repeat domain of TECPR1 selectively binds LC3C which decorates matured autophagosomes. The interaction of LC3C and TECPR1 promotes the recruitment of autophagosomes to lysosomes for degradation. Augmented expression of TECPR1 in neural stem cells reduces the number of protein aggregates by promoting their autophagic clearance, whereas knockdown of LC3C inhibits aggrephagy. The PH domain of TECPR1 selectively interacts with PtdIns(4)P to target TECPR1 to PtdIns(4)P containing lysosomes. Exchanging the PH against a tandem-FYVE domain targets TECPR1 ectopically to endosomes. This leads to an accumulation of LC3C autophagosomes at endosomes and prevents their delivery to lysosomes.

The ongoing pandemic of the new severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused more than one million deaths, overwhelmed many public health systems, and led to a worldwide economic recession.This has raised an unprecedented need to develop antiviral drugs and vaccines, which requires profound knowledge of the fundamental pathology of the virus, including its entry, replication, and release from host cells.The genome of coronaviruses comprises around 30 kb of positive single-stranded RNA, representing one of the largest RNA genomes of viruses.The 5′ part of the genome encodes a large polyprotein, PP1ab, which gives rise to 16 non-structural proteins (nsp1-nsp16).Two proteases encoded in nsp3 and nsp5 cleave the polyprotein into individual proteins.Most nsps belong to the viral replicase complex that promotes replication of the viral genome and translation of structural proteins by producing subgenomic mRNAs.The replicase complexes are found on doublemembrane vesicles (DMVs) that contain viral double-stranded RNA.Expression of a small subset of viral proteins, including nsp3 and nsp4, is sufficient to induce formation of these DMVs in human cells, suggesting that both proteins deform host membranes into such structures.We will discuss the formation of DMVs and provide an overview of other membrane remodeling processes that are induced by coronaviruses.

The homeostasis of cells depends on the selective degradation of damaged or superfluous cellular components. Autophagy is the major pathway that recognizes such components, sequesters them in de novo formed autophagosomes and delivers them to lysosomes for degradation. The recognition of specific cargo and the biogenesis of autophagosomes involve a dedicated machinery of autophagy related (ATG) proteins. Intense research over the past decades has revealed insights into the function of autophagy proteins and mechanisms that govern cargo recognition. Other aspects including the molecular mechanisms involved in the onset of human diseases are less well understood. However, autophagic dysfunctions, caused by age related decline in autophagy or mutations in ATG proteins, are directly related to a large number of human pathologies including neurodegenerative disorders. Here, we review most recent discoveries and breakthroughs in selective autophagy and its relationship to neurodegeneration.

Biological processes essentially all depend on the specific recognition between macromolecules and their interaction partners. Although many such interactions have been characterized both structurally and biophysically, the thermodynamic effects of small atomic changes remain poorly understood. Based on the crystal structure of the bacterial invasion protein internalin (InlA) of Listeria monocytogenes in complex with its human receptor E-cadherin (hEC1), we analyzed the interface to identify single amino acid substitutions in InlA that would potentially improve the overall quality of interaction and hence increase the weak binding affinity of the complex. Dissociation constants of InlA-variant/hEC1 complexes, as well as enthalpy and entropy of binding, were quantified by isothermal titration calorimetry. All single substitutions indeed significantly increase binding affinity. Structural changes were verified crystallographically at < or =2.0-A resolution, allowing thermodynamic characteristics of single substitutions to be rationalized structurally and providing unique insights into atomic contributions to binding enthalpy and entropy. Structural and thermodynamic data of all combinations of individual substitutions result in a thermodynamic network, allowing the source of cooperativity between distant recognition sites to be identified. One such pair of single substitutions improves affinity 5,000-fold. We thus demonstrate that rational reengineering of protein complexes is possible by making use of physically distant hot spots of recognition.

Significance Some intracellular bacteria develop inside a vacuole, which expands during the infection process. We show, here, that the protein ATG16L1 restricts the expansion of the Chlamydia trachomatis vacuole. ATG16L1 is well known for its role in autophagy, a process that contributes to the elimination of intracellular microbes. However, the restriction exerted by ATG16L1 on vacuole expansion relies on a different ATG16L1 function. We demonstrate that the bacteria secrete an effector protein that prevents ATG16L1 binding to TMEM59 and allows rerouting of vesicular traffic to the vacuole. The discovery that one bacterial effector evolved to target ATG16L1’s engagement in intracellular traffic emphasizes the importance this secondary activity of ATG16L1 for maintaining host cell homeostasis.

Autophagy is one of the most versatile recycling systems of eukaryotic cells. It degrades diverse cytoplasmic components such as organelles, protein aggregates, ribosomes and multi-enzyme complexes. Not surprisingly, any failure of autophagy or reduced activity of the pathway contributes to the onset of various pathologies, including neurodegeneration, cancer and metabolic disorders such as diabetes or immune diseases. Furthermore, autophagy contributes to the innate immune response and combats bacterial or viral pathogens. The hallmark of macroautophagy is the formation of a membrane sack that sequesters cytoplasmic cargo and delivers it to lysosomes for degradation. More than 40 autophagy-related (ATG) proteins have so far been identified. A unique protein-conjugation system represents one of the core components of this highly elaborate machinery. It conjugates six homologous ATG8 family proteins to the autophagic membrane. In this review, we summarize the current knowledge regarding the various functions of ATG8 proteins in autophagy and briefly discuss how physical approaches and in vitro reconstitution contributed in deciphering their function.

High-resolution structural analysis has characterized nearly all of the individual domains of ESCRT (endosomal sorting complex required for transport) subunits, all of the core structures of the soluble complexes and many of the interactions involving domains. Recent emphasis in structural studies has shifted towards efforts to integrate these structures into a larger-scale model. Molecular simulations, hydrodynamic analysis, small-angle X-ray scattering and cryo-EM (electron microscopy) techniques have all been brought to bear on the ESCRT system over the last year.

The conjugation of the small ubiquitin (Ub)-like protein Atg8 to autophagic membranes is a key step during the expansion of phagophores. This reaction is driven by 2 interconnected Ub-like conjugation systems. The second system conjugates the Ub-like protein Atg12 to Atg5. The resulting conjugate catalyzes the covalent attachment of Atg8 to membranes. Atg12–Atg5, however, constitutively associates with the functionally less well-characterized coiled-coil protein Atg16. By reconstituting the conjugation of Atg8 to membranes in vitro, we showed that after Atg8 has been attached to phosphatidylethanolamine (PE), it recruits Atg12–Atg5 to membranes by recognizing a noncanonical Atg8-interacting motif (AIM) within Atg12. Atg16 crosslinks Atg8–PE-Atg12–Atg5 complexes to form a continuous 2-dimensional membrane scaffold with meshwork-like architecture. Apparently, scaffold formation is required to generate productive autophagosomes and to deliver autophagic cargo to the vacuole in vivo.

Autophagy is a versatile recycling pathway that delivers cytoplasmic contents to lysosomal compartments for degradation. It involves the formation of a cup-shaped membrane that expands to capture cargo. After the cargo has been entirely enclosed, the membrane is sealed to generate a double-membrane-enclosed compartment, termed the autophagosome. Depending on the physiological state of the cell, the cargo is selected either specifically or non-specifically. The process involves a highly conserved set of autophagy-related proteins. Reconstitution of their action on model membranes in vitro has contributed tremendously to our understanding of autophagosome biogenesis. This review will focus on various in vitro techniques that have been employed to decipher the function of the autophagic core machinery.

Autophagy is one of the most elaborative membrane remodeling systems in eukaryotic cells. Its major function is to recycle cytoplasmic material by delivering it to lysosomes for degradation. To achieve this, a membrane cisterna is formed that gradually captures cargo such as organelles or protein aggregates. The diversity of cargo requires autophagy to be highly versatile to adapt the shape of the phagophore to its substrate. Upon closure of the phagophore, a double-membrane-surrounded autophagosome is formed that eventually fuses with lysosomes. In response to environmental cues such as cytotoxicity or starvation, bulk cytoplasm can be captured and delivered to lysosomes. Autophagy thus supports cellular survival under adverse conditions. During the past decades, groundbreaking genetic and cell biological studies have identified the core machinery involved in the process. In this Review, we are focusing on in vitro reconstitution approaches to decipher the details and spatiotemporal control of autophagy, and how such studies contributed to our current understanding of the pathways in yeast and mammals. We highlight studies that revealed the function of the autophagy machinery at a molecular level with respect to its capacity to remodel membranes.

Activated cell surface receptors are rapidly removed from the plasma membrane through clathrin mediated endocytosis and transported to the endosome where they are either recycled or sorted to the lysosomal pathway to be degraded. Receptors, destined for degradation in the lysosome, are packaged into intraluminal vesicles (ILVs) of endosomes by a reaction that is topologically unrelated to other budding reactions in cells. First, receptors are clustered at the endosomal membrane and receptor-rich membrane patches then bud towards the lumen of the endosome. The nascent membrane buds are finally cleaved from the limiting membrane to release cargo-bearing vesicles into the endosomal interior. The molecular machinery that drives multivesicular body biogenesis, the endosomal sorting complex required for transport (ESCRT) machinery, has been identified through genetic screens. It consists of the cytoplasmic, hetero-multimeric complexes ESCRT-0, -I, -II, and -III, and of the Vps4/VtaI complex. Although the ESCRT machinery has been characterized extensively using cell-biological and biochemical approaches, the molecular mechanism of multivesicular body biogenesis remained unclear. In this chapter, I will present in vitro reconstitution systems that we used to study ESCRT-driven membrane remodeling reactions with purified components on artificial membranes. This includes generation of large and giant unilamellar liposomes, as well as in vitro reconstitution reactions of fluorescently labeled proteins on such membranes. I will discuss both, the potential of in vitro systems to analyze membrane-remodeling events and also their limitations.

Macroautophagy delivers cytoplasmic material to lysosomal/vacuolar compartments for degradation. Conserved multisubunit complexes, composed of autophagy-related (Atg) proteins, initiate the formation of membrane precursors, termed phagophores. Under physiological conditions these cup-shaped structures can capture cytoplasmic material highly selectively. Starvation or cytotoxic stresses, however, initiate the formation of much larger phagophores to enclose cytoplasm nonselectively. The biogenesis of nonselective autophagosomes is initiated by the hierarchical assembly of the Atg1 kinase complex and the recruitment of Atg9 vesicles at the phagophore assembly site (PAS). In this punctum we summarize our recent findings regarding tethering of Atg9 vesicles by the Atg1 kinase complex. We discuss membrane tethering by and activation of its central subunit Atg17 in the context of other canonical membrane tethering factors. Our results show that Atg17 suffices to bind and tether Atg9 vesicles. The Atg31-Atg29 subcomplex inhibits Atg17 activity, and activation of Atg17 depends on the formation of the Atg1 kinase complex that involves recruiting Atg1-Atg13. Our studies lead to a model of unconventional membrane tethering in autophagy.

The hallmark of macroautophagy is the de novo generation of a membrane structure that collects cytoplasmic material and delivers it to lysosomes for degradation. The nucleation of this precursor membrane, termed phagophore, involves the coordinated assembly of the Atg1-kinase complex and the recruitment of Atg9 vesicles. The latter represents one important membrane source in order to produce phagophores in vivo. We explain how the process of phagophore nucleation can be reconstituted from purified components in vitro. We describe the assembly of the ~500 kDa pentameric Atg1-kinase complex from its purified subunits. We also explain how Atg9-donor vesicles are generated in vitro to study the interaction of Atg9 and Atg1-kinase complexes by floatation experiments.

Autophagy is an essential and fundamental pathway that clears unwanted or damaged material from the cell. Initiation of autophagy was previously shown to be dependent on the Ulk1/2 kinase complex. In this issue of The FEBS Journal, Braden and Neufeld investigated the Ulk3 homolog in Drosophila, and proposed a novel, Ulk1/2 independent pathway for autophagy initiation.

Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY-or eukaryotic Sec61-complexes, and are translocated across the membrane during their synthesis 1,2 .Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase [3][4][5] .The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug.During initiation of translocation, a ribosome-nascent chain complex binds to the SecY/Sec61 complex, resulting in insertion of the nascent chain.However, the mechanism of channel opening during translocation is unclear.Here, we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryoelectron microscopy.Non-translating ribosome-SecY channel complexes derived from Methanococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes.The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the N-and C-terminal halves of SecY.In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research,

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity by which the lipidation of members of the LC3 family is facilitated.The crystal structure of the human ATG12~ATG5 conjugate bound to the amino-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues.ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous patch.Mutagenesis data support the importance of both the ATG12-ATG5 interface and the continuous patch for E3 activity.The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high-affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity.These findings provide a foundation for understanding the mechanism of LC3 lipidation.

We have designed a membrane 'staple', which consists of membrane-anchored repeats of the trans-aggregating FM domain that face the lumen of the secretory pathway.In the presence of the disaggregating drug these proteins transit the secretory pathway.When the drug is removed these proteins form electron-dense plaques which we term staples.Unexpectedly, when initially positioned within the cis-Golgi, staples remained at the cis face of the Golgi even after many hours.By contrast, soluble FM-aggregates transited the Golgi.Staples and soluble aggregates placed in cis-Golgi cisternae therefore have different fates.Whereas the membrane staples are located in the flattened, stacked central regions of the cisternae, the soluble aggregates are in the dilated rims.This suggests that while the cisternae are static on the time scale of protein traffic, the dilated rims are mobile and progress in the cis → trans direction via a mechanism that we term 'Rim Progression'.

Abstract Autophagy is a fundamental cellular recycling pathway that captures cytoplasmic material by a phagophore membrane and delivers it to lysosomes for degradation. Nonselective phagophores are generated at specialized ER-domains termed omegasomes. Mechanistically, this process is not well understood. Here, we reconstituted the formation of phagophores from purified components on supported lipid bilayers and by ectopic induction of nonselective autophagy at the plasma membrane. We found that enzymatic conjugation of LC3B to model membranes induces the formation of phagophore-like membrane cups. LC3B functions as membrane anchor, ensuring a sustained interaction of the E3-like ligase complex ATG12¬–ATG5-ATG16L1 with membranes by forming extended membrane coats. ATG16L1 is the major membrane remodeling factor that induces positive membrane curvature, promotes cup formation and stabilizes the rim of membrane cups. Redirecting WIPI2 to the plasma membrane by expressing WIPI2-CAAX induced the formation of omegasome-like membrane cisternae to which LC3B was conjugated. Membrane cups, identical to those observed in vitro, emerged from these cisternae. ATG16L1 variants that did not induce the formation of cups in vitro also failed to promote cup formation in vivo and inhibited the biogenesis of nonselective autophagosomes in cells. We thus propose that ATG16L1 drives the formation of nonselective phagophores by shaping flat donor membranes into membrane cups.

Krankheitserreger besitzen eine charakteristische Ausstattung mit Virulenzfaktoren, die spezifisch mit einzelnen Wirtsmolekulen interagieren. Diese Spezialisierung fuhrt jedoch dazu, dass nicht alle Spezies infiziert werden. Neue Krankheiten werden vorwiegend von bereits existierenden Erregern ausgelost, die durch Mutationen in ihren Virulenzfaktoren zuvor unzugangliche Spezies infizieren konnen. Der mit der Nahrung aufgenommen Krankheitserreger Listeria monocytogenes exprimiert das Invasionsprotein Internalin (InlA), das die Aufnahme der Bakterie in Wirtszellen vermittelt. Die Bindungsspezifitat dieses Molekuls limitiert jedoch die fur L. monocytogenes zuganglichen Spezies. Es kann den Menschen, nicht jedoch die Maus infizieren. Durch rationales Design wurde InlA so verandert, dass es den humanen Rezeptor E-Cadherin (hEC1) mit hoherer Affinitat erkennt was gleichzeitig die Erkennung von E-Cadherin der Maus (mEC1) ermoglicht. Auf atomarer Ebene wurden Regionen mit geringer Komplementaritat in der Interaktionsflache von InlA und hEC1 identifiziert, indem die Kristallstruktur des Erkennungskomplexes analysiert wurde. Einzelne Aminosaure-Substitutionen, die die Bindungsaffinitat zu hEC1 erhohen sollten, wurden in InlA eingefuhrt. Strukturelle Anderungen wurden kristallographisch verifiziert. Die Bindungsaffinitat sowie Bindungsenthalpie und –entropy wurden mittels Isothermer Titrationskalorimetrie bestimmt. Alle vier Substitutionen verstarkten die Affinitat von InlA zu hEC1 betrachtlich. Die Korrelation von Struktur und Biophysik fuhrte zu einem besseren Verstandnis des Beitrages einzelner Atome zur Bindungsenergie. Die Kombination von nur zwei Substitutionen erhohte die Affinitat von InlA dramatisch. Des weiteren erkennt das optimierte InlA mEC1 mit einer Affinitat, die der InlA / hEC1 Interaktion gleicht. Veranderter Bakterien, die beide Mutationen tragen, konnen Mause effizient befallen, was ein vielseitiges Modell zur Erforschung der humanen Listeriose darstellt.

ATG9A is a multispanning membrane protein essential for autophagy.Normally resident in Golgi membranes and endosomes, during amino acid starvation, ATG9A traffics to sites of autophagosome formation.ATG9A is not incorporated into autophagosomes but is proposed to supply so-far-unidentified proteins and lipids to the autophagosome.To address this function of ATG9A, a quantitative analysis of ATG9A-positive compartments immunoisolated from amino acid-starved cells was performed.These ATG9A vesicles are depleted of Golgi proteins and enriched in BAR-domain containing proteins, Arfaptins, and phosphoinositide-metabolizing enzymes.Arfaptin2 regulates the starvation-dependent distribution of ATG9A vesicles, and these ATG9A vesicles deliver the PI4-kinase, PI4KIIIβ, to the autophagosome initiation site.PI4KIIIβ interacts with ATG9A and ATG13 to control PI4P production at the initiation membrane site and the autophagic response.PI4KIIIβ and PI4P likely function by recruiting the ULK1/2 initiation kinase complex subunit ATG13 to nascent autophagosomes...

Close proximities between organelles have been described for decades.However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology.The diversity of approaches warrants a unified vocabulary for the field.Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies.This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites.It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research. In Eukaryotes, intracellular membranes delimit organelles that have distinct biochemical functions.While for decades the organelle field was governed by studies aimed at identifying the unique characteristics of each compartment, the last years have seen a revolution in the field as more focus is being placed on the interactions between the organelles and their role in maintaining cellular homeostasis.Published examples of interactions between two distinct organelles appeared in the late 1950s 1,2 .However, the lack of a perceived physiological role made it hard to envision this as a general and functionally relevant phenomenon.The strong notion at the time was that the physical organization of the cytosol was performed solely by anchoring and movement on

The food-borne pathogen Listeria monocytogenes causes severe infections in immunocompromised patients.At 30% the mortality rate of this pathogen far exceeds that of other foodborne pathogens.The first step of listerial infection involves the uptake of bacteria into epithelial cells of the intestine.This uptake is mediated by the interaction of the major invasin Internalin (InlA) with its human receptor E-Cadherin.We have previously investigated the recognition complex between InlA and human E-cadherin by crystallizing functional fragments of both proteins (InlA' and hEC1).The leucinerich-repeat (LRR) protein InlA is found to bind the N-terminal domain of E-cadherin by its 15 unit LRR-domain.Nevertheless, despite a large interaction surface, the complex is surprisingly weak.Presently, we have undertaken to investigate whether this low affinity is of biological relevance.For this purpose we have substituted individual amino acids in InlA' to increase the affinity for hEC1.We have identified two residues that, when suitably mutated, improve the binding strength 2500-fold.We have furthermore generated strains of L. monocytogenes that incorporate the InlA-mutations within the genome.This allows the effect of the higher affinity InlA-variants on the uptake of L. monocytogenes into human epithelial cells to be analyzed.

The bacterium Listeria monocytogenes is a food-borne human pathogen. It infects humans by inducing its own uptake into epithelial cells of the intestine although these are normally non-phagocytic. Recognition, adhesion and invasion of intestinal epithelial cells is mediated by a single listerial surface protein, Internalin (InlA), through specific interaction with the host cell receptor E-cadherin. We have solved the crystal structure of the functional domain of InlA both uncomplexed and in complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). In the complex between InlA and hEC1, the superhelically twisted leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Site-directed mutagenesis, analytical ultracentrifugation and Biacore experiments indicate that binding affinity is remarkably weak yet highly specific: Pro16 of hEC1, a major determinant for human susceptibility to L. Monocytogenes infection is essential for intermolecular recognition. Ca was found to stabilize the complex. Structurally, this is corroborated by a Ca-binding site bridging the two proteins. This indicates that complex formation in the intestine is favoured by high Ca-concentrations whereas low, intracellular concentrations induce dissociation, freeing the bacterium and allowing it to move through the eukaryotic cell. Our studies thus provide detailed insights into the molecular deception L. monocytogenes employs to exploit the host E-cadherin signal cascade through the expression of a single surface protein.

Read the full review for this Faculty Opinions recommended article: A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA.