The S-palmitoylome and DHHC-PAT interactome of Drosophila melanogaster S2R+ cells indicate a high degree of conservation to mammalian palmitoylomes.

Protein S-palmitoylation, the addition of a long-chain fatty acid to target proteins, is among the most frequent reversible protein modifications in Metazoa, affecting subcellular protein localization, trafficking and protein-protein interactions. S-palmitoylated proteins are abundant in the neuronal system and are associated with neuronal diseases and cancer. Despite the importance of this post-translational modification, it has not been thoroughly studied in the model organism Drosophila melanogaster. Here we present the palmitoylome of Drosophila S2R+ cells, comprising 198 proteins, an estimated 3.5% of expressed genes in these cells. Comparison of orthologs between mammals and Drosophila suggests that S-palmitoylated proteins are more conserved between these distant phyla than non-S-palmitoylated proteins. To identify putative client proteins and interaction partners of the DHHC family of protein acyl-transferases (PATs) we established DHHC-BioID, a proximity biotinylation-based method. In S2R+ cells, ectopic expression of the DHHC-PAT dHip14-BioID in combination with Snap24 or an interaction-deficient Snap24-mutant as a negative control, resulted in biotinylation of Snap24 but not the Snap24-mutant. DHHC-BioID in S2R+ cells using 10 different DHHC-PATs as bait identified 520 putative DHHC-PAT interaction partners of which 48 were S-palmitoylated and are therefore putative DHHC-PAT client proteins. Comparison of putative client protein/DHHC-PAT combinations indicates that CG8314, CG5196, CG5880 and Patsas have a preference for transmembrane proteins, while S-palmitoylated proteins with the Hip14-interaction motif are most enriched by DHHC-BioID variants of approximated and dHip14. Finally, we show that BioID is active in larval and adult Drosophila and that dHip14-BioID rescues dHip14 mutant flies, indicating that DHHC-BioID is non-toxic. In summary we provide the first systematic analysis of a Drosophila palmitoylome. We show that DHHC-BioID is sensitive and specific enough to identify DHHC-PAT client proteins and provide DHHC-PAT assignment for ca. 25% of the S2R+ cell palmitoylome, providing a valuable resource. In addition, we establish DHHC-BioID as a useful concept for the identification of tissue-specific DHHC-PAT interactomes in Drosophila.

Structural characterization of an Arf dimer interface: molecular mechanism of Arf-dependent membrane scission.

Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle.

Epsin but not AP-2 supports reconstitution of endocytic clathrin-coated vesicles.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

GORAB scaffolds COPI at the trans-Golgi for efficient enzyme recycling and correct protein glycosylation.

COPI is a key mediator of protein trafficking within the secretory pathway. COPI is recruited to the membrane primarily through binding to Arf GTPases, upon which it undergoes assembly to form coated transport intermediates responsible for trafficking numerous proteins, including Golgi-resident enzymes. Here, we identify GORAB, the protein mutated in the skin and bone disorder gerodermia osteodysplastica, as a component of the COPI machinery. GORAB forms stable domains at the trans-Golgi that, via interactions with the COPI-binding protein Scyl1, promote COPI recruitment to these domains. Pathogenic GORAB mutations perturb Scyl1 binding or GORAB assembly into domains, indicating the importance of these interactions. Loss of GORAB causes impairment of COPI-mediated retrieval of trans-Golgi enzymes, resulting in a deficit in glycosylation of secretory cargo proteins. Our results therefore identify GORAB as a COPI scaffolding factor, and support the view that defective protein glycosylation is a major disease mechanism in gerodermia osteodysplastica.

Proteomic Profiling of Mammalian COPII and COPI Vesicles.

Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on the formation and fusion of vesicular carriers. Coat protein complex (COP) II vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER), whereas COPI vesicles facilitate traffic from the Golgi to the ER and intra-Golgi transport. Mammalian cells express various isoforms of COPII and COPI coat proteins. To investigate the roles of coat protein paralogs, we have combined in vitro vesicle reconstitution from semi-intact cells with SILAC-based mass spectrometric analysis. Here, we describe the core proteomes of mammalian COPII and COPI vesicles. Whereas the compositions of COPII vesicles reconstituted with various isoforms of the cargo-binding subunit Sec24 differ depending on the paralog used, all of the isoforms of the COPI coat produce COPI-coated vesicles with strikingly similar protein compositions.

Transient Nonlinear Response of Dynamically Decoupled Ionic Conductors.

The present study demonstrates that large electric fields progressively enhance the conductivity of ionic systems up to timescales corresponding to those on which their structural rearrangements take place. Yet, in many ionic materials, some regarded as candidates for electrical energy storage applications, the structural relaxation process can be tremendously slower than (or highly decoupled from) the charge fluctuations. Consequently, nonlinear dielectric spectroscopy may be employed to access rheological information in dynamically decoupled ionic conductors, whereas the combination of large electric power density and good mechanical stability, both technologically highly desired, imposes specific experimental constraints to reliably determine the steady-state conductivity of such materials.

Assembly of COPI and COPII Vesicular Coat Proteins on Membranes.

In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.

9Å structure of the COPI coat reveals that the Arf1 GTPase occupies two contrasting molecular environments.

COPI coated vesicles mediate trafficking within the Golgi apparatus and between the Golgi and the endoplasmic reticulum. Assembly of a COPI coated vesicle is initiated by the small GTPase Arf1 that recruits the coatomer complex to the membrane, triggering polymerization and budding. The vesicle uncoats before fusion with a target membrane. Coat components are structurally conserved between COPI and clathrin/adaptor proteins. Using cryo-electron tomography and subtomogram averaging, we determined the structure of the COPI coat assembled on membranes in vitro at 9 Å resolution. We also obtained a 2.57 Å resolution crystal structure of ßδ-COP. By combining these structures we built a molecular model of the coat. We additionally determined the coat structure in the presence of ArfGAP proteins that regulate coat dissociation. We found that Arf1 occupies contrasting molecular environments within the coat, leading us to hypothesize that some Arf1 molecules may regulate vesicle assembly while others regulate coat disassembly.

Sec24C/D-isoform-specific sorting of the preassembled ER-Golgi Q-SNARE complex.

Secretory proteins are exported from the endoplasmic reticulum in COPII vesicles. SNARE proteins-core machinery for membrane fusion-are incorporated into COPII vesicles by direct interaction with Sec24. Here we report a novel mechanism for sorting of the ER-Golgi Q-SNAREs into COPII vesicles. Different mammalian Sec24 isoforms recruit either the R-SNARE Sec22b or the Q-SNAREs Syntaxin5, GS27, and Bet1. Syntaxin5 is the only Q-SNARE that directly interacts with Sec24C, requiring its "open" conformation. Mutation within the IxM cargo-binding site of Sec24C led to a drastic reduction in sorting of all three Q-SNAREs into COPII vesicles, implying their ER export as a preassembled complex. Analysis of immunoisolated COPII vesicles and intracellular localization of Sec24 isoforms indicate that all ER-Golgi SNAREs are present on the same vesicle. Combined with existing data, our findings yield a general concept of how Sec24 isoforms can recruit fusogenic SNARE subunits to keep them functionally apart and thus prime mammalian COPII vesicles for homotypic fusion.

Inhibition of Ebola virus glycoprotein-mediated cytotoxicity by targeting its transmembrane domain and cholesterol.

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.

Golgi phosphoprotein 3 triggers signal-mediated incorporation of glycosyltransferases into coatomer-coated (COPI) vesicles.

Newly synthesized membrane and secreted proteins undergo a series of posttranslational modifications in the Golgi apparatus, including attachment of carbohydrate moieties. The final structure of so-formed glycans is determined by the order of execution of the different glycosylation steps, which seems intimately related to the spatial distribution of glycosyltransferases and glycosyl hydrolases within the Golgi apparatus. How cells achieve an accurate localization of these enzymes is not completely understood but might involve dynamic processes such as coatomer-coated (COPI) vesicle-mediated trafficking. In yeast, this transport is likely to be regulated by vacuolar protein sorting 74 (Vps74p), a peripheral Golgi protein able to interact with COPI coat as well as with a binding motif present in the cytosolic tails of some mannosyltransferases. Recently, Golgi phosphoprotein 3 (GOLPH3), the mammalian homolog of Vps74, has been shown to control the Golgi localization of core 2 N-acetylglucosamine-transferase 1. Here, we highlight a role of GOLPH3 in the spatial localization of α-2,6-sialyltransferase 1. We show, for the first time, that GOLPH3 supports incorporation of both core 2 N-acetylglucosamine-transferase 1 and α-2,6-sialyltransferase 1 into COPI vesicles. Depletion of GOLPH3 altered the subcellular localization of these enzymes. In contrast, galactosyltransferase, an enzyme that does not interact with GOLPH3, was neither incorporated into COPI vesicles nor was dependent on GOLPH3 for proper localization.

Identification of novel sphingolipid-binding motifs in mammalian membrane proteins.

Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.

Analysis of transmembrane domains and lipid modified peptides with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.

Protein-lipid interactions within the membrane are difficult to detect with mass spectrometry because of the hydrophobicity of tryptic cleavage peptides on the one hand and the noncovalent nature of the protein-lipid interaction on the other hand. Here we describe a proof-of-principle method capable of resolving hydrophobic and acylated (e.g., myristoylated) peptides by optimizing the steps in a mass spectrometric workflow. We then use this optimized workflow to detect a protein-lipid interaction in vitro within the hydrophobic phase of the membrane that is preserved via a covalent cross-link using a photoactivatable lipid. This approach can also be used to map the site of a protein-lipid interaction as we identify the peptide in contact with the fatty acid part of ceramide in the START domain of the CERT protein.

Analysis of Golgi complex functions: in vitro reconstitution systems.

In vitro reconstitution is prerequisite to investigate complex cellular functions at the molecular level. Reconstitution systems range from combining complete cellular cytosol with organelle-enriched membrane fractions to liposomal systems where all components are chemically defined and can be chosen at will. Here, we describe the in vitro reconstitution of COPI-coated vesicles from semi-intact cells. Efficient vesicle formation is achieved by simple incubation of permeabilized cells with the minimal set of coat proteins Arf1 and coatomer, and guanosine trinucleotides. GTP hydrolysis or any mechanical manipulations are not required for efficient COPI vesicle release.

Scission of COPI and COPII vesicles is independent of GTP hydrolysis.

Intracellular transport and maintenance of the endomembrane system in eukaryotes depends on formation and fusion of vesicular carriers. A seeming discrepancy exists in the literature about the basic mechanism in the scission of transport vesicles that depend on GTP-binding proteins. Some reports describe that the scission of COP-coated vesicles is dependent on GTP hydrolysis, whereas others found that GTP hydrolysis is not required. In order to investigate this pivotal mechanism in vesicle formation, we analyzed formation of COPI- and COPII-coated vesicles utilizing semi-intact cells. The small GTPases Sar1 and Arf1 together with their corresponding coat proteins, the Sec23/24 and Sec13/31 complexes for COPII and coatomer for COPI vesicles were required and sufficient to drive vesicle formation. Both types of vesicles were efficiently generated when GTP hydrolysis was blocked either by utilizing the poorly hydrolyzable GTP analogs GTPγS and GMP-PNP, or with constitutively active mutants of the small GTPases. Thus, GTP hydrolysis is not required for the formation and release of COP vesicles.

Differential transport of Influenza A neuraminidase signal anchor peptides to the plasma membrane.

Influenza A Neuraminidase is essential for virus release from the cell surface of host cells. Given differential structures of the N-terminal sequences including the transmembrane domains of neuraminidase subtypes, we investigated their contribution to transport and localization of subtypes N1, N2 and N8 to the plasma membrane. We generated consensus sequences from all protein entries available for these subtypes. We found that 40N-terminal the forty N-terminal amino acids are sufficient to confer plasma membrane localization of fusion proteins, albeit with different efficiencies. Strikingly, subtle differences in the primary structure of the part of the transmembrane domain that resides in the exoplasmic leaflet of the membrane have a major impact on transport efficiency, providing a potential target for the inhibition of virus release.