A global circadian repressor controls antiphasic expression of metabolic genes in Neurospora.

The white-collar complex (WCC), the core transcription factor of the circadian clock of Neurospora, activates morning-specific expression of the transcription repressor CSP1. Newly synthesized CSP1 exists in a transient complex with the corepressor RCM1/RCO1 and the ubiquitin ligase UBR1. CSP1 is rapidly hyperphosphorylated and degraded via UBR1 and its ubiquitin conjugase RAD6. Genes controlled by CSP1 are rhythmically expressed and peak in the evening (i.e., in antiphase to morning-specific genes directly controlled by WCC). Rhythmic expression of these second-tier genes depends crucially on phosphorylation and rapid turnover of CSP1, which ensures tight coupling of CSP1 abundance and function to the circadian activity of WCC. Negative feedback of CSP1 on its own transcription buffers the amplitude of CSP1-dependent oscillations against fluctuations of WCC activity. CSP1 predominantly regulates genes involved in metabolism. It controls ergosterol synthesis and fatty acid desaturases and thereby modulates the lipid composition of membranes.

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.

Molecular recognition of a single sphingolipid species by a protein's transmembrane domain.

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

Sphingomyelin organization is required for vesicle biogenesis at the Golgi complex.

Sphingomyelin and cholesterol can assemble into domains and segregate from other lipids in the membranes. These domains are reported to function as platforms for protein transport and signalling. Do similar domains exist in the Golgi membranes and are they required for protein secretion? We tested this hypothesis by using D-ceramide-C6 to manipulate lipid homeostasis of the Golgi membranes. Lipidomics of the Golgi membranes isolated from D-ceramide-C6-treated HeLa cells revealed an increase in the levels of C6-sphingomyelin, C6-glucosylceramide, and diacylglycerol. D-ceramide-C6 treatment in HeLa cells inhibited transport carrier formation at the Golgi membranes without affecting the fusion of incoming carriers. The defect in protein secretion as a result of D-ceramide-C6 treatment was alleviated by knockdown of the sphingomyelin synthases 1 and 2. C6-sphingomyelin prevented liquid-ordered domain formation in giant unilamellar vesicles and reduced the lipid order in the Golgi membranes of HeLa cells. These findings highlight the importance of a regulated production and organization of sphingomyelin in the biogenesis of transport carriers at the Golgi membranes.

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.

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.

Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication.

Spindle pole bodies (SPBs), like nuclear pore complexes, are embedded in the nuclear envelope (NE) at sites of fusion of the inner and outer nuclear membranes. A network of interacting proteins is required to insert a cytoplasmic SPB precursor into the NE. A central player of this network is Nbp1 that interacts with the conserved integral membrane protein Ndc1. Here, we establish that Nbp1 is a monotopic membrane protein that is essential for SPB insertion at the inner face of the NE. In vitro and in vivo studies identified an N-terminal amphipathic α-helix of Nbp1 as a membrane-binding element, with crucial functions in SPB duplication. The karyopherin Kap123 binds to a nuclear localization sequence next to this amphipathic α-helix and prevents unspecific tethering of Nbp1 to membranes. After transport into the nucleus, Nbp1 binds to the inner nuclear membrane. These data define the targeting pathway of a SPB component and suggest that the amphipathic α-helix of Nbp1 is important for SPB insertion into the NE from within the nucleus.

Distinct role of subcomplexes of the COPI coat in the regulation of ArfGAP2 activity.

COPI vesicles serve for transport of proteins and membrane lipids in the early secretory pathway. Their coat protein (coatomer) is a heptameric complex that is recruited to the Golgi by the small GTPase Arf1. Although recruited en bloc, coatomer can be viewed as a stable assembly of an adaptin-like tetrameric subcomplex (CM4) and a trimeric 'cage' subcomplex (CM3). Following recruitment, coatomer stimulates ArfGAP-dependent GTP hydrolysis on Arf1. Here, we employed recombinant coatomer subcomplexes to study the role of coatomer components in the regulation of ArfGAP2, an ArfGAP whose activity is strictly coatomer-dependent. Within CM4, we define a novel hydrophobic pocket for ArfGAP2 interaction on the appendage domain of γ1-COP. The CM4 subcomplex (but not CM3) is recruited to membranes through Arf1 and can subsequently recruit ArfGAP2. Neither CM3 nor CM4 in itself is effective in stimulating ArfGAP2 activity, but stimulation is regained when both subcomplexes are present. Our findings point to a distinct role of each of the two coatomer subcomplexes in the regulation of ArfGAP2-dependent GTP hydrolysis on Arf1, where the CM4 subcomplex functions in GAP recruitment, while, similarly to the COPII system, the cage-like CM3 subcomplex stimulates the catalytic reaction.

Impaired neurotransmission in ether lipid-deficient nerve terminals.

Isolated defects of ether lipid (EL) biosynthesis in humans cause rhizomelic chondrodysplasia punctata type 2 and type 3, serious peroxisomal disorders. Using a previously described mouse model [Rodemer, C., Thai, T.P., Brugger, B., Kaercher, T., Werner, H., Nave, K.A., Wieland, F., Gorgas, K., and Just, W.W. (2003) Inactivation of ether lipid biosynthesis causes male infertility, defects in eye development and optic nerve hypoplasia in mice. Hum. Mol. Genet., 12, 1881-1895], we investigated the effect of EL deficiency in isolated murine nerve terminals (synaptosomes) on the pre-synaptic release of the neurotransmitters (NTs) glutamate and acetylcholine. Both Ca(2+)-dependent exocytosis and Ca(2+)-independent efflux of the transmitters were affected. EL-deficient synaptosomes respire at a reduced rate and exhibit a lowered adenosin-5'-triphosphate/adenosine diphosphate (ATP/ADP) ratio. Consequently, ATP-driven processes, such as synaptic vesicle cycling and maintenance of Na(+), K(+) and Ca(2+) homeostasis, might be disturbed. Analyzing reactive oxygen species in EL-deficient neural and non-neural tissues revealed that plasmalogens (PLs), the most abundant EL species in mammalian central nervous system, considerably contribute to the generation of the lipid peroxidation product malondialdehyde. Although EL-deficient tissue contains less lipid peroxidation products, fibroblasts lacking ELs are more susceptible to induced oxidative stress. In summary, these results suggest that due to the reduced energy state of EL-deficient tissue, the Ca(2+)-independent efflux of NTs increases while the Ca(2+)-dependent release declines. Furthermore, lack of PLs is mainly compensated for by an increase in the concentration of phosphatidylethanolamine and results in a significantly lowered level of lipid peroxidation products in the brain cortex and cerebellum.

A mitochondrial phosphatase required for cardiolipin biosynthesis: the PGP phosphatase Gep4.

Cardiolipin (CL), a unique dimeric phosphoglycerolipid predominantly present in mitochondrial membranes, has pivotal functions for the cellular energy metabolism, mitochondrial dynamics and the initiation of apoptotic pathways. Perturbations in the mitochondrial CL metabolism cause cardiomyopathy in Barth syndrome. Here, we identify a novel phosphatase in the mitochondrial matrix space, Gep4, and demonstrate that it dephosphorylates phosphatidylglycerolphosphate to generate phosphatidylglycerol, an essential step during CL biosynthesis. Expression of a mitochondrially targeted variant of Escherichia coli phosphatase PgpA restores CL levels in Gep4-deficient cells, indicating functional conservation. A genetic epistasis analysis combined with the identification of intermediates of CL biosynthesis allowed us to integrate Gep4 in the CL-biosynthetic pathway and assign an essential function during early steps of CL synthesis to Tam41, which has previously been shown to be essential for the maintenance of normal CL levels. Our experiments provide the framework for the further dissection of mechanisms that are required for accumulation and maintenance of CL levels in mitochondria.

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.

Phenotypic profiling of the human genome reveals gene products involved in plasma membrane targeting of SRC kinases.

SRC proteins are non-receptor tyrosine kinases that play key roles in regulating signal transduction by a diverse set of cell surface receptors. They contain N-terminal SH4 domains that are modified by fatty acylation and are functioning as membrane anchors. Acylated SH4 domains are both necessary and sufficient to mediate specific targeting of SRC kinases to the inner leaflet of plasma membranes. Intracellular transport of SRC kinases to the plasma membrane depends on microdomains into which SRC kinases partition upon palmitoylation. In the present study, we established a live-cell imaging screening system to identify gene products involved in plasma membrane targeting of SRC kinases. Based on siRNA arrays and a human model cell line expressing two kinds of SH4 reporter molecules, we conducted a genome-wide analysis of SH4-dependent protein targeting using an automated microscopy platform. We identified and validated 54 gene products whose down-regulation causes intracellular retention of SH4 reporter molecules. To detect and quantify this phenotype, we developed a software-based image analysis tool. Among the identified gene products, we found factors involved in lipid metabolism, intracellular transport, and cellular signaling processes. Furthermore, we identified proteins that are either associated with SRC kinases or are related to various known functions of SRC kinases such as other kinases and phosphatases potentially involved in SRC-mediated signal transduction. Finally, we identified gene products whose function is less defined or entirely unknown. Our findings provide a major resource for future studies unraveling the molecular mechanisms that underlie proper targeting of SRC kinases to the inner leaflet of plasma membranes.

Tumor-specific silencing of COPZ2 gene encoding coatomer protein complex subunit ζ 2 renders tumor cells dependent on its paralogous gene COPZ1.

Anticancer drugs are effective against tumors that depend on the molecular target of the drug. Known targets of cytotoxic anticancer drugs are involved in cell proliferation; drugs acting on such targets are ineffective against nonproliferating tumor cells, survival of which leads to eventual therapy failure. Function-based genomic screening identified the coatomer protein complex ζ1 (COPZ1) gene as essential for different tumor cell types but not for normal cells. COPZ1 encodes a subunit of coatomer protein complex 1 (COPI) involved in intracellular traffic and autophagy. The knockdown of COPZ1, but not of COPZ2 encoding isoform coatomer protein complex ζ2, caused Golgi apparatus collapse, blocked autophagy, and induced apoptosis in both proliferating and nondividing tumor cells. In contrast, inhibition of normal cell growth required simultaneous knockdown of both COPZ1 and COPZ2. COPZ2 (but not COPZ1) was down-regulated in the majority of tumor cell lines and in clinical samples of different cancer types. Reexpression of COPZ2 protected tumor cells from killing by COPZ1 knockdown, indicating that tumor cell dependence on COPZ1 is the result of COPZ2 silencing. COPZ2 displays no tumor-suppressive activities, but it harbors microRNA 152, which is silenced in tumor cells concurrently with COPZ2 and acts as a tumor suppressor in vitro and in vivo. Silencing of microRNA 152 in different cancers and the ensuing down-regulation of its host gene COPZ2 offer a therapeutic opportunity for proliferation-independent selective killing of tumor cells by COPZ1-targeting agents.

Recombinant heptameric coatomer complexes: novel tools to study isoform-specific functions.

COPI (coat protein I)-coated vesicles are implicated in various transport steps within the early secretory pathway. The major structural component of the COPI coat is the heptameric complex coatomer (CM). Recently, four isoforms of CM were discovered that may help explain various transport steps in which the complex has been reported to be involved. Biochemical studies of COPI vesicles currently use CM purified from animal tissue or cultured cells, a mixture of the isoforms, impeding functional and structural studies of individual complexes. Here we report the cloning into single baculoviruses of all CM subunits including their isoforms and their combination for expression of heptameric CM isoforms in insect cells. We show that all four isoforms of recombinant CM are fully functional in an in vitro COPI vesicle biogenesis assay. These novel tools enable functional and structural studies on CM isoforms and their subcomplexes and allow studying mutants of CM.

Mutual recognition of sphingolipid molecular species in membranes.

In membranes liquid disordered (l(d)) and liquid ordered (l(o)) domains can exist that differ in fluidity and function. L(o) areas are predominantly composed of cholesterol and sphingomyelin (SM). Study of the formation of such domains is hampered by a lack of methods to analyze specific lipid-lipid interactions at low concentrations of individual molecular lipid species in membranes. Here, we developed a simple biophysical method to experimentally assess the affinity of various molecular species of SM for cholesterol, and for their endogenous counterparts (kin) at physiological concentrations. Fluorescent SM (flc SM) molecular species with a conjugated pentaene system in their fatty acids are employed to monitor their affinity to either cholesterol or their kin by fluorescence unquenching. With this novel method we show that specific interactions of individual SMs with cholesterol or their kin exist, indicating the presence of SM nano-domains in l(d)-phases, strictly based on kin/cholesterol recognition.

Induction of cortical endoplasmic reticulum by dimerization of a coatomer-binding peptide anchored to endoplasmic reticulum membranes.

Cortical endoplasmic reticulum (cER) is a permanent feature of yeast cells but occurs transiently in most animal cell types. Ist2p is a transmembrane protein that permanently localizes to the cER in yeast. When Ist2 is expressed in mammalian cells, it induces abundant cER containing Ist2. Ist2 cytoplasmic C-terminal peptide is necessary and sufficient to induce cER. This peptide sequence resembles classic coat protein complex I (COPI) coatomer protein-binding KKXX signals, and indeed the dimerized peptide binds COPI in vitro. Controlled dimerization of this peptide induces cER in cells. RNA interference experiments confirm that coatomer is required for cER induction in vivo, as are microtubules and the microtubule plus-end binding protein EB1. We suggest that Ist2 dimerization triggers coatomer binding and clustering of this protein into domains that traffic at the microtubule growing plus-end to generate the cER beneath the plasma membrane. Sequences similar to the Ist2 lysine-rich tail are found in mammalian STIM proteins that reversibly induce the formation of cER under calcium control.

Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle formation.

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.

Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome.

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.

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.