Sterols and Sphingolipids as New Players in Cell Wall Building and Apical Growth of Nicotiana tabacum L. Pollen Tubes.

Pollen tubes are tip-growing cells that create safe routes to convey sperm cells to the embryo sac for double fertilization. Recent studies have purified and biochemically characterized detergent-insoluble membranes from tobacco pollen tubes. These microdomains, called lipid rafts, are rich in sterols and sphingolipids and are involved in cell polarization in organisms evolutionarily distant, such as fungi and mammals. The presence of actin in tobacco pollen tube detergent-insoluble membranes and the preferential distribution of these domains on the apical plasma membrane encouraged us to formulate the intriguing hypothesis that sterols and sphingolipids could be a "trait d'union" between actin dynamics and polarized secretion at the tip. To unravel the role of sterols and sphingolipids in tobacco pollen tube growth, we used squalestatin and myriocin, inhibitors of sterol and sphingolipid biosynthesis, respectively, to determine whether lipid modifications affect actin fringe morphology and dynamics, leading to changes in clear zone organization and cell wall deposition, thus suggesting a role played by these lipids in successful fertilization.

Targeting PCSK9 in Liver Cancer Cells Triggers Metabolic Exhaustion and Cell Death by Ferroptosis.

Deregulated lipid metabolism is a common feature of liver cancers needed to sustain tumor cell growth and survival. We aim at taking advantage of this vulnerability and rewiring the oncogenic metabolic hub by targeting the key metabolic player pro-protein convertase subtilisin/kexin type 9 (PCSK9). We assessed the effect of PCSK9 inhibition using the three hepatoma cell lines Huh6, Huh7 and HepG2 and validated the results using the zebrafish in vivo model. PCSK9 deficiency led to strong inhibition of cell proliferation in all cell lines. At the lipid metabolic level, PCSK9 inhibition was translated by an increase in intracellular neutral lipids, phospholipids and polyunsaturated fatty acids as well as a higher accumulation of lipid hydroperoxide. Molecular signaling analysis involved the disruption of the sequestome 1/Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (p62/Keap1/Nrf2) antioxidative axis, leading to ferroptosis, for which morphological features were confirmed by electron and confocal microscopies. The anti-tumoral effects of PCSK9 deficiency were validated using xenograft experiments in zebrafish. The inhibition of PCSK9 was effective in disrupting the oncometabolic process, inducing metabolic exhaustion and enhancing the vulnerability of cancer cells to iron-triggered lipid peroxidation. We provide strong evidence supporting the drug repositioning of anti-PCSK9 approaches to treat liver cancers.

Rewiring Lipid Metabolism by Targeting PCSK9 and HMGCR to Treat Liver Cancer.

Alterations in lipid handling are an important hallmark in cancer. Our aim here is to target key metabolic enzymes to reshape the oncogenic lipid metabolism triggering irreversible cell breakdown. We targeted the key metabolic player proprotein convertase subtilisin/kexin type 9 (PCSK9) using a pharmacological inhibitor (R-IMPP) alone or in combination with 3-hydroxy 3-methylglutaryl-Coenzyme A reductase (HMGCR) inhibitor, simvastatin. We assessed the effect of these treatments using 3 hepatoma cell lines, Huh6, Huh7 and HepG2 and a tumor xenograft in chicken choriorallantoic membrane (CAM) model. PCSK9 deficiency led to dose-dependent inhibition of cell proliferation in all cell lines and a decrease in cell migration. Co-treatment with simvastatin presented synergetic anti-proliferative effects. At the metabolic level, mitochondrial respiration assays as well as the assessment of glucose and glutamine consumption showed higher metabolic adaptability and surge in the absence of PCSK9. Enhanced lipid uptake and biogenesis led to excessive accumulation of intracellular lipid droplets as revealed by electron microscopy and metabolic tracing. Using xenograft experiments in CAM model, we further demonstrated the effect of anti-PCSK9 treatment in reducing tumor aggressiveness. Targeting PCSK9 alone or in combination with statins deserves to be considered as a new therapeutic option in liver cancer clinical applications.

Doxorubicin Inhibits Phosphatidylserine Decarboxylase and Modifies Mitochondrial Membrane Composition in HeLa Cells.

Doxorubicin (DXR) is a drug widely used in chemotherapy. Its mode of action is based on its intercalation properties, involving the inhibition of topoisomerase II. However, few studies have reported the mitochondrial effects of DXR while investigating cardiac toxicity induced by the treatment, mostly in pediatric cases. Here, we demonstrate that DXR alters the mitochondrial membrane composition associated with bioenergetic impairment and cell death in human cancer cells. The remodeling of the mitochondrial membrane was explained by phosphatidylserine decarboxylase (PSD) inhibition by DXR. PSD catalyzes phosphatidylethanolamine (PE) synthesis from phosphatidylserine (PS), and DXR altered the PS/PE ratio in the mitochondrial membrane. Moreover, we observed that DXR localized to the mitochondrial compartment and drug uptake was rapid. Evaluation of other topoisomerase II inhibitors did not show any impact on the mitochondrial membrane composition, indicating that the DXR effect was specific. Therefore, our findings revealed a side molecular target for DXR and PSD, potentially involved in DXR anti-cancer properties and the associated toxicity.

A Combinatorial Lipid Code Shapes the Electrostatic Landscape of Plant Endomembranes.

Membrane surface charge is critical for the transient, yet specific recruitment of proteins with polybasic regions to certain organelles. In eukaryotes, the plasma membrane (PM) is the most electronegative compartment of the cell, which specifies its identity. As such, membrane electrostatics is a central parameter in signaling, intracellular trafficking, and polarity. Here, we explore which are the lipids that control membrane electrostatics using plants as a model. We show that phosphatidylinositol-4-phosphate (PI4P), phosphatidic acidic (PA), and phosphatidylserine (PS) are separately required to generate the electrostatic signature of the plant PM. In addition, we reveal the existence of an electrostatic territory that is organized as a gradient along the endocytic pathway and is controlled by PS/PI4P combination. Altogether, we propose that combinatorial lipid composition of the cytosolic leaflet of organelles not only defines the electrostatic territory but also distinguishes different functional compartments within this territory by specifying their varying surface charges.

Factores de riesgo relacionados con la positividad al mosquito Aedes aegypti en viviendas del área de salud del Policlínico Municipal / Risk factors related to the positivity to Aedes aegypti mosquito in houses of the Municipal Polyclinic health area

Se realizó un estudio de casos y controles representados por 155 y 310 viviendas, respectivamente, para identificar factores de riesgo relacionados con la positividad al mosquito Aedes aegypti en el radio de acción del Policlínico Municipal de Santiago de Cuba, durante el 2012. Entre esos factores de riesgo constituyeron los principales: depósitos bajos no protegidos, patios no saneados, salideros, tanque elevado y otros, acerca de los cuales se obtuvieron el odds ratio, la razón atribuible poblacional y la fracción etiológica. Se concluyó que tales factores perpetuaron la presencia de focos del vector en esos hogares de la mencionada área de salud

A case and control study represented by 155 and 310 houses, respectively, to identify risk factors related to the positivity to Aedes aegypti mosquito in the area of the Municipal Polyclinic in Santiago de Cuba, was carried out during 2012. Among the risk factors there were: unprotected low deposits, dirty yards, likings, high deposits and others, about which the odds ratio, the populational attributable reason and the etiologic fraction were obtained. It was concluded that such factors perpetuated the presence of the vector focuses in those homes of the aforementioned health area

Characterisation of detergent-insoluble membranes in pollen tubes of Nicotiana tabacum (L.).

Pollen tubes are the vehicle for sperm cell delivery to the embryo sac during fertilisation of Angiosperms. They provide an intriguing model for unravelling mechanisms of growing to extremes. The asymmetric distribution of lipids and proteins in the pollen tube plasma membrane modulates ion fluxes and actin dynamics and is maintained by a delicate equilibrium between exocytosis and endocytosis. The structural constraints regulating polarised secretion and asymmetric protein distribution on the plasma membrane are mostly unknown. To address this problem, we investigated whether ordered membrane microdomains, namely membrane rafts, might contribute to sperm cell delivery. Detergent insoluble membranes, rich in sterols and sphingolipids, were isolated from tobacco pollen tubes. MALDI TOF/MS analysis revealed that actin, prohibitins and proteins involved in methylation reactions and in phosphoinositide pattern regulation are specifically present in pollen tube detergent insoluble membranes. Tubulins, voltage-dependent anion channels and proteins involved in membrane trafficking and signalling were also present. This paper reports the first evidence of membrane rafts in Angiosperm pollen tubes, opening new perspectives on the coordination of signal transduction, cytoskeleton dynamics and polarised secretion.

The bifunctional protein TtFARAT from Tetrahymena thermophila catalyzes the formation of both precursors required to initiate ether lipid biosynthesis.

The biosynthesis of ether lipids and wax esters requires as precursors fatty alcohols, which are synthesized by fatty acyl reductases (FARs). The presence of ether glycerolipids as well as branched wax esters has been reported in several free-living ciliate protozoa. In the genome of Tetrahymena thermophila, the only ORF sharing similarities with FARs is fused to an acyltransferase-like domain, whereas, in most other organisms, FARs are monofunctional proteins of similar size and domain structure. Here, we used heterologous expression in plant and yeast to functionally characterize the activities catalyzed by this protozoan protein. Transient expression in tobacco epidermis of a truncated form fused to the green fluorescence protein followed by confocal microscopy analysis suggested peroxisomal localization. In vivo approaches conducted in yeast indicated that the N-terminal FAR-like domain produced both 16:0 and 18:0 fatty alcohols, whereas the C-terminal acyltransferase-like domain was able to rescue the lethal phenotype of the yeast double mutant gat1Δ gat2Δ. Using in vitro approaches, we further demonstrated that this domain is a dihydroxyacetone phosphate acyltransferase that uses preferentially 16:0-coenzyme A as an acyl donor. Finally, coexpression in yeast with the alkyl-dihydroxyacetone phosphate synthase from T. thermophila resulted the detection of various glycerolipids with an ether bond, indicating reconstitution of the ether lipid biosynthetic pathway. Together, these results demonstrate that this FAR-like protein is peroxisomal and bifunctional, providing both substrates required by alkyl-dihydroxyacetone phosphate synthase to initiate ether lipid biosynthesis.

Cell wall constrains lateral diffusion of plant plasma-membrane proteins.

A cell membrane can be considered a liquid-phase plane in which lipids and proteins theoretically are free to diffuse. Numerous reports, however, describe retarded diffusion of membrane proteins in animal cells. This anomalous diffusion results from a combination of structuring factors including protein-protein interactions, cytoskeleton corralling, and lipid organization into microdomains. In plant cells, plasma-membrane (PM) proteins have been described as relatively immobile, but the control mechanisms that structure the PM have not been studied. Here, we use fluorescence recovery after photobleaching to estimate mobility of a set of minimal PM proteins. These proteins consist only of a PM-anchoring domain fused to a fluorescent protein, but their mobilities remained limited, as is the case for many full-length proteins. Neither the cytoskeleton nor membrane microdomain structure was involved in constraining the diffusion of these proteins. The cell wall, however, was shown to have a crucial role in immobilizing PM proteins. In addition, by single-molecule fluorescence imaging we confirmed that the pattern of cellulose deposition in the cell wall affects the trajectory and speed of PM protein diffusion. Regulation of PM protein dynamics by the plant cell wall can be interpreted as a mechanism for regulating protein interactions in processes such as trafficking and signal transduction.

ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma.

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F(0)/F(1)-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F(0)/F(1)-ATP synthase F(1)ß subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F(0)/F(1)-ATP synthase, for ATP production in the mitochondrion.

Biochemical characterization of a chloroplast localized fatty acid reductase from Arabidopsis thaliana.

Primary long-chain fatty alcohols are present in a variety of phyla. In eukaryotes, the production of fatty alcohols is catalyzed by fatty acyl-CoA reductase (FAR) enzymes that convert fatty acyl-CoAs or acyl-ACPs into fatty alcohols. Here, we report on the biochemical properties of a purified plant FAR, Arabidopsis FAR6 (AtFAR6). In vitro assays show that the enzyme preferentially uses 16 carbon acyl-chains as substrates and produces predominantly fatty alcohols. Free fatty acids and fatty aldehyde intermediates can be released from the enzyme, in particular with suboptimal chain lengths and concentrations of the substrates. Both acyl-CoA and acyl-ACP could serve as substrates. Transient expression experiments in Nicotiana tabacum showed that AtFAR6 is a chloroplast localized FAR. In addition, expression of full length AtFAR6 in Nicotiana benthamiana leaves resulted in the production of C16:0-alcohol within this organelle. Finally, a GUS reporter gene fusion with the AtFAR6 promoter showed that the AtFAR6 gene is expressed in various tissues of the plant with a distinct pattern compared to that of other Arabidopsis FARs, suggesting specialized functions in planta.

Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis.

The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.

Glucosylceramide biosynthesis is involved in Golgi morphology and protein secretion in plant cells.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

Blocking ER export of the Golgi SNARE SYP31 affects plant growth.

We recently identified a novel and transplantable di-acidic motif (EXXD) that facilitates ER export of the Golgi syntaxin SYP31 (type IV protein) and which may function also for type I and type II proteins in plants. By mutagenesis of Arabidopsis thaliana SYP31 and live cell imaging experiments in tobacco leaf epidermal cells, we determined that replacing the MELAD sequence of SYP31 with gagag retained SYP31 in the ER, which demonstrates that the di-acidic motif ELAD is critical for SYP31 ER export. To investigate whether blockage of a Golgi SNARE in the ER have consequences for plant growth, we produced tobacco plants stably overexpressing either the wild type MELAD or the mutant gagag form of SYP31. Whereas tobacco plants overexpressing the wild-type SYP31 developed to set seed, tobacco plants overexpressing the mutant form gagag rapidly became chlorotic, ceased their growth and invariably died after several weeks. This indicated that retention of overexpressed SYP31 in the ER is likely toxic for the secretory pathway and, therefore, plant development. Putative explanations for this observation are discussed taking into account SNARE properties and possible interactions.

A novel di-acidic motif facilitates ER export of the syntaxin SYP31.

It is generally accepted that ER protein export is largely influenced by the transmembrane domain (TMD). The situation is unclear for membrane-anchored proteins such as SNAREs, which are anchored to the membrane by their TMD at the C-terminus. For example, in plants, Sec22 and SYP31 (a yeast Sed5 homologue) have a 17 aa TMD but different locations (ER/Golgi and Golgi), indicating that TMD length alone is not sufficient to explain their targeting. To establish the identity of factors that influence SNARE targeting, mutagenesis and live cell imaging experiments were performed on SYP31. It was found that deletion of the entire N-terminus domain of SYP31 blocked the protein in the ER. Several deletion mutants of different parts of this N-terminus domain indicated that a region between the SNARE helices Hb and Hc is required for Golgi targeting. In this region, replacement of the aa sequence MELAD by GAGAG or MALAG retained the protein in the ER, suggesting that MELAD may function as a di-acidic ER export motif EXXD. This suggestion was further verified by replacing the established di-acidic ER export motif DLE of a type II Golgi protein AtCASP and a membrane-anchored type I chimaera, TMcCCASP, by MELAD or GAGAG. The MELAD motif allowed the proteins to reach the Golgi, whereas the motif GAGAG was found to be insufficient to facilitate ER protein export. Our analyses indicate that we have identified a novel and transplantable di-acidic motif that facilitates ER export of SYP31 and may function for type I and type II proteins in plants.

The VLCFA elongase gene family in Arabidopsis thaliana: phylogenetic analysis, 3D modelling and expression profiling.

As precursors of wax compounds, very long chain fatty acids participate in the limitation of non-stomatal water loss and the prevention of pathogen attacks. They also serve as energy storage in seeds and as membrane building blocks. Their biosynthesis is catalyzed by the acyl-CoA elongase, a membrane-bound enzymatic complex containing four distinct enzymes (KCS, KCR, HCD and ECR). Twenty-one 3-ketoacyl-CoA synthase (KCS) genes have been identified in Arabidopsis thaliana genome. In this paper we present an overview of the acyl-CoA elongase genes in Arabidopsis focusing on the entire KCS family. We show that the KCS family is made up of 8 distinct subclasses, according to their phylogeny, duplication history, genomic organization, protein topology and 3D modelling. The analysis of the subcellular localization in tobacco cells of the different subunits of the acyl-CoA elongase shows that all these proteins are localized in the endoplasmic reticulum demonstrating that VLCFA production occurs in this compartment. The expression patterns in Arabidopsis of the acyl-CoA elongase genes suggest several levels of regulations at the tissular or organ level but also under stress conditions suggesting a complex organization of this multigenic family.

Localization of Fas/CD95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway.

Activation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway is known to protect tumor cells from apoptosis and more specifically from the Fas-mediated apoptotic signal. The antitumoral agent edelfosine sensitizes leukemic cells to death by inducing the redistribution of the apoptotic receptor Fas into plasma membrane subdomains called lipid rafts. Herein, we show that inhibition of the PI3K signal by edelfosine triggers a Fas-mediated apoptotic signal independently of the Fas/FasL interaction. Furthermore, similarly to edelfosine, blockade of the PI3K activity, using specific inhibitors LY294002 and wortmannin, leads to the clustering of Fas whose supramolecular complex is colocalized within the lipid rafts. These findings indicate that the antitumoral agent edelfosine down-modulates the PI3K signal to sensitize tumor cells to death through the redistribution of Fas into large platform of membrane rafts.

In tobacco leaf epidermal cells, the integrity of protein export from the endoplasmic reticulum and of ER export sites depends on active COPI machinery.

Trafficking of secretory proteins between the endoplasmic reticulum (ER) and the Golgi apparatus depends on coat protein complexes I (COPI) and II (COPII) machineries. To date, full characterization of the distribution and dynamics of these machineries in plant cells remains elusive. Furthermore, except for a presumed linkage between COPI and COPII for the maintenance of ER protein export, the mechanisms by which COPI influences COPII-mediated protein transport from the ER in plant cells are largely uncharacterized. Here we dissect the dynamics of COPI in intact cells using live-cell imaging and fluorescence recovery after photobleaching analyses to provide insights into the distribution of COPI and COPII machineries and the mechanisms by which COPI influences COPII-mediated protein export from the ER. We found that Arf1 and coatomer are dynamically associated with the Golgi apparatus and that the COPII coat proteins Sec24 and Sec23 localize at ER export sites that track with the Golgi apparatus in tobacco leaf epidermal cells. Arf1 is also localized at additional structures that originate from the Golgi apparatus but that lack coatomer, supporting the model that Arf1 also has a coatomer-independent role for post-Golgi protein transport in plants. When ER to Golgi protein transport is inhibited by mutations that hamper Arf1-GTPase activity without directly disrupting the COPII machinery for ER protein export, Golgi markers are localized in the ER and the punctate distribution of Sec24 and Sec23 at the ER export sites is lost. These findings suggest that Golgi membrane protein distribution is maintained by the balanced action of COPI and COPII systems, and that Arf1-coatomer is most likely indirectly required for forward trafficking out of the ER due to its role in recycling components that are essential for differentiation of the ER export domains formed by the Sar1-COPII system.

Modulation of Fas-mediated apoptosis by lipid rafts in T lymphocytes.

In type I cells, Fas-mediated cell death requires cytoplasmic membrane subdomains called microdomains or lipid rafts. On the contrary, Fas signaling is independent of these structures in type II cells. We report that in human T cells, CD28, CD59, and CD55 are all localized into lipid rafts and that CD28 is concentrated into microdomains enriched in ganglioside GM1, whereas CD59 and CD55 are not. Moreover, CD28 cross-linking leads to the formation of lipid raft clusters which exclude CD59 and CD55, and reciprocally. Coligation of Fas with CD55 or CD59 inhibits the apoptotic signal, whereas CD28 recruitment amplifies the Fas signaling pathway. Therefore, we conclude that 1) different types of microdomains exist on the cell surface, with distinct functional properties and 2) the recruitment of these distinct structures may differentially modulate the Fas pathway. Moreover, our results demonstrate that Fas-induced apoptosis can be controlled at the level of the cytoplasmic membrane.

Sec22 and Memb11 are v-SNAREs of the anterograde endoplasmic reticulum-Golgi pathway in tobacco leaf epidermal cells.

Distinct sets of soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) are distributed to specific intracellular compartments and catalyze membrane fusion events. Although the central role of these proteins in membrane fusion is established in nonplant systems, little is known about their role in the early secretory pathway of plant cells. Analysis of the Arabidopsis (Arabidopsis thaliana) genome reveals 54 genes encoding SNARE proteins, some of which are expected to be key regulators of membrane trafficking between the endoplasmic reticulum (ER) and the Golgi. To gain insights on the role of SNAREs of the early secretory pathway in plant cells, we have cloned the Arabidopsis v-SNAREs Sec22, Memb11, Bet11, and the t-SNARE Sed5, and analyzed their distribution in plant cells in vivo. By means of live cell imaging, we have determined that these SNAREs localize at the Golgi apparatus. In addition, Sec22 was also distributed at the ER. We have then focused on understanding the function of Sec22 and Memb11 in comparison to the other SNAREs. Overexpression of the v-SNAREs Sec22 and Memb11 but not of the other SNAREs induced collapse of Golgi membrane proteins into the ER, and the secretion of a soluble secretory marker was abrogated by all SNAREs. Our studies suggest that Sec22 and Memb11 are involved in anterograde protein trafficking at the ER-Golgi interface.