Regulation of membrane trafficking and organ separation by the NEVERSHED ARF-GAP protein.

Cell separation, or abscission, is a highly specialized process in plants that facilitates remodeling of their architecture and reproductive success. Because few genes are known to be essential for organ abscission, we conducted a screen for mutations that alter floral organ shedding in Arabidopsis. Nine recessive mutations that block shedding were found to disrupt the function of an ADP-ribosylation factor-GTPase-activating protein (ARF-GAP) we have named NEVERSHED (NEV). As predicted by its homology to the yeast Age2 ARF-GAP and transcriptional profile, NEV influences other aspects of plant development, including fruit growth. Co-localization experiments carried out with NEV-specific antiserum and a set of plant endomembrane markers revealed that NEV localizes to the trans-Golgi network and endosomes in Arabidopsis root epidermal cells. Interestingly, transmission electron micrographs of abscission zone regions from wild-type and nev flowers reveal defects in the structure of the Golgi apparatus and extensive accumulation of vesicles adjacent to the cell walls. Our results suggest that NEV ARF-GAP activity at the trans-Golgi network and distinct endosomal compartments is required for the proper trafficking of cargo molecules required for cell separation.

Equine herpesvirus protein E10 induces membrane recruitment and phosphorylation of its cellular homologue, bcl-10.

v-E10, a caspase recruitment domain (CARD)-containing gene product of equine herpesvirus 2, is the viral homologue of the bcl-10 protein whose gene was found to be translocated in mucosa-associated lymphoid tissue (MALT) lymphomas. v-E10 efficiently activates the c-jun NH(2)-terminal kinase (JNK), p38 stress kinase, and the nuclear factor (NF)-kappaB transcriptional pathway and interacts with its cellular homologue, bcl-10, via a CARD-mediated interaction. Here we demonstrate that v-E10 contains a COOH-terminal geranylgeranylation consensus site which is responsible for its plasma membrane localization. Expression of v-E10 induces hyperphosphorylation and redistribution of bcl-10 from the cytoplasm to the plasma membrane, a process which is dependent on the intactness of the v-E10 CARD motif. Both membrane localization and a functional CARD motif are important for v-E10-mediated NF-kappaB induction, but not for JNK activation, which instead requires a functional v-E10 binding site for tumor necrosis factor receptor-associated factor (TRAF)6. Moreover, v-E10-induced NF-kappaB activation is inhibited by a dominant negative version of the bcl-10 binding protein TRAF1, suggesting that v-E10-induced membrane recruitment of cellular bcl-10 induces constitutive TRAF-mediated NF-kappaB activation.

hShroom1 links a membrane bound protein to the actin cytoskeleton.

hShroom1 (hShrm1) is a member of the Apx/Shroom (Shrm) protein family and was identified from a yeast two-hybrid screen as a protein that interacts with the cytoplasmic domain of melanoma cell adhesion molecule (MCAM). The characteristic signature of the Shrm family is the presence of a unique domain, ASD2 (Apx/Shroom domain 2). mRNA analysis suggests that hShrm1 is expressed in brain, heart, skeletal muscle, colon, small intestine, kidney, placenta and lung tissue, as well a variety of melanoma and other cell lines. Co-immunoprecipitation and bioluminescence resonance energy transfer (BRET) experiments indicate that hShrm1 and MCAM interact in vivo and by immunofluorescence microscopy some co-localization of these proteins is observed. hShrm1 partly co-localises with beta-actin and is found in the Triton X-100 insoluble fraction of melanoma cell extracts. We propose that hShrm1 is involved in linking MCAM to the cytoskeleton.

Proteomic analysis of membrane rafts of melanoma cells identifies protein patterns characteristic of the tumor progression stage.

The molecular mechanisms controlling the progression of melanoma from a localized tumor to an invasive and metastatic disease are poorly understood. In the attempt to start defining a functional protein profile of melanoma progression, we have analyzed by LC-MS/MS the proteins associated with detergent resistant membranes (DRMs), which are enriched in cholesterol/sphingolipids-containing membrane rafts, of melanoma cell lines derived from tumors at different stages of progression. Since membrane rafts are involved in several biological processes, including signal transduction and protein trafficking, we hypothesized that the association of proteins with rafts can be regulated during melanoma development and affect protein function and disease progression. We have identified a total of 177 proteins in the DRMs of the cell lines examined. Among these, we have found groups of proteins preferentially associated with DRMs of either less malignant radial growth phase/vertical growth phase (VGP) cells, or aggressive VGP and metastatic cells suggesting that melanoma cells with different degrees of malignancy have different DRM profiles. Moreover, some proteins were found in DRMs of only some cell lines despite being expressed at similar levels in all the cell lines examined, suggesting the existence of mechanisms controlling their association with DRMs. We expect that understanding the mechanisms regulating DRM targeting and the activity of the proteins differentially associated with DRMs in relation to cell malignancy will help identify new molecular determinants of melanoma progression.

Remorin, a solanaceae protein resident in membrane rafts and plasmodesmata, impairs potato virus X movement.

Remorins (REMs) are proteins of unknown function specific to vascular plants. We have used imaging and biochemical approaches and in situ labeling to demonstrate that REM clusters at plasmodesmata and in approximately 70-nm membrane domains, similar to lipid rafts, in the cytosolic leaflet of the plasma membrane. From a manipulation of REM levels in transgenic tomato (Solanum lycopersicum) plants, we show that Potato virus X (PVX) movement is inversely related to REM accumulation. We show that REM can interact physically with the movement protein TRIPLE GENE BLOCK PROTEIN1 from PVX. Based on the localization of REM and its impact on virus macromolecular trafficking, we discuss the potential for lipid rafts to act as functional components in plasmodesmata and the plasma membrane.

Regulation of frizzled receptor activity at the plasma membrane : an interplay of the receptor, the trimeric G protein Go, and RGS protein and a scaffolding protein Kermit

G-protein-signaling pathways convey extracellular signals inside the cells and regulate distinct physiological responses. This type of signaling pathways consists of three major components: G-protein-coupled receptors (GPCRs), heterotrimeric G proteins (G-proteins) and downstream effectors. Upon ligand binding, GPCRs activate heterotrimeric G proteins to initiate the signaling cascade. Dysfunction of GPCR signaling correlates with numerous diseases such as diabetes, nervous and immune system deficiency, and cancer. As the signaling switcher, G-proteins (Gs, Gq/11, G12/13, and Gi/o) have been an appealing topic of research for decades. A heterotrimeric G-protein is composed of three subunits, the guanine nucleotide associated a-subunit, ß and y subunits. In general, the duration of signaling is determined by the lifetime of activated (GTP bound) Ga subunits. Identification of novel communication partners of Ga subunits appears to be an attractive way to understand the machinery of GPCR signaling. In our lab, we mainly focus on Gao, which is abundantly expressed in the nervous system. Here we present two novel interacting partners of Drosophila Gao: Dhit and Kermit, identified through yeast two-hybrid screening and genetic screening respectively. Dhit is characterized by a small size with a conserved RGS domain and an N-terminal cysteine rich motif. The RGS domain possesses the GAP (GTPase activating protein) activity towards G proteins. However, we found that Dhit exerts not only the GAP activity but also the GDI (guanine nucleotide dissociation inhibitor) activity towards Gao. The unexpected GDI activity is preserved in GAIP/RGS19 - a mammalian homologue of Dhit. Further experiments confirmed the GDI activity of Dhit and GAIP/RGS19 in Drosophila and mammalian cell models. Therefore, we propose that Dhit and its mammalian homologues modulate GPCR signaling by a double suppression of Ga subunits - suppression of their nucleotide exchange with GTP and acceleration of their hydrolysis of GTP. Kermit/GEPC was first identified as a binding partner of GAIP/RGS19 in a yeast two- hybrid screen. Instead of interacting with the Drosophila homologue of GAIP/RGS19 (Dhit), Kermit binds to Gao in vivo and in vitro. The functional consequence of Kermit/Gao interaction is the regulation of localization of Vang (one of the planar cell polarity core components) at the apical membrane. Overall, my work elaborated the action of Gao with its two interaction partners in Gao- mediated signaling pathway. Conceivably, the understanding of GPCR signaling including Gao and its regulators or effectors will ultimately shed light on future pharmaceutical research. - Les voies de signalisation médiées par les protéines G transmettent des signaux extracellulaires à l'intérieur des cellules pour réguler des réponses physiologiques distinctes. Cette voie de signalisation consiste en trois composants majeurs : les récepteurs couplés aux protéines G (GPCRs), les protéines G hétérotrimériques (G-proteins) et les effecteurs en aval. Suite à la liaison du ligand, les GPCRs activent les protéines G hétérotrimériques qui initient la cascade de signalisation. Des dysfonctions dans la signalisation médiée par les GPCRs sont corrélées avec de nombreuses maladies comme le diabète, des déficiences immunes et nerveuses, ainsi que le cancer. Puisque la voie de signalisation s'active et se désactive, les protéines G (Gs, Gq/11, G12/13 et Gi/o) ont été un sujet de recherche attrayant pendant des décennies. Une protéine G hétérotrimérique est composée de trois sous-unités, la sous-unité a associée au nucléotide guanine, ainsi que les sous-unités ß et y. En général, la durée du signal est déterminée par le temps de demi-vie des sous-unités Ga activées (Ga liées au GTP). Identifier de nouveaux partenaires de communication des sous-unités Ga se révèle être un moyen attractif de comprendre la machinerie de la signalisation par les GPCRs. Dans notre laboratoire nous nous sommes concentrés principalement sur Gao qui est exprimée de manière abondante dans le système nerveux. Nous présentons ici deux nouveaux partenaires qui interagissent avec Gao chez la drosophile: Dhit et Kermit, qui ont été identifiés respectivement par la méthode du yeast two-hybrid et par criblage génétique. Dhit est caractérisé par une petite taille, avec un domaine RGS conservé et un motif N- terminal riche en cystéines. Le domaine RGS contient une activité GAP (GTPase activating protein) pour les protéines G. Toutefois, nous avons découvert que Dhit exerce non seulement une activité GAP mais aussi une activité GDI (guanine nucleotide dissociation inhibitor) à l'égard de Gao. Cette activité GDI inattendue est préservée dans RGS19 - un homologue de Dhit chez les mammifères. Des expériences supplémentaires ont confirmé l'activité GDI de Dhit et de RGS19 chez Drosophila melanogaster et les modèles cellulaires mammifères. Par conséquent, nous proposons que Dhit et ses homologues mammifères modulent la signalisation GPCR par une double suppression des sous-unités Ga - suppression de leur nucléotide d'échange avec le GTP et une accélération dans leur hydrolyse du GTP. Kermit/GIPC a été premièrement identifié comme un partenaire de liaison de RGS19 dans le criblage par yeast two-hybrid. Au lieu d'interagir avec l'homologue chez la drosophile de RGS19 (Dhit), Kermit se lie à Gao in vivo et in vitro. La conséquence fonctionnelle de l'interaction Kermit/Gao est la régulation de la localisation de Vang, un des composants essentiel de la polarité planaire cellulaire, à la membrane apicale. Globalement, mon travail a démontré l'action de Gao avec ses deux partenaires d'interaction dans la voie de signalisation médiée par Gao. La compréhension de la signalisation par les GPCRs incluant Gao et ses régulateurs ou effecteurs aboutira à mettre en lumière de futurs axes dans la recherche pharmacologique.

Membrane insertion and antibody recognition of a glycosylphosphatidylinositol-anchored protein: an optical study.

The kinetics of binding of a glycolipid-anchored protein (the promastigote surface protease, PSP) to planar lecithin bilayers is studied by an integrated optics technique, in which the bilayer membrane is supported on an optical wave guide and the phase velocities of guided light modes in the wave guide are measured. From these velocities, the optical parameters of the membrane and PSP layers deposited on the waveguide are determined, yielding in particular the mass of PSP bound to the membrane, which is followed in real time. From a comparison of the binding rates of PSP and PSP from which the lipid moiety has been removed, it is shown that the lipid moiety plays a key role in anchoring the protein to the membrane. Specific and nonspecific binding of antibodies to membrane-anchored PSP is also investigated. As little as a fifth of a monolayer of PSP is sufficient to suppress the appreciable nonspecific binding of antibodies to the membrane.

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing.

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.

Profiling of T-cell receptor signaling complex assembly in human CD4 T-lymphocytes using RP protein arrays.

The RP protein (RPP) array approach immobilizes minute amounts of cell lysates or tissue protein extracts as distinct microspots on NC-coated slide. Subsequent detection with specific antibodies allows multiplexed quantification of proteins and their modifications at a scale that is beyond what traditional techniques can achieve. Cellular functions are the result of the coordinated action of signaling proteins assembled in macromolecular complexes. These signaling complexes are highly dynamic structures that change their composition with time and space to adapt to cell environment. Their comprehensive analysis requires until now relatively large amounts of cells (>5 x 10(7)) due to their low abundance and breakdown during isolation procedure. In this study, we combined small scale affinity capture of the T-cell receptor (TCR) and RPP arrays to follow TCR signaling complex assembly in human ex vivo isolated CD4 T-cells. Using this strategy, we report specific recruitment of signaling components to the TCR complex upon T-cell activation in as few as 0.5 million of cells. Second- to fourth-order TCR interacting proteins were accurately quantified, making this strategy specially well-suited to the analysis of membrane-associated signaling complexes in limited amounts of cells or tissues, e.g., ex vivo isolated cells or clinical specimens.

Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63).

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell types. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified leishmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was evident at low enzyme/substrate ratios, over a broad pH range, and was inhibited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus, Leishmania infection may result in leishmanolysin-dependent hydrolysis of MRP, a major protein kinase C substrate in macrophages.

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

The endodermis acts as a "second skin" in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain.

Bacillus subtilis contains two small c-type cytochromes with homologous heme domains but different types of membrane anchors.

We demonstrate that the cccB gene, identified in the Bacillus subtilis genome sequence project, is the structural gene for a 10-kDa membrane-bound cytochrome c(551) lipoprotein described for the first time in B. subtilis. Apparently, CccB corresponds to cytochrome c(551) of the thermophilic bacterium Bacillus PS3. The heme domain of B. subtilis cytochrome c(551) is very similar to that of cytochrome c(550), a protein encoded by the cccA gene and anchored to the membrane by a single transmembrane polypeptide segment. Thus, B. subtilis contains two small, very similar, c-type cytochromes with different types of membrane anchors. The cccB gene is cotranscribed with the yvjA gene, and transcription is repressed by glucose. Mutants deleted for cccB or yvjA-cccB show no apparent growth, sporulation, or germination defect. YvjA is not required for the synthesis of cytochrome c(551), and its function remains unknown.

Ligand binding transmits conformational changes across the membrane-spanning region to the intracellular side of the 5-HT3 serotonin receptor.

Conformational changes of channel activation: Five enhanced green fluorescent protein (EGFP) molecules (green cylinders) were integrated into the intracellular part of the homopentameric ionotropic 5-HT3 receptor. This allowed the detection of extracellular binding of fluorescent ligands (?) to EGFP by FRET, and also enabled the quantification of agonist-induced conformational changes in the intracellular region of the receptor by homo-FRET between EGFPs. The approach opens novel ways for probing receptor activation and functional screening of therapeutic compounds.