RESUMEN
The mechanisms controlling the abundance and sub-cellular distribution of caveolae are not well described. A first step towards determining such mechanisms would be identification of relevant proteins that interact with known components of caveolae. Here, we applied proximity biotinylation (BioID) to identify a list of proteins that may interact with the caveolar protein cavin1. Screening of these candidates using siRNA to reduce their expression revealed that one of them, CSDE1, regulates the levels of mRNAs and protein expression for multiple components of caveolae. A second candidate, CD2AP, co-precipitated with cavin1. Caveolar proteins were observed in characteristic and previously un-described linear arrays adjacent to cell-cell junctions in both MDCK cells, and in HeLa cells overexpressing an active form of the small GTPase Rac1. CD2AP was required for the recruitment of caveolar proteins to these linear arrays. We conclude that BioID will be useful in identification of new proteins involved in the cell biology of caveolae, and that interaction between CD2AP and cavin1 may have an important role in regulating the sub-cellular distribution of caveolae.
Asunto(s)
Caveolas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Perros , Células HeLa , Humanos , Células de Riñón Canino Madin Darby , ARN Interferente Pequeño/genética , Proteína de Unión al GTP rac1/metabolismoRESUMEN
The three Rnd proteins, Rnd1, Rnd2 and RhoE/Rnd3, are a subset of Rho family proteins that are unusual in that they bind but do not hydrolyse GTP, and are therefore not regulated by the classical GTP/GDP conformational switch of small GTPases. Increased expression of each Rnd protein induces loss of stress fibres in cultured fibroblasts and epithelial cells, acting antagonistically to RhoA, which stimulates stress fibre formation. RhoE is farnesylated and localizes partly on membranes, including the Golgi and plasma membrane, and in the cytosol. RhoE inhibits RhoA signalling in part by binding to the RhoA-activated serine/threonine kinase ROCK I (Rho-associated kinase I), thereby preventing it from phosphorylating its targets. RhoE activity is itself regulated by phosphorylation by ROCK I on multiple sites. RhoE phosphorylation enhances its stability, leading to an increase in RhoE levels. In addition, phosphorylation reduces its association with membranes and correlates with its ability to induce loss of stress fibres. RhoE also acts independently of ROCK to inhibit cell cycle progression, in part by preventing translation of cyclin D1, and to inhibit transformation of fibroblasts by oncogenic H-Ras. RhoE is therefore a multifunctional protein whose localization and actions are regulated by phosphorylation.
Asunto(s)
Proteínas Activadoras de GTPasa/fisiología , Animales , Ciclo Celular/fisiología , Citoesqueleto/fisiología , Citoesqueleto/ultraestructura , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/genética , Regulación Enzimológica de la Expresión Génica , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , FosforilaciónRESUMEN
The Sec1-related proteins bind to syntaxin family t-SNAREs with high affinity, thus controlling the interaction of syntaxins with their cognate SNARE partners. Munc18-2 is a Sec1 homologue enriched in epithelial cells and forms a complex with syntaxin 3, a t-SNARE localized to the apical plasma membrane. We generated here a set of Munc18-2 point mutants with substitutions in conserved amino acid residues. The mutants displayed a spectrum of different syntaxin binding efficiencies. The in vitro and in vivo binding patterns were highly similar, and the association of the Munc18-2 variants with syntaxin 3 correlated well with their ability to displace SNAP-23 from syntaxin 3 complexes when overexpressed in Caco-2 cells. Even the Munc18-2 mutants that do not detectably bind syntaxin 3 were membrane associated in Caco-2 cells, suggesting that the syntaxin interaction is not the sole determinant of Sec1 protein membrane attachment. Overexpression of the wild-type Munc18-2 was shown to inhibit the apical delivery of influenza virus hemagglutinin (HA). Interestingly, mutants unable to bind syntaxin 3 behaved differently in the HA transport assay. While one of the mutants tested had no effect, one inhibited and one enhanced the apical transport of HA. This implies that Munc18-2 function in apical membrane trafficking involves aspects independent of the syntaxin 3 interaction.
Asunto(s)
Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Transporte Biológico , Células CACO-2 , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Humanos , Datos de Secuencia Molecular , Proteínas Munc18 , Proteínas/genética , Proteínas Qa-SNARE , Alineación de SecuenciaRESUMEN
The syntaxins are a large protein family implicated in the targeting and fusion of intracellular transport vesicles. A subset of proteins of this family are the four syntaxin 2 splice variants, syntaxins 2A (2), 2B (2'), 2C (2") and 2D. Each syntaxin 2 variant contains an identical, or nearly identical, amino-terminal cytoplasmic domain followed by a distinct hydrophobic (syntaxins 2A and 2B) or hydrophilic (syntaxins 2C and 2D) carboxyl-terminal domain. To investigate whether the difference among the syntaxin 2 variants is functionally important, we have examined comparatively their RNA transcript and protein expression patterns, membrane associations, protein-protein interactions and intracellular localizations. Analysis of the RNA transcript and protein expression patterns demonstrated that syntaxins 2A, 2B and 2C are broadly, but not uniformly, expressed while syntaxin 2D expression is restricted to the brain. Subcellular fractionation studies showed that syntaxins 2A and 2B behave as integral membrane proteins while syntaxin 2C is only partially associated with membranes. In vitro biochemical assays demonstrated that the syntaxin 2 variants exhibit similar yet distinct interactions with other proteins implicated in vesicular trafficking, including SNAP-25, SNAP-23, VAMP-2 and n-sec1. In a variety of nonpolarized cell types, syntaxins 2A and 2B localized to both the plasma membrane and endosomal membranes. However, in two polarized epithelial cell lines, MDCK and Caco-2, syntaxin 2A localized predominantly to the apical plasma membrane while syntaxin 2B was associated with both the apical and the basolateral membranes. These observations indicate that the distinct carboxyl-terminal domains of the syntaxin 2 variants influence their biochemical and localization properties and may therefore confer upon these variants different functional roles in the regulation of intracellular membrane trafficking.
Asunto(s)
Empalme Alternativo , Antígenos de Superficie/genética , Variación Genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/biosíntesis , Antígenos de Superficie/química , Encéfalo/metabolismo , Células COS , Membrana Celular/metabolismo , Clonación Molecular , Regulación de la Expresión Génica , Células HeLa , Humanos , Riñón/metabolismo , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/química , Conformación Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares/metabolismo , Sintaxina 1 , Testículo/metabolismo , Transcripción Genética , TransfecciónRESUMEN
BACKGROUND: Differentiation of epithelial cells involves the assembly of polarized membrane transport machineries necessary for the generation and maintenance of the apical and basolateral membrane domains characteristic of this cell type. We have analyzed the expression patterns of vesicle-docking proteins of the syntaxin family in mouse kidney, focusing on syntaxin 3 and its interaction partner, the Sec1-related Munc-18-2. METHODS: Expression patterns were studied by in situ hybridization and immunocytochemistry and the complex formation of syntaxin 3 and Munc-18-2 by coimmunoprecipitation and Western blotting. RESULTS: We have previously shown by in situ hybridization that Munc-18-2 is present in the proximal tubules and collecting ducts of embryonic day 17 mouse kidney. We compared this with the expression patterns of syntaxin 1A, 2, 3, 4, and 5, and found that syntaxin 3 was enriched in the same epithelial structures in which Munc-18-2 was abundant. By immunocytochemistry, the two proteins colocalized at the apical plasma membrane of proximal tubule and collecting duct epithelial cells, and they were shown to form a physical complex in the kidney. The expression of both proteins was up-regulated during kidney development. The most prominent changes in expression levels coincided with the differentiation of proximal tubules, suggesting a role in the generation of the highly active reabsorption machinery characterizing this segment of the nephron. CONCLUSION: The results show that Munc-18-2 and syntaxin 3 form a complex in vivo and suggest that they participate in epithelial cell differentiation and targeted vesicle transport processes in the developing kidney.
Asunto(s)
Riñón/embriología , Riñón/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Animales , Secuencia de Bases , Diferenciación Celular , Polaridad Celular , Cartilla de ADN/genética , Células Epiteliales/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Inmunohistoquímica , Hibridación in Situ , Riñón/crecimiento & desarrollo , Sustancias Macromoleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos CBA , Proteínas Munc18 , Embarazo , Proteínas/química , Proteínas/genética , Proteínas Qa-SNARE , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sintaxina 1 , Regulación hacia ArribaRESUMEN
The docking/fusion of transport vesicles mediated by the soluble NSF attachment protein receptors (SNAREs) is thought to be regulated by Sec1-related proteins. Munc-18-2, a member of this family, is predominantly expressed in the epithelial cells of several tissues. We demonstrate here that Munc-18-2 colocalizes with syntaxin 3 at the apical plasma membrane of intestinal epithelium and Caco-2 cells. The presence of a physical complex of the two proteins is verified by 2-way coimmunoprecipitation. The quantity of the complex is reduced by treatment of Caco-2 cells with the alkylating agent N-ethylmaleimide which also has an inhibitory effect on the ability of Munc-18-2 to associate with syntaxin 3 in vitro. The amount of Munc-18-2 in the complex increases upon treatment of the cells with the protein kinase C activator phorbol myristate acetate, indicating a functional connection between the complex and cell signalling. Increasing the amount of Munc-18-2 bound to syntaxin 3 by overexpression results in a marked decrease in the SNARE proteins SNAP-23 and cellubrevin bound to the syntaxin. These results define a novel functional complex of Munc-18-2 and syntaxin 3 involved in the regulation of apical membrane transport.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Células CACO-2 , Proteínas Portadoras/análisis , Proteínas Portadoras/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Perros , Expresión Génica , Humanos , Mucosa Intestinal/citología , Proteínas de la Membrana/análisis , Proteínas Munc18 , Proteínas del Tejido Nervioso , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas/análisis , Proteínas/genética , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas SNARE , Transducción de Señal/fisiología , Transfección , Proteína 3 de Membrana Asociada a VesículasRESUMEN
We report the sequence of a human cDNA encoding a deduced 362 amino acid protein with six EF-hand calcium-binding motifs. The protein is a likely human counterpart of the Cab45 protein recently identified in the 3T3-L1 mouse adipocyte cell line [Scherer et al. (1996), J. Cell Biol. 133, 257-268], displaying 87% aa and 83% nt identity with this sequence. The mRNA for human Cab45 is detected ubiquitously in tissue Northern blots.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Humanos , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
Sec1-related proteins are involved in docking and fusion of transport vesicles in eukaryotic cells. Here we report the cloning and molecular characterization of a Sec1-related protein expressed in the MDCK epithelial cell line. This protein represents a canine counterpart of the murine Munc-18-2/Munc-18b/muSec1 protein, displays 93% amino acid identity with these proteins, has a similar tissue mRNA expression pattern, and associates in vitro with syntaxins 1A, 2, and 3. In situ hybridization analysis of embryonic mouse tissues revealed prominent expression of the munc-18-2 mRNA in the epithelia of several tissues. Cell-fractionation studies demonstrated that the majority of Munc-18-2 is membrane associated. Most of the protein is washed off the membranes by sodium carbonate, pH 11.5. However, the protein is poorly solubilized by detergent treatment. The Munc-18-2 protein was localized, by immunofluorescence microscopy, to the plasma membrane of MDCK cells, and is apically distributed in the epithelial cells of mouse tissues. When overexpressed in COS-1 cells, the protein appeared to be largely cytosolic. However, upon expression with syntaxin 1A, it displayed a shift to the plasma membrane, where the two proteins colocalized. These results identified Munc-18-2 as a predominantly epithelial vesicle-transport protein with a polarized distribution and provided novel in vivo evidence for the association of Sec1-related proteins with members of the syntaxin family.