RESUMEN
Multi-PDZ (PSD-95/Discs large/Zonula-occludens-1) domain proteins play a crucial role in the establishment and maintenance of cell polarization. The novel multi-PDZ domain protein FRMPD2 is a potential scaffolding protein consisting of an N-terminal KIND domain, a FERM domain and three PDZ domains. Here we show that FRMPD2 is localized in a polarized fashion in epithelial cells at the basolateral membrane and partially colocalizes with the tight-junction marker protein Zonula-occludens-1. Downregulation of FRMPD2 protein in Caco-2 cells is associated with an impairment of tight junction formation. We find that the FERM domain of FRMPD2 binds phosphatidylinositols and is sufficient for membrane localization. Moreover, we demonstrate that recruitment of FRMPD2 to cell-cell junctions is strictly E-cadherin-dependent, which is in line with our identification of catenin family proteins as binding partners for FRMPD2. We demonstrate that the FERM domain and binding of the PDZ2 domain to the armadillo protein p0071 are required for basolateral restriction of FRMPD2. Moreover, the PDZ2 domain of FRMPD2 is sufficient to partially redirect an apically localized protein to the basolateral membrane. Our results provide novel insights into the molecular function of FRMPD2 and into the targeting mechanism of peripheral membrane proteins in polarized epithelial cells.
Asunto(s)
Polaridad Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas de la Membrana/metabolismo , Dominios PDZ , Secuencia de Aminoácidos , Animales , Proteínas del Dominio Armadillo/metabolismo , Biomarcadores/metabolismo , Cadherinas/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Perros , Técnicas de Silenciamiento del Gen , Humanos , Proteínas de la Membrana/química , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Unión Proteica , Transporte de Proteínas , Proteínas de Uniones Estrechas , Uniones Estrechas/metabolismo , beta Catenina/metabolismoRESUMEN
In the course of macronuclear differentiation in spirotrichous ciliates massive DNA reorganization processes take place, which include splicing, cutting, rearranging and eliminating specific DNA sequences. In order to identify genes involved in these processes we took advantage of suppression subtractive hybridization. We have identified three transcripts that are exclusively expressed during macronuclear development in the ciliate Stylonychia lemnae. Two of the three differentially expressed mRNAs we have analyzed encode for novel proteins. One gene, mdp1 [macronuclear development protein 1 (MDP1)], encodes a homolog of the PIWI protein family. PIWI proteins are involved in germline differentiation processes and RNA silencing in worms, flies, mice, humans and in plants. Possible functions of the S.lemnae PIWI related protein MDP1 in the regulation of macronuclear development will be discussed.
Asunto(s)
Núcleo Celular/genética , Cilióforos/genética , Proteínas Protozoarias/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Núcleo Celular/ultraestructura , Cilióforos/metabolismo , Cilióforos/ultraestructura , Genes Protozoarios , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Protozoarias/biosíntesis , Proteínas Protozoarias/química , ARN Protozoario/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
Protein tyrosine phosphatase-basophil like (PTP-BL) represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the backbone assignments of the PDZ2/PDZ3 tandem domain of PTP-BL. These assignments now provide a basis for the detailed structural investigation of the interaction between the PDZ domains 2 and 3 of PTP-BL. It will lead to a better understanding of the proposed scaffolding function of this tandem domain in multi-protein complexes assembled by PTB-BL.
Asunto(s)
Espectroscopía de Resonancia Magnética/métodos , Proteína Tirosina Fosfatasa no Receptora Tipo 13/química , Secuencia de Aminoácidos , Isótopos de Carbono/química , Peso Molecular , Isótopos de Nitrógeno/química , Estructura Terciaria de Proteína , ProtonesRESUMEN
pEPI-1, a vector in which a chromosomal scaffold/matrix-attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T-antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI-1 by crosslinking with cis-diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south-western analysis, the vector shows exclusive binding to hnRNP-U/SAF-A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA-protein complex demonstrates that pEPI-1 is bound to this protein in vivo. These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.