RESUMO
Four amino acids critical for lactose permease function were altered using site-directed mutagenesis. The resulting Quad mutant (E269Q/R302L/H322Q/E325Q) was expressed at 60% of wild-type levels but found to have negligible transport activity. The Quad mutant was used as a parental strain to isolate suppressors that regained the ability to ferment the alpha-galactoside melibiose. Six different suppressors were identified involving five discrete amino acid changes and one amino acid deletion (Q60L, V229G, Y236D, S306L, K319N and DeltaI298). All of the suppressors transported alpha-galactosides at substantial rates. In addition, the Q60L, DeltaI298 and K319N suppressors regained a small but detectable amount of lactose transport. Assays of sugar-driven cation transport showed that both the Q60L and K319N suppressors couple the influx of melibiose with cations (H(+) or H(3)O(+)). Taken together, the data show that the cation-binding domain in the lactose permease is not a fixed structure as proposed in previous models. Rather, the data are consistent with a model in which several ionizable residues form a dynamic coupling sensor that also may interact directly with the cation and lactose.
Assuntos
Cátions/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Substituição de Aminoácidos , Transporte Biológico , Western Blotting , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Lactose/metabolismo , Melibiose/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Mutagênese Sítio-Dirigida , Plasmídeos/genética , Tiogalactosídeos/metabolismoRESUMO
The orphan nuclear receptor TR2 interacts directly with histone deacetylase HDAC3 and HDAC4. We now report that two domains of HDAC3 are involved in its interaction with TR2. GST pull-down assays show that both the N-terminal (residues 1-135) and the C-terminal (residues 210-428) segments of HDAC3 directly interact with TR2. The interaction is also demonstrated in coimmunoprecipitation experiments. The two TR2-binding sites of HDAC3 compete with each other for binding to TR2. The two receptor-interacting domains (RIDs) of HDAC3 were further dissected and mapped to amino acid residues 1-70 and 270-320. In vivo studies demonstrate that HDAC3 and TR2 can form a complex on the TR2 DNA target and this complex exhibits histone deacetylase activity. These data identify two RIDs of HDAC3 and the biological activity of the complex formed by TR2 and HDAC3 on the TR2 DNA target.
Assuntos
Histona Desacetilases/química , Histona Desacetilases/metabolismo , Receptores dos Hormônios Tireóideos/metabolismo , Animais , Sítios de Ligação , Ligação Competitiva , Células COS , DNA/metabolismo , Histona Desacetilases/fisiologia , Membro 1 do Grupo C da Subfamília 2 de Receptores Nucleares , Testes de Precipitina , Regiões Promotoras Genéticas , Estrutura Terciária de ProteínaRESUMO
A direct interaction between the nuclear receptor TR2 and histone deacetylases (HDACs) 3 and 4 is mediated by the DNA binding domain (DBD) of TR2. To test if this interaction is common to members of the nuclear receptor family, the Cys2-Cys2 type zinc finger (ZF) DBDs were subcloned from several nuclear receptors (mRARalpha, mRXRbeta, mTR2, mTR4, RAR, mPPARdelta, and mPPARgamma2). Using GST pull-downs, both HDACs 3 and 4 were found to interact directly with the core DBD from each receptor. The three-dimensional structure of the ZF domains was essential for this interaction as disruption by zinc chelation precluded interaction with HDACs. The results suggest that the ZFs of nuclear receptors provide a general interaction interface for HDACs 3 and 4. Functional significance of this interaction was demonstrated using ChIP assays where a truncated TR2 protein (lacking the LBD) recruited HDACs 3 and 4 to the target DNA causing demonstrable histone deacetylation. GST pull-downs and mammalian two-hybrid interaction tests were then used to define the interaction domains of HDAC3 with TR2. Both the N- and C-terminal portions of HDAC3 showed interaction with the TR2 DBD. Thus, multiple domains of HDAC3 form the interaction surface for the DBD of nuclear receptors.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Histona Desacetilases/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , Humanos , Camundongos , Membro 1 do Grupo C da Subfamília 2 de Receptores Nucleares , Ligação Proteica , Mapeamento de Interação de Proteínas , Receptores dos Hormônios Tireóideos/metabolismo , Proteínas Repressoras/metabolismo , Dedos de ZincoRESUMO
Receptor interacting protein 140 (RIP140) interacts with retinoic acid receptor (RAR) and retinoid X receptor (RXR) constitutively, but hormone binding enhances this interaction. The ligand-independent interaction is mediated by the amino and central regions of RIP140 which contain a total of nine copies of the LXXLL motif, whereas the agonist-induced interaction is mediated by its carboxyl terminus which contains a novel motif (1063-1076, LTKTNPILYYMLQK). The ligand-independent interaction could be enhanced slightly by agonists, whereas the ligand-dependent interaction was strictly agonist dependent for both RAR and RXR. In the context of heterodimers, ligand occupancy of RXR played a more dominant role for both molecular interaction and biological activity of RIP140. Competition and mutation studies demonstrated an essential role for (1067)Asn and (1073)Met for a ligand-dependent interaction. A model was proposed to address the constitutive and agonist-dependent interaction of RIP140 with RAR/RXR.
