ABSTRACT
Cadherins are calcium-dependent, cell surface glycoproteins involved in cell-cell adhesion. To function in cell-cell adhesion, the transmembrane cadherin molecule must be associated with the cytoskeleton via cytoplasmic proteins known as catenins. Three catenins, alpha-catenin, beta-catenin and gamma-catenin (also known as plakoglobin), have been identified. beta-catenin or plakoglobin is associated directly with the cadherin; alpha-catenin binds to beta-catenin/plakoglobin and serves to link the cadherin/catenin complex to the actin cytoskeleton. The domains on the cadherin and betacatenin/plakoglobin that are responsible for protein-protein interactions have been mapped. However, little is known about the molecular interactions between alpha-catenin and beta-catenin/plakoglobin or about the interactions between alpha-catenin and the cytoskeleton. In this study we have used the yeast two-hybrid system to map the domains on alpha-catenin that allow it to associate with beta-catenin/plakoglobin and with alpha-actinin. We also identify a region on alpha-actinin that is responsible for its interaction with alpha-catenin. The yeast two-hybrid data were confirmed with biochemical studies.
Subject(s)
Actinin/metabolism , Cytoskeletal Proteins/metabolism , Actinin/chemistry , Animals , Binding Sites , Cell Line , Cytoskeletal Proteins/chemistry , Desmoplakins , Humans , Peptide Mapping , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Transfection , alpha Catenin , gamma CateninABSTRACT
The sigmaB subunit of Bacillus subtilis RNA polymerase governs the expression of a large general stress regulon. The results of pulse-chase and immunoprecipitation experiments showed that sigmaB is stable both in the presence and in the absence of the RsbW anti-sigma factor, the principal regulator of sigmaB in response to environmental signals.
Subject(s)
Bacillus subtilis/genetics , Gene Expression Regulation, Bacterial , Sigma Factor/metabolism , Transcription Factors/metabolism , Bacterial Proteins/metabolism , Carrier Proteins/metabolism , Hot Temperature , Macromolecular Substances , Protein BindingABSTRACT
The alternative transcription factor sigma B of Bacillus subtilis is activated during the stationary growth phase by a regulatory network responsive to stationary-phase signals. On the basis of the results reported here, we propose that sigma B controls a general stress regulon that is induced when cells encounter a variety of growth-limiting conditions. Expression of genes controlled by sigma B, including the ctc gene and the sigB operon that codes for sigma B and its associated regulatory proteins, was dramatically induced in both the exponential and stationary phases by environmental challenges known to elicit a general stress response. After cells were subjected to salt stress, the increased expression of lacZ transcriptional fusions to the ctc and sigB genes was entirely dependent on sigma B, and primer extension experiments confirmed that the sigma B-dependent transcriptional start site was used during salt induction of sigB operon expression. Western blotting (immunoblotting) experiments measuring the levels of sigma B protein indicated that ethanol addition and heat stress also induced sigma B activity during logarithmic growth. Salt and ethanol induction during logarithmic growth required RsbV, the positive regulator of sigma B activity that is normally necessary for activity in stationary-phase cells. However, heat induction of sigma B activity was largely independent of RsbV, indicating that there are two distinct pathways by which these environmental signals are conveyed to the transcriptional apparatus.
Subject(s)
Bacillus subtilis/metabolism , Sigma Factor/metabolism , Amino Acid Sequence , Bacillus subtilis/drug effects , Bacillus subtilis/growth & development , Base Sequence , DNA Primers , Ethanol/pharmacology , Genes, Bacterial , Genotype , Hydrogen Peroxide/pharmacology , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Operon , Promoter Regions, Genetic , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/metabolism , Sigma Factor/biosynthesis , Sigma Factor/genetics , Sodium Chloride/pharmacology , Time Factors , beta-Galactosidase/biosynthesis , beta-Galactosidase/metabolismABSTRACT
Transcription factor sigma B of Bacillus subtilis is active during the stationary growth phase, but its physiological role remains unknown. Understanding the function and regulation of genes controlled by sigma B (csb genes) should provide important clues to sigma B function in stationary-phase cells. To this end, we used a genetic approach to identify six new csb genes. This strategy relies on two elements: (i) random transcriptional fusions between the Escherichia coli lacZ gene and genes on the B. subtilis chromosome, generated in vivo with transposon Tn917lacZ, and (ii) a plate transformation technique to introduce a null sigB mutation into the fusion-bearing recipients directly on indicator plates. This strategy allowed the comparison of fusion expression in strains that were isogenic save for the presence or absence of a functional sigma B protein. Beginning with 1,400 active fusions, we identified 11 that were wholly or partly controlled by sigma B. These fusions mapped to six different loci that exhibit substantial contrasts in their patterns of expression in the logarithmic and stationary growth phases, suggesting that they participate in diverse cellular functions. However, for all six loci, the sigma B-dependent component of their expression was manifest largely in the stationary phase. The high frequency of six independent csb loci detected in a random collection of 1,400 fusions screened, the fact that four of the six new loci were defined by a single fusion, and the absence of the previously identified ctc and csbA genes in the present collection strongly suggest that sigma B controls a large stationary-phase regulon.