Signal Transduction During Active Transport

Columbia University, New York NY

Investigators

Abstract

The goal of this project is to understand the mechanism of signaling during active transport. The hypothesis to be evaluated is that information about rates of solute transport into cells is used by regulatory proteins to control gene expression. The rate of substrate transport into a cell may be a better reflection of its availability in the environment than its intracellular concentration. This may be especially true for compounds that are rapidly metabolized. The maltose transport system of E. coli is a well-characterized ATP-binding cassette transporter that has been extensively studied both by genetic and biochemical approaches. Recently it was found that the maltose transporter participates in the regulation of mal gene expression by repressing transcription. This project will investigate the molecular basis for the ability of the transporter to participate in gene regulation using three approaches. Initially the sites on the MalT protein that are required for interaction with the maltose transporter will be identified. A physical interaction between the MalK subunit of the transporter and the MalT protein has been demonstrated. In order to find the region(s) within the MalT protein that are required for interaction with the transporter, malT mutations that interfere with the ability of the MalT protein to interact with the transporter will be isolated. The sites of the mutations will be determined by DNA sequence analysis. Several phenotypes of the malT mutations will be assessed. Second, it will be determined if repression interferes with the ability of the MalT activator to bind to its recognition sites on DNA. The maltose transporter represses mal gene expression by interfering with the function of MalT, the activator of the mal regulon. This could occur by affecting the ability of MalT to bind to MalT sites found upstream from mal promoters. Alternatively, the transporter may affect the ability of MalT to activate transcription without affecting its binding to specific sites on DNA. DNA footprinting of MalT to sites upstream from mal promoters will be measured to assess the ability of MalT to bind to promoter sites in the presence and absence of the purified transporter. Finally, the hypothesis that the regulatory activity of the maltose transporter is coupled to transport activity will be directly tested. The ability of the transporter to repress mal gene expression is inversely correlated with ATP hydrolysis. A mutant transporter that binds but is unable to hydrolyze ATP is a stronger repressor than the wild-type transporter. In addition, mutant transporters with very high levels of ATPase activity are less able to repress and result in high constitutive levels of mal gene expression. The hypothesis that repression is relieved when the maltose transporter is activated and pumping sugars into the cell will be evaluated. Mutant forms of the transporter that are activated by periplasmic maltose binding protein (MBP) without ligand and are able to transport related sugars that are not normally inducers of the mal regulon will be employed. This hypothesis would be supported if, in these strains, activation by MBP or transport of the related sugars results in relief of transporter-mediated repression and constitutive mal gene expression.