Characterization of the membrane-associated components of the ribose high-affinity transport operon in Escherichia coli K12

Charlene Fontanilla Barroga, Purdue University

Abstract

The membrane-associated components of the ribose high-affinity transport, postulated to consist of RbsD, RbsA and RbsC, have been characterized using biochemical, genetic and immunological techniques. RbsD was found to associate with the membrane tightly, and it may even span the membrane. RbsC was confirmed to be an integral membrane protein, binding to the membrane very tightly and can be barely extracted using urea and alkali extraction. RbsD was found to associate with the membrane rather tightly, but it could also be released into the periplasm. In a strain which had a fully induced rbs operon, RbsD was detected at very high levels, RbsA was barely detectible while RbsC was not detected at all. The RbsC antibodies were essential in detecting RbsC. As a prelude to the development of an in vitro reconstitution assay, conditions for solubilizing the ribose transport complex in active form were explored. The extreme hydrophobicity and vast difference in solubility of RbsC compared to RbsA and RbsD prevented the application of the usual octyl glucoside solubilization procedure. Solubilization with dodecyl maltoside was optimized to co-solubilize the three proteins. No complex of all three proteins was detected, indicating that RbsA and RbsC alone could form the ribose transport complex. Experiments indicated that RbsD spans the membrane twice, with its amino terminus exposed in the periplasm, while the bulk of its C-terminal half in the cytoplasm, except the last 21 amino acids which are in the membrane. This orientation of RbsD positions it to interact with the ribose binding protein in the periplasm. RbsA is postulated to have two periplasmically exposed loops, which would mean that RbsA can span the membrane. Since RbsA does not have any obvious hydrobphobic stretches, it may form a hydrophilic pore which is shielded from the membrane by the most hydrophobic component, RbsC. No in vivo fusions have been obtained in rbsC. Topographical models for RbsC are proposed. The product encoded by the rbsA gene has been purified to homogeneity. The pure protein can be specifically labeled with the ATP analog, 5$\sp\prime$-FSBA. Furthermore, the product shows ATPase activity strongly supporting its proposed role of energizing the transport process. (Abstract shortened by UMI.)

Degree

Ph.D.

Advisors

Hermodson, Purdue University.

Subject Area

Biochemistry|Molecular biology

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