Genetic study on the high-affinity transport system for ribose

Abstract

The ribose high-affinity transporter of Escherichia coli is one of the binding protein dependent ABC transporters. It is composed of the periplasmic ribose-binding protein (RBP), the hydrophobic membrane protein (RbsC), and ATP-binding protein (RbsA). To investigate interaction between RBP and permease, regions on the tertiary structure of RBP that interact with the membrane permease were genetically probed by screening a mutation using the chimeric receptor Trz. The mutational changes were repeatedly found in several residues of RBP, concentrating on three surface regions and comprising two domains of the tertiary structure. The two regions, including residues 52 and 166, are specifically involved in the permease interaction while the third region including residues 72, 134, and some others recognizes both the permease and the chemosensory receptor. To understand the structure and function of RbsC, topology of RbsC was investigated by an alkaline phosphatase fusion. Characterization of 64 RbsC-PhoA fusions, isolated either specifically or randomly, revealed that the RbsC protein is composed of six transmembrane helices with three periplasmic loops. In order to confirm the cytoplasmic location of the short C-terminal region (5 a.a.), inside-out or right side-out membrane vesicles were generated in which only in inside-out vesicles the C-terminal region was found to be digested by carboxypeptidase A. This result is consistent with the model, based on the results of alkaline phosphatase fusions, that the protein traverses membrane six times and the N- and C-termini being exposed to the cytoplasm. For the RBP mutations previously characterized (Eym et al., 1996), permease suppressors were isolated in RbsC after a series of enrichments. Based on RbsC topology which contains six transmembrane regions (Park and Park, submitted), suppressors of RbsC for mutations of N- and C-domains of RBP were localized into the last transmembrane region and the cytoplasmic loop I ad...