Structural determination of the haloacid permease Deh4p of Burkholderia sp. MBA4 with a dual-reporter system

Abstract

Introduction: Burkholderia sp. MBA4 produces a haloacid permease, Deh4p, which mediated the uptake of haloacid as a growth substrate. Deh4p contains 552 residues with a mass of 59.4 kDa. Structural prediction of Deh4p suggested the presence of 11 or 12 transmembrane helices. Most of these predictions suggested that the N terminus is located in the cytoplasm. In this study we reported the determination of the topology of Deh4p by using a PhoA-LacZ dual-reporter system. Methods: PhoA is an alkaline phosphatase which only works in the periplasm while beta-galactosidase (LacZ) is an enzyme that works in the cytoplasm. DNA fragments encoding various lengths of deh4p were amplified by PCR and fused in-frame with a phoA-lacZ cassette. These recombinant plasmids were transformed and the fusion proteins expressed in E. coli using a weak constitutive promoter. Enzyme activities of PhoA and LacZ were determined to localize the whereabouts of the reporter and thus the authenticity of the transmembrane domains. Results: The expression of the membrane proteins did not affect the growth of the cells. More than 36 constructs were made and transformed into E. coli. The PhoA/LacZ enzyme activity ratios for these clones have been determined using PNPP and ONPG. The results showed that the first two and the last two predicted transmembrane domains were absent. Conclusions: The current results suggested that there are at most eight transmembrane domains found in Deh4p. The N- and the Cterminals were located in the cytoplasm and a large loop was exposed in the periplasm.