Second, the length of Deh4p, 552 residues, is within Selleckchem INCB018424 the known range of 400 to 600 for MFS [24] and third, it was predicted to have twelve TMS, typical for MFS, by many topology prediction programs such as OCTOPUS [20], TMpro [35], SOSUI [14] and PHDHTM [18]. The monochloroacetate uptake ability of Deh4p was inhibited in the presence of a proton motive force inhibitor, carbonyl cyanide 3-chlorophenyl

hydrazone (Yu, unpublished result). This suggested that Deh4p is most likely a symporter or antiporter. When the topology of Deh4p was predicted using TMHMM [36] and SOSUI [14], the models were different from a typical MFS symmetrical arrangement. Deh4p has a long periplasmic loop, stretching from residues 337 to 454, near the C-terminal. Fig. 1 shows a hydrophobicity plot of Deh4p using ΔGpred algorithm [37]. The prediction showed that there were twelve TMS with the N- and the C-termini located in the cytoplasm. All except TMS 1 and 11 have reliability values of more than 0.75 and the fifth periplasmic loop has a value of 1. These suggested

that the prediction was reasonably good and Deh4p is likely to be a MFS protein. Figure 1 A hydrophobicity plot of Deh4p. A hydrophobicity plot based on the ΔGpred method [37] was produced by the TOPCONS server (topcons.cbr.su.se) [62]. The predicted transmembrane helices are indicated by black (helix from Nin to Cout) and white (helix from Nout to Cin) boxes, respectively. The reliabilities of the helices are also indicated. Topological HSP90 analysis using Deh4p-PhoA-LacZ fusions Although most of the predicted Selleck CAL 101 models of Deh4p exhibited twelve TMS it is necessary to validate these predictions experimentally. The use of find more reporter fusions technique is a commonly used practice. In this study we utilized a dual-reporters system. Bacterial alkaline phosphatase (PhoA) is an enzyme that functions only in the periplasmic space [38] while β-galactosidase (LacZ) is an enzyme that works only in the cytoplasm [39]. The use of these PhoA-LacZ dual-reporters in topology studies gives more reliable results than using just one reporter [33]. Another problem in studying membrane protein is to achieve adequate expression. Some

fusion recombinants do not express [40] while others can be toxic [41]. We have used a ribosomal promoter from Burkholderia sp. MBA4 for successful production of functional membrane protein in E. coli. This S12 promoter is a weak and constitutive promoter in E. coli and has been shown to be ideal for expression of potentially toxic membrane protein [11]. Recombinant proteins made up of Deh4p and truncated derivatives fused with PhoA and LacZα were constructed. The use of LacZα decreased the sizes of the fusion proteins. With an appropriate host that allows α-complementation [42] the LacZα will work normally. DNA fragments containing full-length and truncated deh4p of different lengths were amplified and cloned in-frame with the phoA-lacZα dual reporter genes.