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

TitleStructural determination of the haloacid permease Deh4p of Burkholderia sp. MBA4 with a dual-reporter system
Authors
Issue Date2008
PublisherWiley-Blackwell Publishing Ltd.. The Journal's web site is located at http://www.febsjournal.org/
Citation
The FEBS Journal, 2008, v. 275 n. S1, p. 178 How to Cite?
AbstractIntroduction: 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.
Persistent Identifierhttp://hdl.handle.net/10722/114833
ISSN
2015 Impact Factor: 4.237
2015 SCImago Journal Rankings: 2.141

 

DC FieldValueLanguage
dc.contributor.authorTsang, JSH-
dc.contributor.authorTse, YM-
dc.contributor.authorYu, M-
dc.date.accessioned2010-09-26T05:17:59Z-
dc.date.available2010-09-26T05:17:59Z-
dc.date.issued2008-
dc.identifier.citationThe FEBS Journal, 2008, v. 275 n. S1, p. 178-
dc.identifier.issn1742-464X-
dc.identifier.urihttp://hdl.handle.net/10722/114833-
dc.description.abstractIntroduction: 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.-
dc.languageeng-
dc.publisherWiley-Blackwell Publishing Ltd.. The Journal's web site is located at http://www.febsjournal.org/-
dc.relation.ispartofThe FEBS Journal-
dc.rightsPreprint This is the pre-peer reviewed version of the following article: [FULL CITE], which has been published in final form at [Link to final article]. Authors are not required to remove preprints posted prior to acceptance of the submitted version. Postprint This is the accepted version of the following article: [full citation], which has been published in final form at [Link to final article].-
dc.titleStructural determination of the haloacid permease Deh4p of Burkholderia sp. MBA4 with a dual-reporter system-
dc.typeConference_Paper-
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=1742-464X&volume=275 supplement 1&spage=178&epage=&date=2008&atitle=Structural+determination+of+the+haloacid+permease+Deh4p+of+Burkholderia+sp.+MBA4+with+a+dual-reporter+system.en_HK
dc.identifier.emailTsang, JSH: jshtsang@hkucc.hku.hk-
dc.identifier.emailYu, M: yumanda@graduate.hku.hk-
dc.identifier.authorityTsang, JSH=rp00792-
dc.identifier.doi10.1111/j.1742-4658.2008.06448.x-
dc.identifier.hkuros168047-
dc.identifier.volume275-
dc.identifier.issueS1-
dc.identifier.spage178-
dc.identifier.epage178-
dc.publisher.placeUnited Kingdom-

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