Functional Analysis of the Cation Diffusion Facilitator ZitB from Escherichia coli

Persistent Link:
http://hdl.handle.net/10150/195975
Title:
Functional Analysis of the Cation Diffusion Facilitator ZitB from Escherichia coli
Author:
Haney, Christopher J
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
This research sought to elucidate the molecular mechanism by which a recently described family of ion transport proteins, the cation diffusion facilitators (CDFs), transfer ions across biomembranes. Using the Escherichia coli homologs ZitB, and FieF, as well as CzcD from Cupriavidus metallidurans CH34 as models, the amino acids essential to CDF function were identified, and the transport behavior of ZitB and its homolog FieF, were described.Site-directed ZitB mutants were used to determine the necessity of individual amino acids. The mutation of several well-conserved acidic residues resulted in the loss of a ZitB-mediated zinc-resistant phenotype in the zinc-sensitive E. coli strain GG48. ZitB also complemented the potassium uptake deficient strain TK2420, suggesting that ZitB works as an antiporter, possibly allowing potassium into the cell while effluxing zinc.This result was further investigated in experiments using everted membrane vesicles(EMVs). Vesicles bearing ZitB accumulated 65Zn(II) in a NADH-dependent manner, with an apparent KM of 1 micromolar. This accumulation was inhibitable by the protonophore FCCP, suggesting CDF dependence upon the proton motive force. Similar results were obtained using both EMVs and proteoliposomes containing the CDF homologs CzcD, from C. metallidurans CH34, and FieF from E. coli. Despite facilitating 65Zn(II) uptakeinto EMVs, fieF transcription was zinc and iron-dependent, but independent of the ironuptake regulator Fur. FieF expression in trans complemented the iron-sensitive phenotypeof a strain lacking fur, causing it to accumulate less 55Fe than wild type. Reconstituted proteoliposomes containing FieF also accumulated less 55Fe than those without.This research confirms that CDF proteins likely depend on the cell's proton gradient, effluxing substrate in a metal:proton antiport arrangement. Substrate acquisition and throughput is facilitated by a set of acidic amino acids and histidines. The relatively lowapparent KM of ZitB suggests a homeostatic role for the protein; however, the iron-inducibilityof fieF hints at a role in iron detoxification, so the cellular functions of the CDF family may be quite diverse, even within the bacteria.
Type:
text; Electronic Dissertation
Keywords:
ZitB; CDF; Cation Diffusion Facilitator; Zinc
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Soil, Water & Environmental Science; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Rensing, Christopher

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleFunctional Analysis of the Cation Diffusion Facilitator ZitB from Escherichia colien_US
dc.creatorHaney, Christopher Jen_US
dc.contributor.authorHaney, Christopher Jen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThis research sought to elucidate the molecular mechanism by which a recently described family of ion transport proteins, the cation diffusion facilitators (CDFs), transfer ions across biomembranes. Using the Escherichia coli homologs ZitB, and FieF, as well as CzcD from Cupriavidus metallidurans CH34 as models, the amino acids essential to CDF function were identified, and the transport behavior of ZitB and its homolog FieF, were described.Site-directed ZitB mutants were used to determine the necessity of individual amino acids. The mutation of several well-conserved acidic residues resulted in the loss of a ZitB-mediated zinc-resistant phenotype in the zinc-sensitive E. coli strain GG48. ZitB also complemented the potassium uptake deficient strain TK2420, suggesting that ZitB works as an antiporter, possibly allowing potassium into the cell while effluxing zinc.This result was further investigated in experiments using everted membrane vesicles(EMVs). Vesicles bearing ZitB accumulated 65Zn(II) in a NADH-dependent manner, with an apparent KM of 1 micromolar. This accumulation was inhibitable by the protonophore FCCP, suggesting CDF dependence upon the proton motive force. Similar results were obtained using both EMVs and proteoliposomes containing the CDF homologs CzcD, from C. metallidurans CH34, and FieF from E. coli. Despite facilitating 65Zn(II) uptakeinto EMVs, fieF transcription was zinc and iron-dependent, but independent of the ironuptake regulator Fur. FieF expression in trans complemented the iron-sensitive phenotypeof a strain lacking fur, causing it to accumulate less 55Fe than wild type. Reconstituted proteoliposomes containing FieF also accumulated less 55Fe than those without.This research confirms that CDF proteins likely depend on the cell's proton gradient, effluxing substrate in a metal:proton antiport arrangement. Substrate acquisition and throughput is facilitated by a set of acidic amino acids and histidines. The relatively lowapparent KM of ZitB suggests a homeostatic role for the protein; however, the iron-inducibilityof fieF hints at a role in iron detoxification, so the cellular functions of the CDF family may be quite diverse, even within the bacteria.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectZitBen_US
dc.subjectCDFen_US
dc.subjectCation Diffusion Facilitatoren_US
dc.subjectZincen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineSoil, Water & Environmental Scienceen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairRensing, Christopheren_US
dc.contributor.committeememberRensing, Christopheren_US
dc.contributor.committeememberMaier, Rainaen_US
dc.contributor.committeememberPierson, Lelanden_US
dc.identifier.proquest2271en_US
dc.identifier.oclc659748115en_US
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