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Structure/Function Analysis of Amino Acid Permeases in Higher Plants

Structure/Function Analysis of Amino Acid Permeases in Higher Plants
Structure/Function Analysis of Amino Acid Permeases in Higher Plants

Nitrogen is essential in all living organisms for growth and development. Nitrogen is redistributed in the plant mainly in the form of amino acids. An essential part of this redistribution is the ability to transport amino acids across plasma membranes. The proteins responsible for this transport are of significant physiological importance in plants. The aim of the work described in this thesis has been to further characterise the amino acid permeases, RcAAP1 and RcAAP3, from Ricinus communis and to correlate the structural properties of these transporters with their functional characteristics.

RcAAP3 was expressed in Xenopus laevis oocytes. Radiolabelled uptake analysis and electrophysiological methods were used to determine the kinetic parameters of this permease. A transport model was presented in which the permease operates as an amino acid:proton symport for neural amino acids, with a transport ratio of two protons to two amino acid molecules. Binding of amino acid and proton is random and positively co-operative.

Random mutagenesis combined with a suitable selection procedure is a useful technique for isolating mutants with desired functional properties. Three random mutagenesis protocols were investigated in this project with the aim of isolating mutated amino acid permeases. DNA-shuffling (a technique that involves the random fragmentation of a pool of related sequences and then recombination using self-primered PCR) and error-prone PCR (a method in which Mn2+ is added to the PCR or the dNTP ratio is altered) failed to produce DNA in sufficient yield or of sufficient quality for ligation and subsequent transformation into yeast. Chemical mutagenesis, a procedure involving EMS mutagenesis of yeast expressing wild type transporters, was the most successful in terms of isolating potential mutants.

University of Southampton
Beech, Ian David
4e6ce1fd-5ed3-4faf-bec9-dfa90c326420
Beech, Ian David
4e6ce1fd-5ed3-4faf-bec9-dfa90c326420

Beech, Ian David (2001) Structure/Function Analysis of Amino Acid Permeases in Higher Plants. University of Southampton, Doctoral Thesis.

Record type: Thesis (Doctoral)

Abstract

Nitrogen is essential in all living organisms for growth and development. Nitrogen is redistributed in the plant mainly in the form of amino acids. An essential part of this redistribution is the ability to transport amino acids across plasma membranes. The proteins responsible for this transport are of significant physiological importance in plants. The aim of the work described in this thesis has been to further characterise the amino acid permeases, RcAAP1 and RcAAP3, from Ricinus communis and to correlate the structural properties of these transporters with their functional characteristics.

RcAAP3 was expressed in Xenopus laevis oocytes. Radiolabelled uptake analysis and electrophysiological methods were used to determine the kinetic parameters of this permease. A transport model was presented in which the permease operates as an amino acid:proton symport for neural amino acids, with a transport ratio of two protons to two amino acid molecules. Binding of amino acid and proton is random and positively co-operative.

Random mutagenesis combined with a suitable selection procedure is a useful technique for isolating mutants with desired functional properties. Three random mutagenesis protocols were investigated in this project with the aim of isolating mutated amino acid permeases. DNA-shuffling (a technique that involves the random fragmentation of a pool of related sequences and then recombination using self-primered PCR) and error-prone PCR (a method in which Mn2+ is added to the PCR or the dNTP ratio is altered) failed to produce DNA in sufficient yield or of sufficient quality for ligation and subsequent transformation into yeast. Chemical mutagenesis, a procedure involving EMS mutagenesis of yeast expressing wild type transporters, was the most successful in terms of isolating potential mutants.

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Published date: 2001

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Local EPrints ID: 464603
URI: http://eprints.soton.ac.uk/id/eprint/464603
PURE UUID: dbe54548-6df9-4ba7-bc26-959cab1f7139

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Date deposited: 04 Jul 2022 23:50
Last modified: 16 Mar 2024 19:38

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Author: Ian David Beech

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