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Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1

Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1
Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1
We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.
1040-4651
3069-3083
Swarup, Ranjan
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Kargul, Joanna
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Marchant, Alan
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Zadik, Daniel
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Rahman, Abidur
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Mills, Rebecca
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Yemm, Anthony
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May, Sean
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Williams, Lorraine
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Millner, Paul
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Tsurumi, Seiji
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Moore, Ian
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Napier, Richard
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Kerr, Ian D.
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Bennett, Malcolm J.
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Swarup, Ranjan
01a7c8f9-9165-4aa8-8d7f-f84879894f97
Kargul, Joanna
68fea4ac-1699-4b59-8d25-9462048713a1
Marchant, Alan
f39b76e6-4e95-4d78-9923-59e7ceb60bfd
Zadik, Daniel
ff36375c-a3ee-4972-8fb8-7855c7a09217
Rahman, Abidur
c63b9f91-bf29-49ed-b7e0-4355652a07e2
Mills, Rebecca
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Yemm, Anthony
d30cc281-1506-4ff3-b74e-44f8eaab1bef
May, Sean
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Williams, Lorraine
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Millner, Paul
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Tsurumi, Seiji
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Moore, Ian
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Napier, Richard
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Kerr, Ian D.
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Bennett, Malcolm J.
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Swarup, Ranjan, Kargul, Joanna, Marchant, Alan, Zadik, Daniel, Rahman, Abidur, Mills, Rebecca, Yemm, Anthony, May, Sean, Williams, Lorraine, Millner, Paul, Tsurumi, Seiji, Moore, Ian, Napier, Richard, Kerr, Ian D. and Bennett, Malcolm J. (2004) Structure-Function Analysis of the Presumptive Arabidopsis Auxin Permease AUX1. The Plant Cell, 16 (11), 3069-3083. (doi:10.1105/tpc.104.024737).

Record type: Article

Abstract

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

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Submitted date: 4 June 2004
Published date: 14 October 2004

Identifiers

Local EPrints ID: 35546
URI: http://eprints.soton.ac.uk/id/eprint/35546
ISSN: 1040-4651
PURE UUID: acf0a0f2-f979-4c26-8c2d-319cddae121f

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Date deposited: 18 May 2006
Last modified: 07 Jan 2022 22:24

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Contributors

Author: Ranjan Swarup
Author: Joanna Kargul
Author: Alan Marchant
Author: Daniel Zadik
Author: Abidur Rahman
Author: Rebecca Mills
Author: Anthony Yemm
Author: Sean May
Author: Paul Millner
Author: Seiji Tsurumi
Author: Ian Moore
Author: Richard Napier
Author: Ian D. Kerr
Author: Malcolm J. Bennett

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