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.
School of Biosciences, Sutton Bonington Campus, University of Nottingham, United Kingdom.
Ranjan
R
Swarup
Joanna
J
Kargul
Alan
A
Marchant
Daniel
D
Zadik
Abidur
A
Rahman
Rebecca
R
Mills
Anthony
A
Yemm
Sean
S
May
Lorraine
L
Williams
Paul
P
Millner
Seiji
S
Tsurumi
Ian
I
Moore
Richard
R
Napier
Ian D
ID
Kerr
Malcolm J
MJ
Bennett
D000483:Alleles; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018522:Gravitropism / Q000502:physiology; D007210:Indoleacetic Acids / Q000378:metabolism*; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000502:physiology; D030821:Plants, Genetically Modified; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship
C100794:AUX1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D007210:Indoleacetic Acids