Project Number: 1907-21000-024-08
Project Type: Reimbursable

Start Date: Sep 01, 2007
End Date: Aug 31, 2010

Objective:
This proposal builds upon our group¿s recent progress working with the only Al tolerance gene cloned to date, ALMT1, which was isolated from wheat roots by our collaborators in Dr. Matsumoto¿s lab at Okayama University. ALMT1 (for aluminum-activated malate transporter) encodes a novel membrane transporter that mediates Al-activated root malate efflux underlying wheat Al tolerance. We have cloned and characterized ALMT1 and homologs from Arabidopsis and maize and have shown that although highly similar, their functional properties (Al activation and malate selective transport) critical for conferring a role in Al tolerance, differ significantly. The overall goal of this research is to initiate a structure-function analysis of ALMT-type transporters to identify amino acid domains and associated structural motifs underlying the transporters¿ functional properties. The specific objectives are: 1) To conduct a general functional (biophysical) characterization of ALMT-type transporters. 2) The second objective will involve an interdisciplinary approach using molecular biology, protein biochemistry and computational analysis of protein sequence and structure in combination with functional analysis via our of the heterologous expression systems.

Approach:
To conduct a general functional (biophysical) characterization of ALMT-type transporters. This will involve expression of ALMT cRNAs in Xenopus oocytes followed by 2 microelectrode current-voltage analysis to elucidate the transport properties of different ALMT transporters. The specific transport properties to be studied include: A. A comparative characterization of ALMT anion permeability to determine what solutes the members of the ALMT family from maize, wheat and Arabidopsis transport, including organic acid and mineral anions. B. A pharmacological profiling of ALMT-type transporters using anion channel inhibitors to help elucidate the functional (e.g. pore structure and ion interactions) and structural (e.g. selectivity filter) features of the ion permeation pathway. C. Investigation into the regulation of these transporters including specificity if Al activation, identification of cytoplasmic factors that alter function (e.g., phosphorylation, different cytoplasmic ions, etc). For the second objective looking at the relationship between ALMT protein structure and function, the investigation of functional domains involved in ion selectivity, gating, Al-binding, and channel modulation will be conducted. This will involve the construction of ALMT chimeras to facilitate domain swapping as well as sequential deletions and site-directed mutagenesis followed by heterologous expression in oocytes for functional electrophysiological analysis of ALMT transport properties.