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The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters.

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters.
 
Creator Thakur, Anil
Bachhawat, Anand K
 
Subject QR Microbiology
 
Description Hgt1p, a high-affinity glutathione transporter from Saccharomyces cerevisiae belongs to the recently described family of OPTs (oligopeptide transporters), the majority of whose members still have unknown substrate specificity. To obtain insights into substrate recognition and translocation, we have subjected all 21 residues of TMD9 (transmembrane domain 9) to alanine-scanning mutagenesis. Phe523 was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in Km without affecting expression or localization. Phe523 and the previously identified polar residue Gln526 were on the same face of the helix suggesting a joint participation in glutathione recognition, whereas two other polar residues, Ser519 and Asn522, of TMD9, although also orientated on the same face, did not appear to be involved. The size and hydrophobicity of Phe523 were both key features of its functionality, as seen from mutational analysis. Sequence alignments revealed that Phe523 and Gln526 were conserved in a cluster of OPT homologues from different fungi. A second cluster contained isoleucine and glutamate residues in place of phenylalanine and glutamine residues, residues that are best tolerated in Hgt1p for glutathione transporter activity, when introduced together. The critical nature of the residues at these positions in TMD9 for substrate recognition was exploited to assign substrate specificities of several putative fungal orthologues present in these and other clusters. The presence of either phenylalanine and glutamine or isoleucine and glutamate residues at these positions correlated with their function as high-affinity glutathione transporters based on genetic assays and the Km of these transporters towards glutathione.
 
Publisher Portland Press
 
Date 2010-08-01
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/516/1/bachhawat10.1.pdf
Thakur, Anil and Bachhawat, Anand K (2010) The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters. The Biochemical journal, 429 (3). pp. 593-602. ISSN 1470-8728
 
Relation http://www.biochemj.org/bj/429/bj4290593.htm
http://crdd.osdd.net/open/516/