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Eukaryotic Cell, February 2005, p. 476-483, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.476-483.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

DNA-Binding Specificity of the IDI-4 Basic Leucine Zipper Factor of Podospora anserina Defined by Systematic Evolution of Ligands by Exponential Enrichment (SELEX)

Karine Dementhon{dagger} and Sven J. Saupe*

Laboratoire de Génétique Moléculaire des Champignons, Institut de Biochimie et de Génétique Cellulaires, UMR 5095 CNRS-Université de Bordeaux 2, Bordeaux, France

Received 18 October 2004/ Accepted 1 December 2004

Heterokaryon incompatibility is a cell destruction process that occurs when fungal cells of unlike genotype fuse. In Podospora anserina, autophagy is engaged during cell death by incompatibility and a number of genes are induced at the transcriptional level. These genes are termed idi (induced during incompatibility) genes. Among these is idi-4, a gene encoding a basic leucine zipper (bZIP) factor. IDI-4 displays similarity to the GCN4/cross-pathway control (CPC) factors that control gene expression in response to amino acid starvation in fungi. The overexpression of idi-4 triggers autophagy, leads to cell death, and also increases the expression of a number of idi genes, in particular idi-7, a gene involved in autophagy. Herein, we determined the in vitro target sequence of IDI-4. We have purified the recombinant IDI-4 bZIP domain and show that this 83-amino-acid-long peptide dimerizes in vitro and adopts an {alpha}-helical fold. We have then used a systematic evolution of ligands by exponential enrichment procedure to identify the sequence bound by the IDI-4 bZIP domain. The IDI-4 binding site consensus sequence corresponds to the ATGANTCAT pseudopalindrome. IDI-4 binding sites are present in the promoter region of the idi-7 gene, and the bZIP IDI-4 peptide binds to the idi-7 promoter in vitro. The identified IDI-4 consensus binding sequence is very similar to the GCN4/CPC binding site, raising the possibility of an interplay and/or partial functional redundancy between IDI-4 and CPC-type bZIP factors in fungi.

* Corresponding author. Mailing address: Laboratoire de Génétique Moléculaire des Champignons, Institut de Biochimie et de Génétique Cellulaires, UMR 5095 CNRS-Université de Bordeaux 2, 1 rue Camille St Saëns, 33077 Bordeaux Cedex, France. Phone: 33 (0) 5 56 99 90 27. Fax: 33 (0) 5 56 99 90 67. E-mail: sven.saupe{at}ibgc.u-bordeaux2.fr.

{dagger} Present address: Plant and Microbial Biology Department, University of California, Berkeley, CA 94720-3102.

Eukaryotic Cell, February 2005, p. 476-483, Vol. 4, No. 2
1535-9778/05/$08.00+0     doi:10.1128/EC.4.2.476-483.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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