KlAft, the Kluyveromyces lactis Ortholog of Aft1 and Aft2, Mediates Activation of Iron-Responsive Transcription Through the PuCACCC Aft-Type Sequence

Abstract

Iron homeostasis in fungi is regulated at the transcriptional level by two different mechanisms. It is mediated by a conserved GATA-type repressor in most fungi except in the yeast Saccharomyces cerevisiae, where it is controlled by the transcription activators Aft1 and Aft2. These activators are encoded by the paralogous genes AFT1 and AFT2, which result from the whole-genome duplication. Here, we explore regulation of iron homeostasis in the yeast Kluyveromyces lactis that diverged from S. cerevisiae before this event. We identify an ortholog of AFT1/AFT2, designated KlAFT, whose deletion leads to the inability to grow under iron limitation. We show with quantitative real-time PCR analysis that KlAft activates the transcription of all homologs of the Aft1-target genes involved in the iron transport at the cell surface in response to iron limitation. However, homologs of Aft2-specific target genes encoding intracellular iron transporters are regulated neither by KlAft nor by iron. Both bioinformatic and DNA binding and transcription analyses demonstrate that KlAft activates iron-responsive gene expression through the PuCACCC Aft-type sequence. Thus, K. lactis is the first documented species with a positive irontranscriptional control mediated by only one copy of the Aft-type regulator. This indicates that this function was acquired before the whole-genome duplication and was then diversified into two regulators in S. cerevisiae.

Candida albicans Hap43 Is a Repressor Induced under Low-Iron Conditions and Is Essential for Iron-Responsive Transcriptional Regulation and Virulence

“…Notably, these target mRNAs are mostly transcribed from genes that encode iron-dependent proteins, and thereby, degradation of these mRNAs decreases the consumption and facilitates the efficient usage of limited amounts of iron. Although Pfam prediction indicates that the Aft-type proteins (PF08731) exist in most hemiascomycota yeasts, only S. cerevisiae and Kluyveromyces lactis Aft orthologs have so far been demonstrated to possess iron-responsive transcriptional activity via the PuCACCC binding motif in the promoter region of the iron regulon (12).…”

The Basis for Evolution of DNA-Binding Specificity of the Aft1 Transcription Factor in Yeasts

“…Previous work indicated that Aft1 in S. cerevisiae binds to the sequence TGCACCC whereas KlAft in K. lactis binds to the sequence PuCACCC (Yamaguchi-Iwai et al 1996;Conde e Silva et al 2009). Here, we compared the ability of KlAft and Aft1 to bind to the TGCACCC Aft1-binding site of the FET3 promoter in the same S. cerevisiae cellular context.…”

“…The DNA-binding domain of the Aft proteins has been identified as a WRKY-GCM1 zincfinger domain (Babu et al 2006). It belongs to the subgroup of the WRKY-GCM1 domains with insert because it contains an insert of 20-90 residues between two conserved cysteine residues that have been suggested to be involved in a zincfinger domain (Babu et al 2006;Conde e Silva et al 2009). We examined the amino acid sequence of the insert and identified a variable sequence (VS) within the insert that is predicted to be disordered and highly phosphorylated.…”

“…The TGCACCC sequence is widespread among promoter regions of genes of the iron regulon in hemiascomycetes Previous computer analyses identified TGCACCC and PuCACCC Aft-type sequences as consensus sequences in the promoters of iron-regulated genes in S. cerevisiae and K. lactis, respectively (Rutherford et al 2003;Courel et al 2005;Conde e Silva et al 2009). To study Aft-type cisregulatory sequences and their evolution, we extended our analyses to diverse species belonging to hemiascomycete clades and for which the genome sequences are available.…”

“…In most fungi, the iron-regulatory pathway is mediated by a Zn-finger GATA-type factor that represses the transcription of the iron-uptake genes under high-iron conditions (Haas et al 2008). In contrast, in the hemiascomycete yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, the transcription of the same group of genes is activated by an Aft-type transcription factor under conditions of low iron availability (Yamaguchi-Iwai et al 1995;Conde e Silva et al 2009). An AFT gene has been identified in the genome of all hemiascomycetes studied except Yarrowia lipolityca.…”