Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts

Abstract

Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to selected CSREs and provide evidence that KlSip4 counteracts KlCat8-mediated transcription activation by competing for binding to some but not all CSREs. The finding that the hierarchical relationship of these transcription factors differs between K. lactis and S. cerevisiae and that the sets of target genes have diverged contributes to explaining the phenotypic differences in metabolic life-style.

Two homologs of the Cat8 transcription factor are involved in the regulation of ethanol utilization in Komagataella phaffii

“…However, cat8-1Δ and cat8-2Δ had significantly lower growth rates than the wild-type Full circles (•) represent the positions of the 6 functionally characterized Cat8 and Sip4 as well as K. phaffii Cat8-1 and Cat8-2 protein sequences that were used for BLAST search: 1. Mehlgarten et al 2015, 2. Lesage et al 1996, 3.…”

“…Growth on gluconeogenic carbon sources such as glycerol and ethanol requires enzymes from gluconeogenesis and the glyoxylate cycle, among others. In the yeasts Saccharomyces cerevisiae and Kluyveromyces lactis , the genes encoding these enzymes are activated through upstream activation sites (UAS) found in their promoters, such as the carbon source responsive elements (CSREs) (Mehlgarten et al 2015 ; Turcotte et al 2010 ). The CSREs are under the control of two transcriptional regulators, which are members of the binuclear zinc cluster family: Cat8 and Sip4 (Roth et al 2004 ; Vincent and Carlson 1998 ).…”

“…Cat8 (CATabolite repression) and Sip4 (Snf1 interacting protein) possess a highly similar N-terminal zinc cluster (Zn(II) 2 Cys 6 ) binding domain (Rahner et al 1996 ), but they share only little similarity in the rest of their protein sequences (Mehlgarten et al 2015 ; Turcotte et al 2010 ). Although both Cat8 and Sip4 were shown to bind the CSRE (consensus sequence: YCCRTTNRNCGG) (Roth et al 2004 ; Vincent and Carlson 1998 ), Sip4 recognizes and binds to a more specific CSRE motif than Cat8, which probably explains why Cat8 and Sip4 contribute unequally to gene activation via binding to this motif (Hiesinger et al 2001 ).…”

“…Although both Cat8 and Sip4 were shown to bind the CSRE (consensus sequence: YCCRTTNRNCGG) (Roth et al 2004 ; Vincent and Carlson 1998 ), Sip4 recognizes and binds to a more specific CSRE motif than Cat8, which probably explains why Cat8 and Sip4 contribute unequally to gene activation via binding to this motif (Hiesinger et al 2001 ). In S. cerevisiae and K. lactis , Cat8 and Sip4 were described to be activators of transcription, but their mechanism of action in these two yeast are slightly different (Mehlgarten et al 2015 ).…”

“…The Klcat8 knock-out mutants can grow on glycerol but not on C2 carbon sources such as acetate and ethanol, which shows that unlike in S. cerevisiae , the gluconeogenesis encoding genes are not regulated by Cat8 nor Sip4 in K. lactis (Georis et al 2000 ). On the other hand, contrary to S. cerevisiae where Cat8 but not Sip4 is required for growth on ethanol, both Klcat8 and Klsip4 knock-out mutants exhibit a growth defect on C2 carbon sources (Mehlgarten et al 2015 ). It was also shown that only Kl Sip4 binds to the CSRE motifs in the promoters of the glyoxylate pathway genes and the carnitine shuttle encoding genes (Mehlgarten et al 2015 ; Rodicio et al 2008 ).…”