The FOX transcription factor Hcm1 regulates oxidative metabolism in response to early nutrient limitation in yeast. Role of Snf1 and Tor1/Sch9 kinases

Abstract

Within Saccharomyces cerevisiae, Hcm1is a member of the forkhead transcription factor family with a role in chromosome organization. Our group recently described its involvement in mitochondrial biogenesis and stress resistance, and reports here that Hcm1 played a role in adaptation to respiratory metabolism when glucose or nitrogen was decreased. Regulation of Hcm1 activity occurs in at least three ways: i) protein quantity, ii) subcellular localization, and iii) transcriptional activity. Transcriptional activity was measured using a reporter gene fused to a promoter that contains a binding site for Hcm1. We also analyzed the levels of several genes whose expression is known to be regulated by Hcm1 levels and the role of the main kinases known to respond to nutrients. Lack of sucrose-nonfermenting (Snf1) kinase increases cytoplasmic localization of Hcm1, whereas Δtor1 cells showed a mild increase in nuclear Hcm1. In vitro experiments showed that Snf1 clearly phosphorylates Hcm1 while Sch9 exerts a milder phosphorylation. Although in vitroTor1 does not directly phosphorylate Hcm1, in vivo rapamycin treatment increases nuclear Hcm1. We conclude that Hcm1 participates in the adaptation of cells from fermentation to respiratory metabolism during nutrient scarcity. According to our hypothesis, when nutrient levels decrease, Snf1 phosphorylates Hcm1. This results in a shift from the cytoplasm to the nucleus and increased transcriptional activity of genes involved in respiration, use of alternative energy sources, NAD synthesis and oxidative stress resistance.

The SNF1 Kinase Ubiquitin-associated Domain Restrains Its Activation, Activity, and the Yeast Life Span

“…It was recently reported that Snf1 phosphorylates the yeast Forkhead protein, Hcm1, in response to stress, facilitating shuttling of Hcm1 from the cytosol to the nucleus (55). Hcm1 is tightly linked to Fkh1/2 activity as it transcribes FKH1 and FKH2 during late S phase (70).…”

“…An evolutionarily conserved stress response network utilizing AMPK and Forkhead proteins has been described (53,55,57). In yeast, it was shown that the Forkhead protein Hcm1, but not Fkh1 or Fkh2, was phosphorylated by Snf1 under stress conditions (55).…”

“…Furthermore, combined disruption of both FKH1 and FKH2 was required to decrease CLS, and the double mutants no longer responded to caloric restriction, indicating that Fkh1 and Fkh2 are critical responders to caloric restriction, a metabolic stress that also results in AMPK/SNF1 kinase activation. To date, however, Snf1 has only been shown to interact with the Hcm1 Forkhead protein (55) and not Fkh1 or Fkh2.…”

The Opposing Actions of Target of Rapamycin and AMP-Activated Protein Kinase in Cell Growth Control

“…Thus, Snf1 and TORC1 may act at similar steps in the regulation of Msn2 (Mayordomo et al 2002). Finally, TORC1 and Snf1 phosphorylate Hcm1 to inhibit and stimulate, respectively, nuclear translocation of this transcription factor (Rodriguez-Colman et al 2013). The nuclear translocation of Hcm1 leads to a shift in metabolism from fermentation to respiration.…”

The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

“…Protein extracts were prepared for gel electrophoresis. Phos-tag gel electrophoresis was conducted as described previously (31). Briefly, ϳ2 ϫ 10 8 to 4 ϫ 10 8 cells for each sample or time point were used for extraction.…”