# A genetic screen reveals a key role for Reg1 in 2-deoxyglucose sensing and yeast AMPK inhibition

## TL;DR

This study shows that the Reg1 protein in yeast plays a key role in sensing glucose and its analog 2DG, influencing how yeast regulate their metabolism.

## Contribution

The study identifies novel Reg1 mutations that reveal its role in glucose and 2DG sensing, offering new insights into yeast metabolic regulation.

## Key findings

- Mutations in Reg1 allow yeast to maintain glucose-regulated Snf1 activity but fail to respond to 2DG.
- Resistance to 2DG arises from reduced phosphorylation or constitutive Snf1 activation.
- Reg1 is positioned as a central mediator of glucose sensing in yeast.

## Abstract

The yeast Saccharomyces cerevisiae thrives in sugar-rich environments by rapidly consuming glucose and favoring alcoholic fermentation. This strategy is tightly regulated by the glucose repression pathway, which prevents the expression of genes required for the utilization of alternative carbon source. Central to this regulatory network is the yeast ortholog of the heterotrimeric 5′AMP-activated protein kinase (AMPK), which adjusts gene expression in response to glucose availability. The activity of the yeast AMPK complex is primarily regulated by the phosphorylation state of its catalytic subunit Snf1, a process orchestrated by a balance between upstream kinases and phosphatases. Among the latter, the Protein Phosphatase 1 (PP1) complex Reg1/Glc7 plays a critical role in inhibiting Snf1 activity under glucose-rich conditions. Despite its importance, the precise mechanism by which glucose availability leads to Snf1 inhibition remains incompletely understood. Evidence suggests that hexokinase 2 (Hxk2) participates in this pathway, potentially coupling the early steps of glucose metabolism to Snf1 signaling. Notably, the toxic glucose analog 2-deoxyglucose (2DG)- which is phosphorylated by Hxk2 but not further metabolized- mimics glucose in its ability to repress Snf1, implicating glucose or 2DG phosphorylation as a key regulatory signal. Additionally, yeast AMPK activity correlates with 2DG resistance through mechanisms that are incompletely described. In this study, we performed a large-scale 2DG-resistance genetic screen to explore both the molecular basis of 2DG resistance and AMPK regulation in yeast. The identified mutations confer resistance either by reducing 2DG phosphorylation (e.g., mutations in HXK2) or by enhancing constitutive Snf1 activity, via gain-of-function alleles in AMPK subunits or loss-of-function mutations in REG1 and GLC7. We also describe a novel series of REG1 missense mutations, including reg1-W165G, that maintain basal, glucose-regulated Snf1 activity but fail to mediate 2DG-induced Snf1 inhibition. These findings position Reg1 as a central mediator in glucose sensing, possibly by sensing 2DG-derived -and by extension, glucose-derived- metabolites.

Yeast such as Saccharomyces cerevisiae thrive in sugar-rich environments by rapidly consuming glucose and converting it to ethanol, even when oxygen is present. This strategy relies on a pathway named “glucose repression pathway”, which blocks the use of alternative carbon sources. At the core of this pathway is Snf1, the yeast equivalent of mammalian AMPK, whose activity is regulated by upstream kinases and the Reg1/Glc7 phosphatase complex. Despite extensive research, how glucose metabolism inhibits Snf1 remains unresolved.

A useful tool to probe this pathway is the toxic sugar mimic 2-deoxyglucose (2DG), which, once phosphorylated by hexokinase 2, mimics glucose in shutting down Snf1, although it cannot be metabolized further. Here, we performed a large-scale screen for yeast mutants resistant to 2DG. Resistance arose either from reduced 2DG phosphorylation or from mutations that keep Snf1 active despite glucose or 2DG. Crucially, we identified novel Reg1 missense mutations that preserve glucose regulation but fail to mediate 2DG-dependent Snf1 inhibition. These findings reveal Reg1 as a central mediator of glucose sensing, and suggest it may directly interpret signals from early sugar metabolism—a previously unrecognized role that reshapes our understanding of how yeast couples nutrient detection to metabolic control.