2-deoxyglucose transiently inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity

2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export - thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation. Author summaryIn this article, we study the resistance to a drug named 2-deoxyglucose (2DG). 2DG is an efficient inhibitor of several metabolic pathways, particularly glycolysis which is of prime importance for tumor cell proliferation. Cancer cells but also cells from other organisms can develop various resistance strategies that only being unraveled. Here, using bakers yeast as a model organism, we document the resistance mechanism of a mutant lacking the arrestin gene ROD1, known to regulate nutrient transporter endocytosis. First, we show that 2DG triggers the endocytosis of many nutrient transporters. Mechanistically, 2DG leads to a transient inhibition of the AMPK signaling pathway, which in turn promotes the activation of Rod1, a pre-requisite for 2DG-induced endocytosis. Consequently, cells lacking Rod1 maintain various proteins at the plasma membrane including hexose transporters, the latter being essential for 2DG resistance. Hexose transporter stabilization at the cell surface may sustain glucose import and help cells cope with the 2DG-induced metabolic blockade, while also allowing 2DG detoxication.