A Snf1-related nutrient-responsive kinase antagonizes endocytosis in yeast

Abstract

Endocytosis is regulated in response to changing environmental conditions to adjust plasma membrane (PM) protein composition for optimal cell growth. Protein networks involved in cargo capture and sorting, membrane sculpting and deformation, and vesicle scission have been well-characterized, but less is known about the networks that sense extracellular cues and relay signals to trigger endocytosis of specific cargo. Hal4 and Hal5 are yeast Snf1related kinases that were previously reported to regulate nutrient transporter stability by an unknown mechanism. Here we demonstrate that loss of Hal4 and Hal5 activates endocytosis of many different kinds of PM proteins, including Art1-mediated and Art1-independent endocytic events. Acute inhibition of Hal5 in the absence of Hal4 triggers rapid endocytosis, suggesting that Hal kinases function in a nutrient-sensing relay upstream of the endocytic response. Interestingly, Hal5 localizes to the PM, but shifts away from the cell surface in response to stimulation with specific nutrients. We propose that Hal5 functions as a nutrientresponsive regulator of PM protein stability, antagonizing endocytosis and promoting stability of endocytic cargos at the PM in nutrient-limiting conditions.

Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast

“…We found that co-expression of ubiquitin with the kinase Vhs1 resulted in immunodetection of Ser57 phosphorylated ubiquitin ( Figure 3A and Figure 3—figure supplement 1 ). Vhs1 is part of the yeast family of Snf1-related kinases ( Tumolo et al, 2020 ), and additional screening revealed three other kinases in this family that phosphorylated ubiquitin at the Ser57 position: Sks1 (which is 43% identical to Vhs1) ( Figure 3B ), Gin4 and Kcc4 ( Figure 3—figure supplement 2 ). A previous study reported consensus phosphorylation motifs for Vhs1, Gin4, and Kcc4, and all bear similarity to the amino acid sequence surrounding Ser57 in ubiquitin ( Supplementary file 2 ; Mok et al, 2010 ).…”

“…The family of Snf1-related kinases in yeast share homology with the human AMPK-related kinases ( Tumolo et al, 2020 ). To test if human AMPK-related kinases exhibit activity toward ubiquitin, we performed in vitro kinase assays and found that a subset of this family phosphorylated ubiquitin at the Ser57 position specifically on tetramers ( Figure 3G ).…”

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids

“…While both Art1 and Art2 lead to the degradation of AATs, they answer to distinct metabolic cues and are thus wired into distinct signaling pathways. Activation of Art1 by amino acid influx requires the coordinated interplay of TORC1 signaling to inactivate Npr1 (a kinase that negatively regulates Art1) and the action of phosphatases (Gournas et al, 2017, Lee et al, 2019, MacGurn et al, 2011, Tumolo et al, 2020. In response to amino acid limitation, TORC1 is no longer active.…”

Enhancing lifespan of budding yeast by pharmacological lowering of amino acid pools

“…On the other hand, SDC culture medium contains Met (537 μM) which represses Mup1 production. To determine if Myr treatment effects Mup1 in the same way in these two types of media, we examined cells harboring chromosomal MUP1 tagged with ecliptic pHluorin (a GFP variant that loses fluorescence at low pH) which does not fluoresce in the vacuole and late endosomal compartments and thus can be used to quantify the abundance of Mup1 at the PM and early endosomal compartments [68][69][70]. Following transfer of stationary phase cells into fresh SC medium, we observed very low Mup1-pHluorin signal that increased until plateauing at about 4 hr ( Figure 4C).…”

“…Mup1 localization and trafficking were analyzed by fluorescence microscopy and flow cytometry as previously described [69,70]…”