Independent evolution of polymerization in the Actin ATPase clan regulates hexokinase activity

Abstract

Yeast glucokinase activity is limited by its polymerization, which is critical for cell viability during glucose refeeding. AbstractThe actin protein fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, like the carbohydrate kinases, do not polymerize. We find that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with unique ultrastructure, distinct from that of cytoskeletal polymers. In cells, Glk1 polymerizes upon sugar addition and depolymerizes upon sugar withdrawal. Glk1 polymerization inhibits its enzymatic activity, thus the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation eliminating Glk1 polymerization alleviates concentration-dependent enzyme inhibition, causing glucokinase activity to become unconstrained. Polymerization-based regulation of Glk1 activity serves an important function in vivo: yeast containing non-polymerizing Glk1 are less fit when growing on sugars and more likely to die when refed glucose. Glucokinase polymerization arose within the ascomycete fungi and is conserved across a group of divergent (150-200 mya) yeast. We show that Glk1 polymerization arose independently from other actin-related filaments and allows yeast to rapidly modulate glucokinase activity as nutrient availability changes. crystallographic advice, along with Christina Zimanyi, Lukas Bane, and Elizabeth May. We thank Jim Wilhelm for helpful discussions. We thank the Arnold and Mabel Beckman Cryo-EM Center at the University of Washington for access to electron microscopes. This work used NE-CAT beamlines (GM124165) at the APS (DE-AC02-06CH11357). Authors Contributions: S. cerevisiae strains were cloned by PRS and QAJ. Fluorescence microscopy was done by PRS and QAJ. PRS measured the relative expression of Glk1 by flow cytometry. Plasmid construction and protein purification, in vitro measurements of polymerization and enzymatic activity, Xray crystallography, Glk1 crystal structure refinement, and negative stain EM were done by PRS. PRS measured the viability of cells during glucose refeeding and measured the fitness of competed strains by flow cytometry. CryoEM sample preparation and data collection were

Ensemble cryo-EM structures demonstrate human IMPDH2 filament assembly tunes allosteric regulation

“…Most of these metabolic filaments are assembled from important regulatory enzymes, which suggests polymerization may play a role in modulating flux through these pathways. Recently, in just a few cases, structural and biochemical studies have provided insight into the functional consequences of enzyme filament assembly, suggesting that one role of polymers is to regulate activity by locking enzymes into active or inactive conformations through assembly contacts (Hunkeler et al 2018; Barry et al 2014; Lynch et al 2017; Webb et al 2018; Stoddard et al 2019). Thus, it was surprising when our initial characterization of IMPDH2 filaments showed that assembly did not affect enzymatic activity or the ability to switch between active or inactive conformations (Anthony et al 2017).…”

Structures, functions, and mechanisms of filament forming enzymes: a renaissance of enzyme filamentation

“…The enzyme is induced in the absence of glucose and expressed in high glucose (Maitra 1970). A fusion protein of Glk1 and GFP is found to be diffused throughout the cytoplasm in the absence of glucose, but formed fiber-like self-assemblies in the presence of glucose (Stoddard et al 2019). These bundles rapidly disassemble when glucose was removed from the growth media (Stoddard et al 2019).…”

“…A fusion protein of Glk1 and GFP is found to be diffused throughout the cytoplasm in the absence of glucose, but formed fiber-like self-assemblies in the presence of glucose (Stoddard et al 2019). These bundles rapidly disassemble when glucose was removed from the growth media (Stoddard et al 2019). Purified Glk1 at 7.5 μM is non-filamentous in the absence of ligands, but was found to form helical filaments in vitro in the presence of its substrates (ATP and glucose, mannose, glucosamine) or products (ADP and sugar-6-phosphate) (Stoddard et al 2019).…”

“…These bundles rapidly disassemble when glucose was removed from the growth media (Stoddard et al 2019). Purified Glk1 at 7.5 μM is non-filamentous in the absence of ligands, but was found to form helical filaments in vitro in the presence of its substrates (ATP and glucose, mannose, glucosamine) or products (ADP and sugar-6-phosphate) (Stoddard et al 2019). No filaments were formed in response to pH, fructose, or galactose (fructose and galactose result in fiber formation in vivo ) (Stoddard et al 2019).…”

“…Purified Glk1 at 7.5 μM is non-filamentous in the absence of ligands, but was found to form helical filaments in vitro in the presence of its substrates (ATP and glucose, mannose, glucosamine) or products (ADP and sugar-6-phosphate) (Stoddard et al 2019). No filaments were formed in response to pH, fructose, or galactose (fructose and galactose result in fiber formation in vivo ) (Stoddard et al 2019). Some polymerization is found with N-acetylglucosamine-6-phosphate and N-acetylglucosamine, inhibitors of Glk1 (Stoddard et al 2019).…”