Identification of a multienzyme complex for glucose metabolism in living cells

Abstract

Sequential metabolic enzymes in glucose metabolism have long been hypothesized to form multienzyme complexes that regulate glucose flux in living cells. However, it has been challenging to directly observe these complexes and their functional roles in living systems. In this work, we have used wide-field and confocal fluorescence microscopy to investigate the spatial organization of metabolic enzymes participating in glucose metabolism in human cells. We provide compelling evidence that human liver-type phosphofructokinase 1 (PFKL), which catalyzes a bottleneck step of glycolysis, forms various sizes of cytoplasmic clusters in human cancer cells, independent of protein expression levels and of the choice of fluorescent tags. We also report that these PFKL clusters colocalize with other rate-limiting enzymes in both glycolysis and gluconeogenesis, supporting the formation of multienzyme complexes. Subsequent biophysical characterizations with fluorescence recovery after photobleaching and FRET corroborate the formation of multienzyme metabolic complexes in living cells, which appears to be controlled by post-translational acetylation on PFKL. Importantly, quantitative high-content imaging assays indicated that the direction of glucose flux between glycolysis, the pentose phosphate pathway, and serine biosynthesis seems to be spatially regulated by the multienzyme complexes in a cluster-size-dependent manner. Collectively, our results reveal a functionally relevant, multienzyme metabolic complex for glucose metabolism in living human cells.

RNA promotes phase separation of glycolysis enzymes into yeast G bodies in hypoxia

“…Similar structures also form in the neurons of C. elegans, where enzyme clustering in response to hypoxia was associated with proper synaptic function, suggesting that glycolysis enzymes coalesce to increase glycolysis and meet the local high energy demand during synapses (Jang et al, 2016). Furthermore, cancer cell lines form small aggregates of glycolysis enzymes even in the presence of oxygen, suggesting that concentrating glycolysis enzymes is a highly conserved process (Kohnhorst et al, 2017). Additionally, pyrenoids, which enhance the rate of carbon fixation in Chlamydomonas by concentrating CO 2 in the presence of ribulose 1,5 bisphosphate carboxylase oxygenase (RuBisCo), were recently shown to form via phase separation (Freeman Rosenzweig et al, 2017).…”

The Glycolytic Protein Phosphofructokinase Dynamically Relocalizes into Subcellular Compartments with Liquid-like Properties in vivo

“…Immunohistochemical studies on isolated Drosophila flight muscle revealed a distinct pattern of co-localization of glycolytic protein isoforms to muscle sarcomeres (Sullivan et al, 2003) and vertebrate endothelial cells display enrichment of glycolytic proteins to lamellipodia and filopodia, where mitochondria are mostly absent (De Bock et al, 2013). Together, these studies and others suggest that glycolytic proteins could be compartmentalized in specific tissues (Kastritis and Gavin, 2018;Kohnhorst et al, 2017). Yet the notion of a subcellular organization for glycolytic proteins has remained controversial, largely due to the lack of studies examining the dynamic distribution of these enzymes in vivo (Brooks and Storey, 1991;Menard et al, 2014).…”

“…Glycolytic enzymes were also shown to co-localize into subcellular compartments termed "G-bodies" in yeast, the formation of which was important for cellular division during conditions of persistent hypoxia (Jin et al, 2017). Similar observations in mammalian tissue culture cells also demonstrated that glycolytic enzymes co-localize into clusters (Kohnhorst et al, 2017). A human isoform of phosphofructokinase (PFK) was recently shown to assemble into tetramers that oligomerize into higher-ordered filamentous structures in vitro (Webb et al, 2017).…”

Illuminating the Cell’s Biochemical Activity Architecture

“…For example, by expressing fluorescently tagged proteins in living cells, An and colleagues previously found that the six metabolic enzymes that catalyze the ten-step pathway responsible for de novo purine biosynthesis all colocalize within a multi-enzyme complex – the “purinosome” – that dynamically forms visible cytosolic clusters directly associated with purine synthesis 30 . In a recent study, Kohnhorst et al similarly investigated the behavior of fluorescent protein-tagged glucose metabolic enzymes in living cells and observed the formation of a “glucosome” containing four enzymes that catalyze rate-limiting steps in glycolysis and gluconeogenesis 31 . These complexes also formed visible clusters that were distinct from other known cytosolic bodies and whose sizes varied dynamically in response to treatments that shifted glucose flux to favor specific pathways (e.g., the pentose phosphate pathway, serine biosynthesis), suggesting a mechanism for compartmentalizing metabolic processes and controlling cellular metabolic states 31 .…”

“…In a recent study, Kohnhorst et al similarly investigated the behavior of fluorescent protein-tagged glucose metabolic enzymes in living cells and observed the formation of a “glucosome” containing four enzymes that catalyze rate-limiting steps in glycolysis and gluconeogenesis 31 . These complexes also formed visible clusters that were distinct from other known cytosolic bodies and whose sizes varied dynamically in response to treatments that shifted glucose flux to favor specific pathways (e.g., the pentose phosphate pathway, serine biosynthesis), suggesting a mechanism for compartmentalizing metabolic processes and controlling cellular metabolic states 31 . Additional imaging studies have revealed that purinosomes localize to the mitochondrial surface 32 and are regulated by GPCR 33 and mTOR 32 signaling; biosensor-based imaging may therefore be useful in revealing additional spatial links between signaling enzyme activity and purinosome function.…”