Studies on the structure of aldolase bound to F-actin and its role in endocytosis
Ho, Nhu Quynh
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Fructose 1,6-bisphosphate aldolase, or simply aldolase, is a key enzyme involved in the glucose and fructose metabolic pathways. Vertebrate aldolases exists in nature as extremely stable tetramers. While essential for energy metabolism, aldolase is involved in numerous “moonlighting functions,” many of which involve the ability to bind F-actin. Evidence suggests that the quaternary structure of aldolase may have evolved for such moonlighting activities in the cell. Though aldolase was one of the first actin-binding proteins discovered, the exact structure and biological functions of this interaction have remained elusive. Electron microscopy studies demonstrate that a minimum of a dimer is required to bind and decorate actin filaments, but only tetrameric aldolase is capable of binding and crosslinking F-actin into bundles. Experiments are described that will lead to a high-definition structure of the aldolase-F-actin complex and resolution of the binding interface used by aldolase. These include efforts to create chimeric aldolase tetramers using in vitro protein hybridization and modular cloning approaches. Understanding this critical interaction is necessary for mechanistic models explaining aldolase moonlighting functions. The significance of aldolase’s association with F-actin in vivo has only more recently been explored. Studies with two other aldolase binding partners—Wiskott-Aldrich Syndrome protein (WASP) and Sorting Nexin 9 (SNX9), suggest that aldolase may be moonlighting as a scaffold protein during late stage endocytic events in the cell. The hypothesis that aldolase has a regulatory role in endocytosis is tested. Precise coordination and efficiency of recruited proteins are hallmarks of clathrin-mediated endocytosis, and a scaffolding role for aldolase could serve at least two functions: 1) to sequester the active domains of WASP and SNX9 prior to vesicle scission, and 2), enable sufficient concentration of WASP and SNX9 at sites of endocytosis via association with F-actin. This type of regulation would sequester each protein until the exact function is needed, allowing release at the appropriate time points as well as in the proper sequence. Uptake assays performed using two different markers and cell lines show upregulation of endocytosis after knockdown of aldolase. A model is proposed for participation by aldolase in the dynamics of this process.