Abstract
The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.
Highlights
The Vps13 proteins are conserved in all eukaryotic organisms
Our results suggest that functioning of Vps13 is regulated by its interaction with phosphatidylinositol 3-phosphate (PI3P), and losing PI3P-binding ability by hVps13-I2749R mutant protein accompanied with its abnormal cellular cycling might be a reason for its pathogenicity in some patients suffering for ChAc
Vps13-I2749R mutation, an equivalent of human mutation I2771R found in patient suffering from ChAc, impairs the function of the yeast Vps13 protein
Summary
The Vps proteins are conserved in all eukaryotic organisms. In the human genome, Vps proteins are encoded by four genes VPS13A, VPS13B, VPS13C and VPS13D [1]. Most mutations found in ChAc patients lead to the generation of a premature stop codon and cause a marked reduction of the mutant protein level in cells probably due a premature termination of translation and degradation of the hVPS13A transcript via nonsense mediated decay [3]. The hVps13A is apparently involved in actin cytoskeleton organization and dynamics, since red blood cells and platelets from ChAc patients display depolymerization of the cortical actin cytoskeleton probably causing the acanthocytosis [5,6]. The hVps13A protein, plays a role in metabolism of phospholipids, as it was shown that knockout of human hVPS13A led to a reduction in the level of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus and plasma membrane in the neuronal model PC12 cells [8].
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