Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing..
Fungi such as Candida species are a major cause of hospital-acquired infections especially in immuno-compromised patients, and invasive candidiasis is associated with a high mortality rate. Central to Candida albicans virulence is its ability to switch between budding (yeast) and filamentous (hyphal) growth, and to colonise different body niches. In each distinct environment it must remodel its cell surface to ensure appropriate levels of transporters and cell wall biosynthesis enzymes. Endocytosis is known to be a critical pathway in surface remodelling, allowing cells to internalise proteins that are no longer needed at the plasma membrane. Endocytosis is also crucial to highly polarised hyphal growth, where endocytic recycling of key membrane proteins is essential to maintain their polarised location at the hyphal tip. The aim of this study is to investigate the role of the AP-2 endocytic adaptor complex in endocytosis within C. albicans. Homozygous deletions were generated in an essential subunit of the AP-2 complex. The deletion did not affect rates of cell growth or fluid phase endocytosis, but using fluorescence and electron microscopy, defects were observed in hyphal polarisation and in cell wall organisation. We have shown that the AP-2 complex is required for the recycling of the key cell wall biosynthesis enzyme Chs3, via its Yxxφ internalisation motif(s). We demonstrate this interaction is critical for correct cell wall deposition and polarised growth. Thus, AP-2 mediated endocytic recycling is a key step in the regulation of the fungal cell wall.
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