Abstract
ABSTRACTPoint mutations in the human MYO1E gene, encoding class I myosin Myo1e, are associated with focal segmental glomerulosclerosis (FSGS), a primary kidney disorder that leads to end-stage kidney disease. In this study, we used a simple model organism, fission yeast Schizosaccharomyces pombe, to test the effects of FSGS-associated mutations on myosin activity. Fission yeast has only one class I myosin, Myo1, which is involved in actin patch assembly at the sites of endocytosis. The amino acid residues mutated in individuals with FSGS are conserved between human Myo1e and yeast Myo1, which allowed us to introduce equivalent mutations into yeast myosin and use the resulting mutant strains for functional analysis. Yeast strains expressing mutant Myo1 exhibited defects in growth and endocytosis similar to those observed in the myo1 deletion strain. These mutations also disrupted Myo1 localization to endocytic actin patches and resulted in mis-localization of Myo1 to eisosomes, linear membrane microdomains found in yeast cells. Although both mutants examined in this study exhibited loss of function, one of these mutants was also characterized by the decreased protein stability. Thus, using the yeast model system, we were able to determine that the kidney-disease-associated mutations impair myosin functional activity and have differential effects on protein stability.
Highlights
Missense mutations in the human MYO1E gene, encoding molecular motor protein myosin 1e (Myo1e), are associated with kidney disease (Al-Hamed et al, 2013; Mele et al, 2011; SannaCherchi et al, 2011)
In order to better understand the pathogenesis of focal segmental glomerulosclerosis (FSGS) and guide the identification of potential therapy targets for FSGS, it will be important to determine how myosin 1e motor activity contributes to the function of the glomerular filtration barrier
Mutations in the motor domain of Myo1, equivalent to the FSGS-associated Myo1e mutations, result in yeast growth defects Based on sequence alignment (Fig. 1B), we identified conserved residues T140 and A181 in the S. pombe Myo1 motor domain as homologous to the residues T119 and A159 in human Myo1e, which are disrupted by the T119I and A159P mutations in humans with FSGS
Summary
Missense mutations in the human MYO1E gene, encoding molecular motor protein myosin 1e (Myo1e), are associated with kidney disease (Al-Hamed et al, 2013; Mele et al, 2011; SannaCherchi et al, 2011). These mutations have been predicted to disrupt Myo1e activity, testing this prediction directly in a vertebrate model organism is challenging, owing to the need for complex genetic modifications and because of the limitations of microscopic and biochemical characterization in such a model.
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