Steroid-Resistant Nephrotic Syndrome-Associated <i>MYO1E</i> Mutations Have Differential Effects on Myosin 1e Localization, Dynamics, and Activity.

Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in <i>MYO1E</i> are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the <i>MYO1E</i> variants identified by genomic sequencing have not been functionally characterized. Here, we set out to analyze two mutations in the Myo1e motor domain, T119I and D388H, which were selected on the basis of protein sequence conservation. EGFP-tagged human <i>Myo1e</i> constructs were delivered into the Myo1e-KO mouse podocyte-derived cells <i>via</i> adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated <i>Myo1e</i> constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity <i>in vitro</i>. Both mutants were expressed as full-length proteins in the Myo1e-KO cells. However, unlike wild-type (WT) Myo1e, the T119I variant was not enriched at the cell junctions or clathrin-coated vesicles (CCVs). In contrast, D388H variant localization was similar to that of WT. The rate of dissociation of the D388H variant from cell-cell junctions and CCVs was decreased, suggesting this mutation affects Myo1e interactions with binding partners. ATPase activity and ability to translocate actin filaments were drastically reduced for the D388H mutant, supporting findings from cell-based experiments. T119I and D388H mutations are deleterious to Myo1e functions. The experimental approaches used in this study can be applied to future characterization of novel <i>MYO1E</i> variants associated with SRNS.