Myo1e is a nonmuscle motor protein enriched in podocytes. Mutations in MYO1E are associated with steroid-resistant nephrotic syndrome (SRNS). Most of the MYO1E 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 Myo1e constructs were delivered into the Myo1e-KO mouse podocyte–derived cells via adenoviral infection to analyze Myo1e protein stability, Myo1e localization, and clathrin-dependent endocytosis, which is known to involve Myo1e activity. Furthermore, truncated Myo1e constructs were expressed using the baculovirus expression system and used to measure Myo1e ATPase and motor activity in vitro.

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 MYO1E variants associated with SRNS.

Myo1e 是一种富含足细胞的非肌肉运动蛋白。MYO1E突变与类固醇抵抗性肾病综合征 (SRNS) 相关。大多数通过基因组测序鉴定的MYO1E变体尚未进行功能表征。在这里，我们着手分析 Myo1e 运动结构域中的两个突变 T119I 和 D388H，它们是根据蛋白质序列保守性选择的。

通过腺病毒感染将EGFP 标记的人Myo1e构建体递送至 Myo1e-KO 小鼠足细胞衍生细胞中，以分析 Myo1e 蛋白稳定性、Myo1e 定位和网格蛋白依赖性内吞作用（已知与 Myo1e 活性有关）。此外，使用杆状病毒表达系统表达截短的Myo1e构建体，并用于测量 Myo1e ATP 酶和体外运动活性。

两种突变体在 Myo1e-KO 细胞中均表达为全长蛋白。然而，与野生型 (WT) Myo1e 不同，T119I 变体在细胞连接处或网格蛋白包被的囊泡 (CCV) 处并未富集。相比之下，D388H 变异定位与 WT 相似。D388H 变体从细胞-细胞连接和 CCV 的解离率降低，表明该突变影响 Myo1e 与结合伴侣的相互作用。D388H 突变体的 ATP 酶活性和易位肌动蛋白丝的能力大大降低，支持了基于细胞的实验的结果。

T119I 和 D388H 突变对 Myo1e 功能有害。本研究中使用的实验方法可应用于未来与 SRNS 相关的新型MYO1E变体的表征。