Rho GTPases play a key role in various membrane trafficking processes. RhoU is an atypical small Rho GTPase related to Rac/Cdc42, which possesses unique N- and C-terminal domains that regulate its function and its subcellular localization. RhoU localizes at the plasma membrane, on endosomes and in cell adhesion structures where it governs cell signaling, differentiation and migration. However, despite its endomembrane localization, RhoU function in vesicular trafficking has been unexplored. Here, we identified intersectins (ITSNs) as new binding partners for RhoU and showed that the second PxxP motif at the N terminus of RhoU mediated interactions with the SH3 domains of ITSNs. To evaluate the function of RhoU and ITSNs in vesicular trafficking, we used fluorescent transferrin as a cargo for uptake experiments. We showed that silencing of either RhoU or ITSN2, but not ITSN1, increased transferrin accumulation in early endosomes, resulting from a defect in fast vesicle recycling. Concomitantly, RhoU and ITSN2 colocalized to a subset of Rab4-positive vesicles, suggesting that a RhoU–ITSN2 interaction may occur on fast recycling endosomes to regulate the fate of vesicular cargos.

Rho GTPases 在各种膜运输过程中发挥着关键作用。RhoU 是一种与 Rac/Cdc42 相关的非典型小 Rho GTPase，具有独特的 N 端和 C 端结构域，可调节其功能和亚细胞定位。RhoU 定位于质膜、核内体和细胞粘附结构，控制细胞信号传导、分化和迁移。然而，尽管其在内膜定位，RhoU 在囊泡运输中的功能尚未被探索。在这里，我们确定了交叉蛋白（ITSN）作为 RhoU 的新结合伴侣，并表明 RhoU N 末端的第二个 PxxP 基序介导与 ITSN 的 SH3 结构域的相互作用。为了评估 RhoU 和 ITSN 在囊泡运输中的功能，我们使用荧光转铁蛋白作为摄取实验的货物。我们发现，RhoU 或 ITSN2 的沉默（而非 ITSN1）会增加早期内体中转铁蛋白的积累，这是由于快速囊泡回收的缺陷造成的。同时，RhoU 和 ITSN2 共定位于 Rab4 阳性囊泡的子集，表明 RhoU-ITSN2 相互作用可能发生在快速回收的内体上，以调节囊泡货物的命运。