Biochemistry section, Department of Biology/Chemistry, Osnabrück University, 49076 Osnabrück, Germany.
Zoology and Developmental Biology section, Department of Biology/Chemistry, Osnabrück University, 49076 Osnabrück, Germany.
Proc Natl Acad Sci U S A. 2023 Jul 25;120(30):e2303750120. doi: 10.1073/pnas.2303750120. Epub 2023 Jul 18.
Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-GTPase exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here, we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro and alterations in early endosomal structures in nephrocytes. A shift from Rab5 to more Rab7-positive structures in yeast suggests faster endosomal maturation. Using modeling, we further identify a conserved Rab5-binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Ras-related in brain (Rab) conversion and organelle maturation along the endomembrane system.
从早期到晚期内涵体的成熟依赖于它们的标记蛋白 Rab5 到 Rab7 的交换。这需要 Rab7 通过其特异性鸟嘌呤核苷酸交换因子（GEF） Mon1-Ccz1 激活。该复合物在膜上的有效 GEF 活性依赖于 Rab5，从而驱动内涵体上 Rab-GTPase 的交换。然而，Rab5 在 Mon1-Ccz1 激活中的作用的分子细节尚不清楚。在这里，我们确定了 Mon1 中参与 GEF 调节的关键特征。我们表明，Mon1 的固有无序 N 端结构域自动抑制膜上 Rab5 依赖性 GEF 活性。因此，Mon1 的截断导致体外 GEF 活性更高，并且在肾细胞中早期内涵体结构发生改变。在酵母中从 Rab5 到更多 Rab7 阳性结构的转变表明内涵体成熟更快。通过建模，我们进一步确定了 Mon1 中保守的 Rab5 结合位点。破坏 Rab5 相互作用的突变导致膜上的 GEF 活性差和体内生长缺陷。我们的分析为理解 Ras 相关 in brain (Rab) 转换和沿内体系统的细胞器成熟的机制提供了一个框架。
