Coordination between ESCRT function and Rab conversion during endosome maturation

The endosomal pathway is essential for regulating cell signaling and cellular homeostasis. Rab5-positive early endosomes receive proteins from the plasma membrane. Dependent on a ubiquitin mark on the protein, they will be either recycled or sorted into intraluminal vesicles (ILVs) by endosomal sorting complex required for transport (ESCRT) proteins. During endosome maturation Rab5 is replaced by Rab7 on endosomes that are able to fuse with lysosomes to form endolysosomes. However, whether ESCRT-driven ILV formation and Rab5-to-Rab7 conversion are coordinated remains unknown. Here we show that loss of early ESCRTs led to enlarged Rab5-positive endosomes and prohibited Rab conversion. Reduction of ubiquitinated cargo alleviated this phenotype. Moreover, ubiquitinated proteins on the endosomal limiting membrane prevented the displacement of the Rab5 guanine nucleotide exchange factor (GEF) RABX-5 by the GEF for Rab7, SAND-1/CCZ-1. Overexpression of Rab7 could partially overcome this block, even in the absence of SAND-1 or CCZ1, suggesting the presence of a second Rab7 GEF. Our data reveal a hierarchy of events in which cargo corralling by ESCRTs is upstream of Rab conversion, suggesting that ESCRT-0 and ubiquitinated cargo could act as timers that determine the onset of Rab conversion.

内体通路（endosomal pathway）对于调控细胞信号转导与细胞稳态至关重要。Rab5阳性早期内体（Rab5-positive early endosomes）接收来自细胞质膜（plasma membrane）的蛋白质。根据蛋白质上的泛素标记（ubiquitin mark），这些蛋白质要么被循环转运，要么被转运必需内体分选复合物（endosomal sorting complex required for transport, ESCRT）分选进入管腔内囊泡（intraluminal vesicles, ILVs）。在内体成熟过程中，可与溶酶体融合形成内溶酶体的内体表面的Rab5会被Rab7替换。然而，ESCRT介导的ILV形成与Rab5向Rab7的转换是否存在协同调控，目前仍未明确。本研究显示，早期ESCRT复合物的缺失会导致Rab5阳性内体体积增大，并阻断Rab转换过程。减少泛素化转运货物（ubiquitinated cargo）可缓解该表型。此外，内体限制膜上的泛素化蛋白质会阻碍Rab7鸟苷酸交换因子（guanine nucleotide exchange factor, GEF）SAND-1/CCZ-1对Rab5鸟苷酸交换因子RABX-5的置换。即使在缺失SAND-1或CCZ1的情况下，过表达Rab7也可部分克服这一阻滞，这表明存在第二种Rab7鸟苷酸交换因子。我们的研究结果揭示了一套事件层级：由ESCRT介导的转运货物聚集过程发生于Rab转换的上游，这提示ESCRT-0与泛素化转运货物可作为调控Rab转换起始的计时器。