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.