Ubiquitinated plasma membrane proteins (cargo) are delivered to endosomes and sorted by endosomal sorting complex required for transport (ESC RT) machinery into endosome intralumenal vesicles (ILVs) for degradation. In contrast to the current model that postulates that ILVs form individually from inward budding of the endosomal limiting membrane, plant ILVs form as networks of concatenated vesicle buds by a novel vesiculation mechanism. We ran computational simulations based on experimentally derived diffusion coefficients of an ESC RT cargo protein and electron tomograms of Arabidopsis thaliana endosomes to measure cargo escape from budding ILVs. We found that 50% of the ESC RT cargo would escape from a single budding profile in 5-20 ms and from three concatenated ILVs in 80-200 ms. These short cargo escape times predict the need for strong diffusion barriers in ILVs. Consistent with a potential role as a diffusion barrier, we find that the ESC RT-III protein SNF7 remains associated with ILVs and is delivered to the vacuole for degradation.
