vesicle concatenation in plant endosomes

Vesicle trafficking pathways are highly regulated processes that require specific machineries to select vesicle cargo, deform and fission membranes, and mediate the transport and fusion of the resulting vesicle with the acceptor compartment. The initial sorting and clustering of cargo macromolecules in a vesicle require their concentration in a small region of the donor membrane. In the case of clathrin-mediated endocytosis and the anterograde and retrograde trafficking between the endoplasmic reticulum and Golgi via COP-II and COP-I vesicles, respectively, the cargo molecules directly or through adapters recruit a protein coat that aids in the local deformation of the membrane into a bud (Traub and Bonifacino, 2013; Gomez-Navarro and Miller, 2016). The cargo remains physically associated with these coats after the vesicle is free in the cytoplasm and until the coat disassembles, greatly limiting the possibility of cargo escape back to the donor membrane by diffusion during vesicle formation. The degradation of plasma membrane proteins depends on different membrane vesiculation events along the endocytic and endosomal pathways. At the plasma membrane, linear amino acid motifs, conformational motifs, and/or posttranslational modifications such as phosphorylation and ubiquitination of plasma membrane proteins are recognized by adapter and accessory proteins (Traub and Bonifacino, 2013) that also facilitate the assembly of the clathrin coat and the formation of endocytic vesicles. Once released from the plasma membrane, clathrin-coated vesicles lose their coats and fuse with endosomes. At endosomes, the endocytosed cargo proteins can be recycled back to the plasma membrane or internalized into endosomal intralumenal vesicles (ILVs) for their degradation upon the fusion of multivesicular endosomes (MVEs) with vacuoles/lysosomes (Henne et al., 2011). The major sorting signal for endosomal-mediated vesiculation is ubiquitin (MacDonald et al., 2012). Ubiquitinated cargo proteins on the endosomal limiting membrane are recognized and sorted into ILVs by endosomal sorting complex required for transport (ESC RT) proteins (Schuh and Audhya, 2014; Paez Valencia et al., 2016). The ESC RT machinery mediates negative membrane bending (away from the cytoplasm), leading to a vesiculation process in the reverse topology to most membranedeformation events, including clathrin-coated endocytosis. In fungi and metazoans, five multimeric ESC RT complexes have been identified (ESC RT-0, -I, -II, and -III and VPS4-VTA1; Henne et 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. ESC RT-mediated vesicle concatenation in plant endosomes