No abstract

The sequential action of the Vps27/HRS complex, ESCRT-I, -II, and -III is required to sort ubiquitinated transmembrane proteins to the lumen of lysosomes via the multivesicular body (MVB) pathway. While Vps27/HRS, ESCRT-I, and -II are recruited to endosomes as preformed complexes, the ESCRT-III subunits Vps20, Snf7, Vps24, and Vps2 only assemble into a complex on endosomes. We have addressed the pathway and the regulation for ESCRT-III assembly. Our findings indicate the ordered assembly of a transient 450 kDa ESCRT-III complex on endosomes. Despite biochemical and structural similarity, each subunit contributes a specific function. Vps20 nucleates transient oligomerization of Snf7, which appears to sequester MVB cargo. Vps24 terminates Snf7 oligomerization by recruiting Vps2, which subsequently engages the AAA-ATPase Vps4 to dissociate ESCRT-III. We propose that the ordered assembly and disassembly of ESCRT-III delineates an MVB sorting domain to sequester cargo and complete the last steps of MVB sorting.

Summary Receptor down-regulation in the MVB pathway is mediated by the ESCRT complexes. ESCRT-III is composed of four protein subunits that are monomeric in the cytosol and oligomerize into a protein lattice only upon membrane binding. Recent studies have shown that the ESCRT-III protein Snf7 can form a filament by undergoing homo-oligomerization. To examine the role of membrane binding and of interactions with other ESCRT components in initiating Snf7 oligomerization, we used fluorescence spectroscopy to directly detect and characterize the assembly of the Snf7 oligomer on liposomes using purified ESCRT components. The observed fluorescence changes reveal an obligatory sequence of membrane-protein and protein-protein interactions that generate the active conformation of Snf7. Also, we demonstrate that ESCRT-III assembly drives membrane deformation. Furthermore, using an in vitro disassembly assay, we directly demonstrate that Vps24 and Vps2 function as adaptors in the ATP-dependent membrane disassembly of the ESCRT-III complex by recruiting the AAA ATPase Vps4.

Eukaryotic cells use the extracellular signal regulated kinase (ERK) cascade to connect cell-surface receptors to intracellular targets. Although various signals are routed through the ERK pathway, cells respond accordingly to a given stimulus. To regulate proper signal transduction, scaffolds and adaptors are employed to organize specific signaling units. The scaffold protein MP1 (MEK1 partner) assembles a scaffold complex in the ERK cascade. We show that p14 functions as an adaptor protein, which is required and sufficient to localize MP1 to endosomes. Reduction of MP1 or p14 protein levels by siRNAi results in defective signal transduction. Therefore, our results suggest that the endosomal localization of the p14/MP1-MAPK scaffold complex is crucial for signal transduction.

SummaryThe endosomal sorting complexes required for transport (ESCRT) assemble into a multisubunit machinery that performs a topologically unique membrane bending and scission reaction away from the cytoplasm. This evolutionarily highly conserved process is required for the multivesicular body (MVB) pathway, cytokinesis and HIV budding. The modular setup of the machinery with five distinct ESCRT complexes (ESCRT-0, -I, -II, -III and the Vps4 complex) that have a clear division of tasks — from interaction with ubiquitinated membrane proteins to membrane deformation and abscission — allows them to be flexibly integrated into these three very different biological processes (Figure 1).

Axon growth across the Drosophila midline requires Comm to downregulate Robo, the receptor for the midline repellent Slit. We show here that comm is required in neurons, not in midline cells as previously thought, and that it is expressed specifically and transiently in commissural neurons. Comm acts as a sorting receptor for Robo, diverting it from the synthetic to the late endocytic pathway. A conserved cytoplasmic LPSY motif is required for endosomal sorting of Comm in vitro and for Comm to downregulate Robo and promote midline crossing in vivo. Axon traffic at the CNS midline is thus controlled by the intracellular trafficking of the Robo guidance receptor, which in turn depends on the precisely regulated expression of the Comm sorting receptor.

The extracellular signal-regulated kinase (ERK) cascade regulates proliferation, differentiation, and survival in multicellular organisms. Scaffold proteins regulate intracellular signaling by providing critical spatial and temporal specificity. The scaffold protein MEK1 (mitogen-activated protein kinase and ERK kinase 1) partner (MP1) is localized to late endosomes by the adaptor protein p14. Using conditional gene disruption of p14 in mice, we now demonstrate that the p14–MP1-MEK1 signaling complex regulates late endosomal traffic and cellular proliferation. This function its essential for early embryogenesis and during tissue homeostasis, as revealed by epidermis-specific deletion of p14. These findings show that endosomal p14–MP1-MEK1 signaling has a specific and essential function in vivo and, therefore, indicate that regulation of late endosomal traffic by extracellular signals is required to maintain tissue homeostasis.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3–45 s lifetimes, the ESCRT-III assemblies accumulated 75–200 Snf7 and 15–50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.