The endocytic recycling compartment maintains segregation of cargo after sorting in peripheral endosomes, and cargo is recycled by distinct pathways to the plasma membrane. In addition, tubular recycling endosomes can be generated from sorting endosomes and preferentially traffic clathrin-independent cargo.
Background: Vesiculation of tubular recycling endosomes is essential for the recycling of receptors and lipids to the plasma membrane. Results: A novel vesiculation assay was used to demonstrate a role for endocytic regulatory proteins in vesiculation. Conclusion: EHD family proteins play significant roles in both the vesiculation and generation of tubular recycling endosomes. Significance: This provides the first direct evidence of differential EHD function in vesiculation and tubulation.
Mitochondria play essential roles in cellular energy processes, including ATP production, control of reactive oxygen species (ROS) and apoptosis. While mitochondrial function is regulated by the dynamics of fusion and fission, mitochondrial homeostasis remains incompletely understood. Recent studies implicate dynamin-2 and dynamin-related protein-1 (Drp1, also known as DNM1L), as GTPases involved in mitochondrial fission. Here, we identify the ATPase and endocytic protein EHD1 as a novel regulator of mitochondrial fission. EHD1 depletion induces a static and elongated network of mitochondria in the cell. However, unlike dynamin-2 and Drp1, whose depletion protects cells from staurosporine-induced mitochondrial fragmentation, EHD1-depleted cells remain sensitive to staurosporine, suggesting a different mechanism for EHD1 function. Recent studies have demonstrated that VPS35 and the retromer complex influence mitochondrial homeostasis either by Mul1-mediated ubiquitylation and degradation of the fusion protein Mfn2, or by removal of inactive Drp1 from the mitochondrial membrane. We demonstrate that EHD1 and its interaction partner rabankyrin-5 interact with the retromer complex to influence mitochondrial dynamics, likely by inducing VPS35-mediated removal of inactive Drp1 from mitochondrial membranes. Our study sheds light on mitochondrial dynamics, expanding a new paradigm of endocytic protein regulation of mitochondrial homeostasis.
Dynamic trafficking of G protein-coupled receptors (GPCRs) out of cilia is mediated by the BBSome. In concert with its membrane recruitment factor, the small GTPase ARL6/BBS3, the BBSome ferries GPCRs across the transition zone, a diffusion barrier at the base of cilia. Here, we present the near-atomic structures of the BBSome by itself and in complex with ARL6GTP, and we describe the changes in BBSome conformation induced by ARL6GTP binding. Modeling the interactions of the BBSome with membranes and the GPCR Smoothened (SMO) reveals that SMO, and likely also other GPCR cargoes, must release their amphipathic helix 8 from the membrane to be recognized by the BBSome.
An elaborate network of dynamic lipid membranes, termed tubular recycling endosomes (TRE), coordinates the process of endocytic recycling in mammalian cells. The C-terminal Eps15 homology domain (EHD)-containing proteins have been implicated in the bending and fission of TRE, thus regulating endocytic recycling. EHD proteins have an EH domain that interacts with proteins containing an NPF motif. We found that NPFcontaining EHD1 interaction partners such as molecules interacting with CasL-like1 (MICAL-L1) and Syndapin2 are essential for TRE biogenesis. Also crucial for TRE biogenesis is the generation of phosphatidic acid, an essential lipid component of TRE that serves as a docking point for MICAL-L1 and Syndapin2. EHD1 and EHD3 have 86% amino acid identity; they homo-and heterodimerize and partially co-localize to TRE. Despite their remarkable identity, they have distinct mechanistic functions. EHD1 induces membrane vesiculation, whereas EHD3 supports TRE biogenesis and/or stabilization by an unknown mechanism. While using phospholipase D inhibitors (which block the conversion of glycerophospholipids to phosphatidic acid) to deplete cellular TRE, we observed that, upon inhibitor washout, there was a rapid and dramatic regeneration of MICAL-L1-marked TRE. Using this "synchronized" TRE biogenesis system, we determined that EHD3 is involved in the stabilization of TRE rather than in their biogenesis. Moreover, we identify the residues Ala-519/Asp-520 of EHD1 and Asn-519/ Glu-520 of EHD3 as defining the selectivity of these two paralogs for NPF-containing binding partners, and we present a model to explain the atomic mechanism and provide new insight for their differential roles in vesiculation and tubulation, respectively.
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