Collapsin Response Mediator Protein-2 (Crmp2) Regulates Trafficking by Linking Endocytic Regulatory Proteins to Dynein Motors

Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are important regulatory proteins that control key endocytic transport steps. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. Here we have identified the collapsin response mediator protein-2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells have remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin (Tf) from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, Tf relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein-based transport upon depletion of Crmp2, MICAL-L1, or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose overexpression inhibits dynein activity, prevented the relocalization of internalized Tf to the ERC upon depletion of Crmp2, MICAL-L1, or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1·EHD1-based vesicular transport to dynein motors.     

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients, lipids and receptors is a crucial process for all eukaryotic cells. Accordingly, endocytosis is highly conserved across cell types and species. Once internalized, small cargo-containing vesicles fuse with early endosomes (also known as sorting endosomes), where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway. Although the mechanisms that regulate this sorting are still poorly understood, some receptors are directed to late endosomes and lysosomes for degradation, whereas other receptors are recycled back to the plasma membrane; either directly or through recycling endosomes. The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways. Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membrane-associated proteins, as a consequence of the activity of multiple specific GTPase-activating proteins (GAPs) and GTP exchange factors (GEFs). Once bound to GTP, Rabs interact with a multitude of effector proteins that carry out Rab-specific functions. Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology (EHD) proteins, which are also intimately involved in endocytic regulation. A particularly interesting example of common Rab-EHD interaction partners is the MICAL-like protein, MICAL-L1. MICAL-L1 and its homolog, MICAL-L2, belong to the larger MICAL family of proteins, and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins, as well as controlling cytoskeletal rearrangement and neurite outgrowth. In this review, we summarize the functional roles of MICAL and Rab proteins, and focus on the significance of their interactions and the implications for endocytic transport.     

Interactions between EHD proteins and Rab11-FIP2: a role for EHD3 in early endosomal transport

Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport.     

MICAL-L1 is a tubular endosomal membrane hub that connects Rab35 and Arf6 with Rab8a

Endocytosis is a conserved process across species in which cell surface receptors and lipids are internalized from the plasma membrane. Once internalized, receptors can either be degraded or be recycled back to the plasma membrane. A variety of small GTP-binding proteins regulate receptor recycling. Despite our familiarity with many of the key regulatory proteins involved in this process, our understanding of the mode by which these proteins co-operate and the sequential manner in which they function remains limited. In this study, we identify two GTP-binding proteins as interaction partners of the endocytic regulatory protein molecule interacting with casl-like protein 1 (MICAL)-L1. First, we demonstrate that Rab35 is a MICAL-L1-binding partner in vivo. Over-expression of active Rab35 impairs the recruitment of MICAL-L1 to tubular recycling endosomes, whereas Rab35 depletion promotes enhanced MICAL-L1 localization to these structures. Moreover, we demonstrate that Arf6 forms a complex with MICAL-L1 and plays a role in its recruitment to tubular endosomes. Overall, our data suggest a model in which Rab35 is a critical upstream regulator of MICAL-L1 and Arf6, while both MICAL-L1 and Arf6 regulate Rab8a function.     

EHD1 and Eps15 interact with phosphatidylinositols via their Eps15 homology domains

The C-terminal Eps15 homology domain-containing protein, EHD1, regulates the recycling of receptors from the endocytic recycling compartment to the plasma membrane. In cells, EHD1 localizes to tubular and spherical recycling endosomes. To date, the mode by which EHD1 associates with endosomal membranes remains unknown, and it has not been determined whether this interaction is direct or via interacting proteins. Here, we provide evidence demonstrating that EHD1 has the ability to bind directly and preferentially to an array of phospholipids, preferring phosphatidylinositols with a phosphate at position 3. Previous studies have demonstrated that EH domains coordinate calcium binding and interact with proteins containing the tripeptide asparagine-proline-phenylalanine (NPF). Using two-dimensional nuclear magnetic resonance analysis, we now describe a new function for the Eps15 homology (EH) domain of EHD1 and show that it is capable of directly binding phosphatidylinositol moieties. Moreover, we have expanded our studies to include the C-terminal EH domain of EHD4 and the second of the three N-terminal EH domains of Eps15 and demonstrated that phosphatidylinositol binding may be a more general property shared by certain other EH domains. Further studies identified a positively charged lysine residue (Lys-483) localized within the third helix of the EH domain, on the opposite face of the NPF-binding pocket, as being critical for the interaction with the phosphatidylinositols.     

EHD1 regulates cholesterol homeostasis and lipid droplet storage

Endocytic transport is critical for the subcellular distribution of free cholesterol and the endocytic recycling compartment (ERC) is an important organelle that stores cholesterol and regulates its trafficking. The C-terminal EHD protein, EHD1, controls receptor recycling through the ERC and affects free cholesterol distribution in the cell. We utilized embryonic fibroblasts from EHD1 knockout mice (Ehd1(-/-)MEF) and SiRNA in normal MEF cells to assess the role of EHD1 in intracellular transport of cholesterol. Surprisingly, Ehd1(-/-)MEFs displayed reduced levels of esterified and free cholesterol, which returned to normal level upon re-introduction of wild-type, but not dysfunctional EHD1. Moreover, triglyceride and cholesterol storage organelles known as 'lipid droplets' were smaller in size in cells lacking EHD1, indicating that less esterified cholesterol and triglycerides were being stored. Decreased cellular cholesterol and reduced lipid droplet size in Ehd1(-/-)MEFs correlated with ineffectual cholesterol uptake via LDL receptor, suggesting involvement of EHD1 in LDL receptor internalization.     

MICAL-L1 Links EHD1 to Tubular Recycling Endosomes and Regulates Receptor Recycling

Endocytic recycling of receptors and lipids occurs via a complex network of tubular and vesicular membranes. EHD1 is a key regulator of endocytosis and associates with tubular membranes to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with preexisting tubules in vivo. How EHD1 is recruited to these tubular endosomes remains unclear. We have determined that the Rab8-interacting protein, MICAL-L1, associates with EHD1, with both proteins colocalizing to long tubular membranes, in vitro and in live cells. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs, sequences that typically interact with EH-domains. Our data show that the MICAL-L1 C-terminal coiled-coil region is necessary and sufficient for its localization to tubular membranes. Moreover, we provide unexpected evidence that endogenous MICAL-L1 can link both EHD1 and Rab8a to these structures, as its depletion leads to loss of the EHD1-Rab8a interaction and the absence of both of these proteins from the membrane tubules. Finally, we demonstrate that MICAL-L1 is essential for efficient endocytic recycling. These data implicate MICAL-L1 as an unusual type of Rab effector that regulates endocytic recycling by recruiting and linking EHD1 and Rab8a on membrane tubules.     

Common and distinct roles for the binding partners Rabenosyn-5 and Vps45 in the regulation of endocytic trafficking in mammalian cells

In several invertebrate organisms, the Sec1p/Munc18-like protein Vps45 interacts with the divalent Rab4/Rab5 effector, Rabenosyn-5 and carries out multiple functions in the endocytic/secretory pathways. In mammalian cells, Vps45 and Rabenosyn-5 also interact, but the molecular characterization of this binding, and the functional relationship between these two proteins has not been well defined. Here we identify a novel sequence within Rabenosyn-5 required for its interaction with Vps45. We demonstrate that hVps45-depletion decreases expression of Rabenosyn-5, likely resulting from Rabenosyn-5 degradation through the proteasomal pathway. Furthermore, we demonstrate that similar to Rabenosyn-5-depletion, hVps45-depletion causes impaired recycling of β1 integrins, and a subsequent delay in human fibroblast cell migration on fibronectin-coated plates. Moreover, β1 integrin recycling could be rescued by reintroduction of siRNA-resistant wild-type Rabenosyn-5, but not a mutant deficient in Vps45 binding. However, unlike Rabenosyn-5-depletion, which induces Golgi fragmentation and decreased recruitment of sorting nexin retromer subunits to the Golgi, hVps45-depletion induces Golgi condensation and accumulation of retromer subunits in the vicinity of the Golgi. In part, these phenomena could be attributed to reduced Syntaxin16 expression and altered localization of both Syntaxin16 and Syntaxin6 upon Vps45-depletion. Overall, these findings implicate hVps45 and Rabenosyn-5 in post early endosome transport, and we propose that their interaction serves as a nexus to promote bidirectional transport along the endosome-to-recycling compartment and endosome-to-Golgi axes.