| Recent studies have identified the C-terminal Eps15 homology domain (EHD) - containing proteins as critical regulators of endocytic trafficking. Four highly homologous C-terminal EHDs (EHD1-EHD4) have been identified in mammals. Thus far EHD4, the most divergent C-terminal EHD protein, has been poorly characterized. We demonstrate here that endogenous EHD4 localizes to Rab5- and EEA1-containing endosomes. Knock-down of EHD4 expression leads to the generation of enlarged early endosomal (EE) structures that are enriched in levels of the activated GTP-bound Rab5. Moreover, loss of EHD4 impairs the trafficking of the transfen-in receptor and MHC class I molecule to the endocytic recycling compartment (ERC). Also, cargo destined for degradation, such as internalized low density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Finally, we found that endogenous E HD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis.;Most receptors are sorted from the EHD4-regulated E E to the ERC from where they recycle back to the PM. Receptor recycling occurs via a complex network of tubular and vesicular membranes. EHD1, the best characterized EHD protein, associates with tubular membranes emanating from the ERC to facilitate recycling. Although EHD proteins tubulate membranes in vitro, EHD1 primarily associates with pre-existing tubules in vivo. Accordingly, the mechanism regulating EHD1 association with tubules remains unclear. We have determined that the Raba-interacting protein, MICAL-L1, associates with EHD1, with both proteins co-localizing to long tubular membranes. MICAL-L1 is a largely uncharacterized member of the MICAL-family of proteins that uniquely contains two asparagine-proline-phenylalanine motifs. 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 links 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. |
