EHBP 1 L 1 coordinates Rab 8 and Bin 1 to regulate apical-directed transport in polarized epithelial cells

In polarized epithelial cells, the transport pathway is directed to the apical or basolateral plasma membrane, which differ in protein and lipid composition (Rodriguez-Boulan et al., 2005). Several findings suggest that newly synthesized protein exported from the TGN is delivered to the endocytic recycling compartment (ERC), which is regarded as a recycling endosome, and sorted to the apical or basolateral plasma membrane (Ang et al., 2004; Thuenauer et al., 2014). Rab GTPases belong to the Ras small GTPase superfamily (Wennerberg et al., 2005). More than 60 mammalian Rab proteins define vesicle and organelle identity by recruiting various binding proteins to the membrane. The Rab protein acts upstream of SNA RE-mediated fusion to the target membrane (Barr, 2013). Rab8 is a highly conserved small GTPase in eukaryotic cells and regulates exocytic transport to a polarized plasma membrane (Peränen, 2011). The mammalian genome encodes two Rab8 isoforms: Rab8a and Rab8b. Small intestine cells in both Rab8a knockout (KO) and Rab8a/8b double-knockout (DKO) mice show accumulated apical cargo proteins in lysosomes, which suggests that Rab8 is involved in apical transport (Sato et al., 2007, 2014). Previous studies provide insight into the molecular mechanisms related to Rab8. In Saccharomyces cerevisiae, the Rab8 homolog Sec4 plays a role in exocytic vesicle motility with Myo2, an actin-based motor protein, and mediates vesicle docking to a polarized plasma membrane by binding to a tethering complex, the exocyst (Guo et al., 1999; He and Guo, 2009; Jin et al., 2011). Importantly, small intestines from microvillus inclusion disease patients with a gene mutation for Myo5B, a mammalian homolog of yeast Myo2, exhibit short microvilli and microvillus inclusion bodies, which are also observed in Rab8 KO mouse intestine cells (Sato et al., 2007; Ruemmele et al., 2010). Despite its role in exocytic vesicle motility and tethering, Rab8 is mainly localized to the ERC in mammals and Caenorhabditis elegans, indicating unknown functions of Rab8 at the ERC (Hattula et al., 2006; Sharma et al., 2009; Shi et al., 2010). Emerging evidence suggests that Rab proteins may coordinate with Bin/Amphiphysin/Rvs (BAR) domain proteins, which are membrane curvature sensing/generating proteins on the ERC (Pant et al., 2009; Giridharan et al., 2013). In this study, we identified a novel Rab8-binding protein complex, EHBP1L1–Bin1–dynamin, and demonstrated its role in apical transport. Our results suggest that this complex localizes to the ERC and functions in the generation of vesicles/tubules containing apical cargo proteins. In small intestine The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1or Bin1-depleted or dynamininhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport. EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells