Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-α-containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells

Transforming growth factor-a (TGF-alpha) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-a is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-a and that TGF-a is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of mu 1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-a. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-a and increased cytosolic TGF-a immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-alpha-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.