TY - JOUR
T1 - 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
AU - Li, Cunxi
AU - Hao, Mingming
AU - Cao, Zheng
AU - Ding, Wei
AU - Graves-Deal, Ramona
AU - Hu, Jianyong
AU - Piston, David W.
AU - Coffey, Robert J.
PY - 2007/8
Y1 - 2007/8
N2 - Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α 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-α and that TGF-α 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 μ1B 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-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.
AB - Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α 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-α and that TGF-α 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 μ1B 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-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α-containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.
UR - http://www.scopus.com/inward/record.url?scp=34547743772&partnerID=8YFLogxK
U2 - 10.1091/mbc.E07-02-0172
DO - 10.1091/mbc.E07-02-0172
M3 - Article
C2 - 17553928
AN - SCOPUS:34547743772
SN - 1059-1524
VL - 18
SP - 3081
EP - 3093
JO - Molecular biology of the cell
JF - Molecular biology of the cell
IS - 8
ER -