Naked2 acts as a cargo recognition and targeting protein to ensure proper delivery and fusion of TGF-alpha containing exocytic vesicles at the lower lateral membrane of polarized MDCK cells.
Transforming growth factor-alpha (TGF-alpha) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-alpha 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-alpha and that TGF-alpha 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 mu1B 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-alpha. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-alpha and increased cytosolic TGF-alpha 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.
Li
Cunxi
C
Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA.
Hao
Mingming
M
Cao
Zheng
Z
Ding
Wei
W
Graves-Deal
Ramona
R
Hu
Jianyong
J
Piston
David W
DW
Coffey
Robert J
RJ
D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D016764:Cell Polarity / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004285:Dogs; D005089:Exocytosis / Q000187:drug effects; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects; D008856:Microscopy, Fluorescence; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D013552:Swine; D013634:Tannins / Q000494:pharmacology; D016211:Transforming Growth Factor alpha / Q000378:metabolism*; D022161:Transport Vesicles / Q000187:drug effects / Q000378:metabolism*
D033961:Adaptor Protein Complex 1; D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C449092:NKD2 protein, human; D034741:RNA, Small Interfering; D013634:Tannins; D016211:Transforming Growth Factor alpha