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Vol. 18, Issue 8, 3081-3093, August 2007

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Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

Cunxi Li^*, Mingming Hao, Zheng Cao^*, Wei Ding^*, Ramona Graves-Deal^*, Jianyong Hu^*, David W. Piston, and Robert J. Coffey^*,

Departments of *Medicine and Cell and Developmental Biology and Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, TN 37232; and Department of Veterans Affairs Medical Center, Nashville, TN 37232-2279

Submitted February 26, 2007; Revised May 18, 2007; Accepted May 24, 2007
Monitoring Editor: Jennifer Lippincott-Schwartz

Transforming growth factor- (TGF-) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF- 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- and that TGF- 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-. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF- and increased cytosolic TGF- immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Robert J. Coffey (robert.coffey{at}vanderbilt.edu).

Abbreviations used: TGF, transforming growth factor-; HA, hemagglutinin epitope tag; MDCK, Madin-Darby canine kidney; LLC-PK1, Lilly Laboratories Culture-Pig Kidney type1; EGF, epidermal growth factor; PDZ, PSD-95/SAP90, Discs large and Zona Occludens-1; G2A, 2nd residue glycine replaced by alanine; TGN, trans-Golgi network; LDLR, low-density lipoprotein receptor; VSV-G, vesicular stomatitis virus-glycoprotein.