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

This Article Full Text Full Text (PDF) Supplemental Material All Versions of this Article: E07-02-0172v1 18/8/3081    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Li, C. Articles by Coffey, R. J. Search for Related Content PubMed PubMed Citation Articles by Li, C. Articles by Coffey, R. J.

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.

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				Z. Cao, C. Li, J. N. Higginbotham, J. L. Franklin, D. L. Tabb, R. Graves-Deal, S. Hill, K. Cheek, W. G. Jerome, L. A. Lapierre, et al. Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis Mol. Cell. Proteomics, September 1, 2008; 7(9): 1651 - 1667. [Abstract] [Full Text] [PDF]