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This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: M700155-MCP200v1 7/9/1651    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Google Scholar Articles by Cao, Z. Articles by Coffey, R. J. PubMed PubMed Citation Articles by Cao, Z. Articles by Coffey, R. J. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 7:1651-1667, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Use of Fluorescence-activated Vesicle Sorting for Isolation of Naked2-associated, Basolaterally Targeted Exocytic Vesicles for Proteomics Analysis^*,S

Zheng Cao, Cunxi Li, James N. Higginbotham, Jeffrey L. Franklin, David L. Tabb^¶,||, Ramona Graves-Deal, Salisha Hill^**, Kristin Cheek^**, W. Gray Jerome, Lynne A. Lapierre^,¶¶, James R. Goldenring^,,¶¶, Amy-Joan L. Ham^¶,** and Robert J. Coffey^,,¶¶,||||

From the Departments of Medicine, Cell and Developmental Biology, ^¶ Biochemistry, ^|| Biomedical Informatics, Surgery, and Pathology and ^** The Proteomics Laboratory of the Mass Spectrometry Research Center, Vanderbilt University, Nashville, Tennessee 37232 and the ^¶¶ Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232

By interacting with the cytoplasmic tail of a Golgi-processed form of transforming growth factor- (TGF), Naked2 coats TGF-containing exocytic vesicles and directs them to the basolateral corner of polarized epithelial cells where the vesicles dock and fuse in a Naked2 myristoylation-dependent manner. These TGF-containing Naked2-associated vesicles are not directed to the subapical Sec6/8 exocyst complex as has been reported for other basolateral cargo, and thus they appear to represent a distinct set of basolaterally targeted vesicles. To identify constituents of these vesicles, we exploited our finding that myristoylation-deficient Naked2 G2A vesicles are unable to fuse at the plasma membrane. Isolation of a population of myristoylation-deficient, green fluorescent protein-tagged G2A Naked2-associated vesicles was achieved by biochemical enrichment followed by flow cytometric fluorescence-activated vesicle sorting. The protein content of these plasma membrane de-enriched, flow-sorted fluorescent G2A Naked2 vesicles was determined by LC/LC-MS/MS analysis. Three independent isolations were performed, and 389 proteins were found in all three sets of G2A Naked2 vesicles. Rab10 and myosin IIA were identified as core machinery, and Na⁺/K⁺-ATPase 1 was identified as an additional cargo within these vesicles. As an initial validation step, we confirmed their presence and that of three additional proteins tested (annexin A1, annexin A2, and IQGAP1) in wild-type Naked2 vesicles. To our knowledge, this is the first large scale protein characterization of a population of basolaterally targeted exocytic vesicles and supports the use of fluorescence-activated vesicle sorting as a useful tool for isolation of cellular organelles for comprehensive proteomics analysis.

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