D

D. of exocyst function, likely through interactions with Sec15A. INTRODUCTION The generation and maintenance of epithelial polarity, which is indispensable for the functional integrity of epithelial tissues, requires sorting, transport, and delivery of newly synthesized and endocytosed proteins to the correct apical or basolateral plasma membrane domain (Yeaman epithelial cells (Langevin Rab11 interaction to a single helix in the C-terminal region of Sec15 (Wu for 3 h at 4C. The endosome-enriched fraction (1 ml) at the 25%/35% sucrose interface was collected with a needle. Sheep anti-rabbit magnetic Dynabeads (50 l; Invitrogen) were washed with 0.2% (wt/vol) bovine serum albumin (BSA) in PBS two times and incubated with 1 ml 5% (wt/vol) BSA in PBS overnight at 4C. The following day the beads were recovered with a magnetic particle concentrator (Dynal, Oslo, Norway) and resuspended in 1 ml 5% BSA in PBS containing 5 g of Rab11 polyclonal antibody (ab3612) or nonspecific rabbit IgG and incubated overnight at 4C. The beads were washed with 1% GNE-617 (wt/vol) BSA in PBS, resuspended in 2 ml of 5% BSA in PBS, and incubated with 1 ml of the endosome fraction 3 h at 4C on a rotator. The Rab11-positive endosomes associated with the Dynabeads were collected using a magnetic plate and washed two times with 0.2% BSA in PBS, and then one additional time with PBS. The endosome suspension was transferred to a new tube, magnetic beads were collected using a magnetic particle concentrator, and PBS was removed by aspiration. The endosomes bound to beads were boiled in Laemlli sample buffer and resolved on 15% SDS PAGE gel. Western blots were performed as described previously (Maples test. p 0.05 was considered significant. RESULTS The Intracellular Pool of Exocyst Subunits Is Associated in Part with EEA1-, Tf-, and Rab11a-positive Endosomes But Not the TGN of Polarized MDCK Cells Initial studies of exocyst subunit distribution in MDCK cells showed that Sec6 and Sec8 were localized at or near the tight junctions of cells after initiation of cell-to-cell contact or tubulogenesis (Grindstaff proteins (Wu Sec15CT blocked its interaction with Rab11 (Wu has previously established that the distribution of individual exocyst components is not MAD-3 always identical (Beronja (Beronja Sec15 C-terminus also binds to Rab3, Rab8, and Rab27 in two-hybrid assays (Wu below for an alternative possibility). Unfortunately, you will find few reagents to study Sec15B at present, so the part of this isoform in endocytic traffic is remaining to future studies. The lack of effect of expressing GFP-Sec15CT or pSuper-Sec15A on apical IgA recycling was amazing as both apical recycling and basolateral-to-apical transcytosis are regulated by Rab11a (Wu (http://www.molbiolcell.org/cgi/doi/10.1091/10.1091/mbc.E07-02-0097) about August 8, 2007. ?The online version of this article contains supplemental material at (http://www.molbiolcell.org). Recommendations Altschuler Y., Kinlough C. L., Poland P. A., Bruns J. B., Apodaca G., Weisz O. A., Hughey R. P. Clathrin-mediated endocytosis of MUC1 is definitely modulated by its glycosylation state. Mol. Biol. Cell. 2000;11:819C831. [PMC free article] [PubMed] [Google Scholar]Ang A. L., Folsch H., Koivisto U. GNE-617 M., Pypaert M., Mellman I. The Rab8 GTPase selectively regulates AP-1B-dependent basolateral transport in polarized Madin-Darby canine kidney cells. J. Cell Biol. 2003;163:339C350. [PMC free article] [PubMed] [Google Scholar]Ang A. L., Taguchi T., Francis S., Folsch H., Murrells L. J., Pypaert M., Warren G., Mellman I. Recycling endosomes can serve as intermediates during transport from your Golgi to the plasma membrane of MDCK cells. J. Cell Biol. 2004;167:531C543. [PMC free article] [PubMed] [Google Scholar]Apodaca G., Cardone M. H., Whiteheart S. W., DasGupta B. R., Mostov K. E. Reconstitution of transcytosis in SLO-permeabilized MDCK cells: living of an NSF-dependent fusion mechanism with the apical surface of MDCK cells. EMBO J. 1996;15:1471C1481. [PMC free article] [PubMed] [Google Scholar]Apodaca G., Katz L. A., Mostov K. E. Receptor-mediated transcytosis of IgA in MDCK cells is definitely via apical recycling endosomes. J. Cell Biol. 1994;125:67C86. [PMC free article] [PubMed] [Google Scholar]Bacallao R., Stelzer E. H. Preservation of biological specimens for observation inside a confocal fluorescence microscope and operational principles of confocal fluorescence microscopy. Methods Cell Biol. 1989;31:437C452. [PubMed] [Google Scholar]Barile M., Pisitkun T., Yu M. J., Chou C. L., Verbalis M. J., Shen R. GNE-617 F., Knepper M. A. Large scale protein recognition in intracellular aquaporin-2 vesicles from renal.