Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells.
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
Oztan
Asli
A
Laboratory of Epithelial Cell Biology/Renal Electrolyte Division of the Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Silvis
Mark
M
Weisz
Ora A
OA
Bradbury
Neil A
NA
Hsu
Shu-Chan
SC
Goldenring
James R
JR
Yeaman
Charles
C
Apodaca
Gerard
G
D000818:Animals; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D004285:Dogs; D015536:Down-Regulation / Q000235:genetics; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism*; D007070:Immunoglobulin A / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D007070:Immunoglobulin A; D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033921:Vesicular Transport Proteins; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C109899:Exoc6 protein, rat; C093846:early endosome antigen 1