Identification of syntenin as a protein of the apical early endocytic compartment in Madin-Darby canine kidney cells

Abstract

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after membrane internalization of the lipophilic fluorescent probe trimethylamino-diphenylhexatriene. Western blot analysis of sorted fractions showed enrichment of the early endosomal markers transferrin receptor and the small GTPase Rab5. Two-dimensional gel analysis indicated that the apical and basolateral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolateral endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin. This protein contains two PDZ domains (PSD-95, Dlg, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic domains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localized to both plasma membrane domains and to an intracellular vesicular compartment. Syntenin positive vesicles colocalized with internalized transferrin in the perinuclear region. In addition, syntenin colocalized in the apical supranuclear region with Rab5 and Rab11; the latter is a marker for the apical recycling endosomes in MDCK cells.