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

We used flow cytometry to sort and analyze apical and basolateral endocytic vesicles from filter-grown Madin-Darby canine kidney (MDCK) cells after me mbrane internalization of the lipophilic fluorescent probe trimethylamino-d iphenylhexatriene. Western blot analysis of sorted fractions showed enrichm ent of the early endosomal markers transferrin receptor and the small GTPas e Rab5, Two-dimensional gel analysis indicated that the apical and basolate ral early endosomes differed significantly in their protein composition. We found nine polypeptides to be specifically enriched in apical or basolater al endocytic vesicles. An apical protein identified by microsequencing was the adaptor molecule syntenin, This protein contains two PDZ domains (PSD-9 5, Dig, and ZO-1 homology) that bind syndecan and ephrin-B2 cytoplasmic dom ains. In MDCK cells, transiently overexpressed Myc-tagged syntenin localize d to both plasma membrane domains and to an intracellular vesicular compart ment, 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 api cal recycling endosomes in MDCK cells.