Cytoplasmic signals mediate apical early endosomal targeting of endotubin in MDCK cells.
Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin-Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.
Gokay
K E
KE
Department of Cell Biology and Anatomy, University of Arizona, Tucson, AZ, USA.
Young
R S
RS
Wilson
J M
JM
D000818:Animals; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D007668:Kidney; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D015375:Receptors, Interleukin-2 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection
D008562:Membrane Glycoproteins; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; C427264:endotubin protein, Canis familiaris