Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions

Prominin is a recently identified polytopic membrane protein expressed in v arious epithelial cells, where it is selectively associated with microvilli . When expressed in non-epithelial cells, prominin is enriched in plasma me mbrane protrusions., This raises the question of whether the selective asso ciation of prominin with microvilli in epithelial cells is solely due to it s preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enr ichment in plasma membrane protrusions, To investigate this question, we ha ve generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescenc e and domain-selective cell surface biotinylation experiments on transfecte d MDCK cells grown on permeable supports demonstrated the virtually exclusi ve apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed tha t newly synthesized prominin was directly targeted to the apical plasma mem brane domain, Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvil li, Both the apical-specific localization of prominin and its selective ret ention in microvilli were maintained when MDCK cells mere cultured in low-c alcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for di rect delivery to the apical plasma membrane domain and for the enrichment i n the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and r etention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial ce lls.