Prestin topology: localization of protein epitopes in relation to the plasma membrane

Computer modeling of the outer hair cell (OHC) motor protein prestin produc es ambiguous results regarding transmembrane regions and localization of it s termini. To determine the location of prestin's N- and C-termini, we crea ted prestin constructs with synthetic epitopes located immediately upstream or downstream of prestin. The spatial distribution of these epitopes was s tudied in prestin-transfected cells using immunofluorescence. In permeabili zed cells, antibodies label the plasma membrane of 30% of the cells, reflec ting transfection efficiency. Under non-permeabilizing conditions, the few labeled cells also displayed a lack of plasma membrane integrity. These dat a suggest that prestin's N-and C-termini are cytoplasmic. Furthermore, pres tin staining in OHCs was observed only under permeabilizing conditions. The se results implicate prestin's N- and C-termini as portions that may intera ct with other cytoplasmic proteins. A model of prestin membrane topology is also considered based on the results. NeuroReport 12:1929-1935 (C) 2001 Li ppincott Williams & Wilkins.