DIRECT TARGETING OF NEUTRAL ENDOPEPTIDASE (EC-3.4.24.11) TO THE APICAL CELL-SURFACE OF TRANSFECTED LLC-PK1 CELLS AND UNPOLARIZED SECRETION OF ITS SOLUBLE FORM

LLC-PK1 cells were transfected with a cDNA encoding rabbit neutral end opeptidase (NEP;EC 3.4.24.11), an abundant enzyme of the kidney proxim al brush border. Clones of cells expressing high levels of the protein were isolated. Selective biotinylation and radioimmunolabelling were used to determine that 85-95% of NEP was localized in the apical domai n of filter-grown LLC-PK1 cells. Pulse-chase and selective biotinylati on studies revealed that the majority (85%) of newly made NEP was dire ctly targeted to the apical membrane. However, a soluble form of NEP w as found to be secreted in approximately equal amounts from both sides of the monolayer when expressed in LLC-PK1 cells. Transfected pro-opi omelanocortin, a pituitary hormone precursor, was secreted almost excl usively into the basolateral medium, suggesting that the bulk flow is to the basolateral membrane, This behaviour contrasts with that observ ed in MDCK cells, where both the transmembrane and secreted forms of N EP are directly targeted to the apical membrane and where the secretio n of pro-opiomelanocortin is unpolarized.