BIOTINYLATION AND ASSESSMENT OF MEMBRANE POLARITY - CAVEATS AND METHODOLOGICAL CONCERNS

Studies of epithelial membrane polarity have been greatly facilitated through the use of the N-hydroxysuccinimide-biotin surface labeling te chnique (M. Sargiacomo, M. Lisanti, L. Graeve, A. Le Bivic, and E. Rod riguez-Boulan. J. Membr. Biol. 107: 277-286, 1989). We have used this technique in studies on the sorting and targeting of ion-transporting adenosinetriphosphatase molecules in polarized epithelial cells. Throu gh efforts to optimize this technique in our experimental system, we h ave encountered several experimental conditions and circumstances wher e biotinylation is extremely inefficient and the assessment of membran e polarity which it provides is misleading. We demonstrate that the pH and ionic strength of the biotinylation buffer can dramatically affec t biotin incorporation and that protocol-dependent variations in the r ecovery of biotinylated proteins can result in misrepresentation of th e actual apical/basolateral distribution of a protein. Conditions and protocols that may improve the sensitivity and accuracy of this techni que are discussed.