Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells.
Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous "peripheral" and "integral" membrane proteins. "Peripheral" proteins were found to be approximately fivefold more abundant with this procedure than "integral" membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.
Sargiacomo
M
M
Department of Cell Biology, Cornell University Medical College, New York, New York 10021.
Lisanti
M
M
Graeve
L
L
Le Bivic
A
A
Rodriguez-Boulan
E
E
D000818:Animals; D001710:Biotin; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004848:Epithelium / Q000378:metabolism; D007365:Intercellular Junctions / Q000378:metabolism; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*
D008565:Membrane Proteins; D001710:Biotin