(Na ÷ + K+)-ATPase and Plasma Membrane Polarity of Intestinal Epithelial Cells: Presence of a Brush Border Antigen in the Distal Large Intestine that Is Immunologically Related to/3 Subunit

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the/3 subunit of (Na + + K÷)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the ct-/~ enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the c~ subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na + + K÷)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting/~-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na + + K÷)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the fl subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled/3 subunit or a ~-subunit-like protein is also transported to the apical cell pole. T HE (Na + + K÷)-ATPase is a heterodimeric surface membrane protein complex that is composed of a catalytic 100-kD ot subunit and a 50-kD/~ subunit of unknown function (for reviews see Cantley, 1981; JCrgensen, 1982, 1986; Rossier, 1984; Glynn, 1985). This enzyme catalyzes the transmembrane exchange of Na ÷ ions for K ÷ ions, a process requiring ATP. Biochemical studies and the amino acid sequence deduced from complementary DNA suggest that the catalytic t~ subunit spans the bilayer several times (Shull et al., 1985; Kawakami et al., 1985; Ovchinnikov et al., 1986, 1988; Chehab, 1987). A binding site for cardiac glycosides, such as ouabain, is located on the extracytoplasmic side while both the ATP-binding site and the phosphorylation site are located on the cytoplasmic side of the ct subunit. Contrary to the ot subunit which appears to lack covalently bound carbohydrates, the/3 subunit is a glycosylated transmembrane protein whose principal mass protrudes into the intercellular space (Girardet et al., 1983; Noguchi et al., 1986; Shull et al., 1986; Ovchinnikov et al., 1986; Brown et al., 1987). A. Marxer's present address is Hoffmann-La Roche, Diagnostica, CH-4303 Kaiseraugst, Switzerland. B. Stieger's present address is Department of Clinical Pharmacology, University Hospital, 8091 Ziirich, Switzerland. The two enzyme subunits are synthesized independently from separate mRNA species (Geering et al., 1985). Some studies have suggested that the c~ subunit might be synthesized on free polysomes before its posttranslational assembly with the lipid bilayer, a process that may be mediated by the cotranslationally inserted/3 subunit (Sabatini et al., 1982; Hiatt et al., 1984). However, most authors now agree that both the/3 and a subunits are membrane associated during their synthesis (Geering et al., 1985) and that subunit assembly occurs very rapidly (Fambrough and Bayne, 1983; Tamkun and Fambrough, 1986). There is little doubt that in polarized epithelial cells functionally active (Na ÷ + K÷)-ATPase must be localized in the basolateral membrane domain of the cell surface under physiological conditions that are characterized by a luminal-toserosal transport of Na + (Schultz, 1981; Ernst and Schreiber, 1981). However, immunolocalization studies with polyclonal or even monoclonal antibodies have led to conflicting results. While a majority of studies confirmed the principal basolateral localization of (Na + + K+)-ATPase (Fambrough and Bayne, 1983; Kashgarian et al., 1985; Gerard et al., 1985; Gorvel et al., 1983; Yamamoto et al., 1984; Almers and Stirling, 1984), some authors have found opposite localiza© The Rockefeller University Press, 0021-9525/89/09/1057/13 $2.00 The Journal of Cell Biology, Volume 109, September 1989 1057-1069 1057 on Jauary 7, 2016 jcb.rress. org D ow nladed fom Published September 1, 1989