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J. Cell Biol.,
Volume 139, Number 4, November 17, 1997 941-949
* Department of Cell Biology and Anatomy, Cornell Medical College, New York 10021; and Although many polarized proteins are sorted
to the same membrane domain in all epithelial tissues,
there are some that exhibit a cell type-specific polarity.
We recently found that band 3 (the anion exchanger
AE1) was present in the apical membrane of a renal intercalated cell line when these cells were seeded at low
density, but its targeting was reversed to the basolateral
membrane under the influence of an extracellular matrix protein secreted when the cells were seeded at high
density. Because apical and basolateral lipids differ in
epithelia, we asked what effect might these lipids have
on band 3 function. This question is especially interesting since apical anion exchange in these cells is resistant
to disulfonic stilbene inhibitors while basolateral anion
exchange is quite sensitive. Furthermore, the apical anion exchanger cannot be stained by antibodies that
readily identify the basolateral protein.
We used short chain sphingolipid analogues and
found that sphingomyelin was preferentially targeted to
the basolateral domain in the intercalated cell line. The
ganglioside GM1 (Gal 1 Departments of Medicine and
Physiology, College of Physicians and Surgeons of Columbia University, New York 10032
1, 3GalNAc1, 4Gal-NeuAc
2,
3Gal1, 4Glc ceramide) was confined to the apical membrane as visualized by confocal microscopy after
addition of fluorescent cholera toxin to filter grown
cells. We reconstituted erythrocyte band 3 into liposomes using apical and basolateral types of lipids and
examined the inhibitory potency of 4,4
-dinitorsostilbene-2,2-disulfonic acid (DNDS; a reversible stilbene) on 35SO4/SO4 exchange. Although anion exchange in sphingomyelin liposomes was sensitive to
inhibition, the addition of increasing amounts of the
ganglioside GM1 reduced the potency of the inhibitor
drastically. Because these polarized lipids are present in
the exofacial surface of the bilayer, we propose that the
lipid structure might influence the packing of the transmembrane domains of band 3 in that region, altering
the binding of the stilbenes to these chains. These results highlight the role of polarized lipids in changing
the function of unpolarized proteins or of proteins
whose locations differ in different epithelia.
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