The effect of apical and basolateral lipids on the function of the band 3 anion exchange protein.
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 1beta1, 3GalNAcbeta1, 4Gal-NeuAcalpha2, 3Galbeta1, 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.
van't Hof
W
W
Department of Cell Biology and Anatomy, Cornell Medical College, New York 10021, USA.
Malik
A
A
Vijayakumar
S
S
Qiao
J
J
van Adelsberg
J
J
Al-Awqati
Q
Q
D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation; D016764:Cell Polarity; D002478:Cells, Cultured; D002518:Ceramides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D007700:Kinetics; D008563:Membrane Lipids / Q000502:physiology*; D011510:Proteolipids; D011817:Rabbits; D013267:Stilbenes / Q000494:pharmacology; D013431:Sulfates / Q000378:metabolism
D001457:Anion Exchange Protein 1, Erythrocyte; D002518:Ceramides; D008563:Membrane Lipids; D011510:Proteolipids; D013267:Stilbenes; D013431:Sulfates; C020485:proteoliposomes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid