Influenza M2 proton channel activity selectively inhibits trans-Golgi network release of apical membrane and secreted proteins in polarized Madin-Darby canine kidney cells

The function of acidification in protein sorting along the biosynthetic pat hway has been difficult to elucidate, in part because reagents used to alte r organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in prot ein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We express ed the influenza virus M2 protein, an acid-activated ion channel that equil ibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral protei ns. M2 activity affects the pH of only a subset of acidified organelles, an d its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G.Apodaca, Y.Altschuler, S. Hardy, and O.A. Weisz. 1998. Mel. Biol. C ell. 8:2477-2490; Henkel, J.R., J.L. Popovich, G.A.Gibson, S.C. Watkins, an d O.A. Weisz. 1999. J. Biol. Chem. 274:9854-9860). M2 expression significan tly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered pol ymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of gamma-glutamyl-transpeptidase were reduced with no ef fect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human gro wth hormone [hGH]) that was secreted equally from both surfaces. However, M 2 slowed apical secretion of a glycosylated mutant of hGH that was secreted predominantly apically. Our results suggest a role for acidic trans-Golgi network pH in signal-mediated loading of apical cargo into forming vesicles .