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© The Rockefeller University Press, 0021-9525/2000//495 $5.00
The Journal of Cell Biology, Volume 148, Number 3, , 2000 495-504

Influenza M2 Proton Channel Activity Selectively Inhibits Trans-Golgi Network Release of Apical Membrane and Secreted Proteins in Polarized Madin-Darby Canine Kidney Cells

^a Laboratory of Epithelial Cell Biology, Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, Pennsylvania 15261
Renal-Electrolyte Division, University of Pittsburgh, 3550 Terrace St., Pittsburgh, PA 15261.(412) 383-8956(412) 383-8891

weisz{at}msx.dept-med.pitt.edu

The function of acidification in protein sorting along the biosynthetic pathway has been difficult to elucidate, in part because reagents used to alter organelle pH affect all acidified compartments and are poorly reversible. We have used a novel approach to examine the role of acidification in protein sorting in polarized Madin-Darby canine kidney (MDCK) cells. We expressed the influenza virus M2 protein, an acid-activated ion channel that equilibrates lumenal and cytosolic pH, in polarized MDCK cells and examined the consequences on the targeting and delivery of apical and basolateral proteins. M2 activity affects the pH of only a subset of acidified organelles, and its activity can be rapidly reversed using ion channel blockers (Henkel, J.R., G. Apodaca, Y. Altschuler, S. Hardy, and O.A. Weisz. 1998. Mol. Biol. Cell. 8:2477–2490; Henkel, J.R., J.L. Popovich, G.A. Gibson, S.C. Watkins, and O.A. Weisz. 1999. J. Biol. Chem. 274:9854–9860). M2 expression significantly decreased the kinetics of cell surface delivery of the apical membrane protein influenza hemagglutinin, but not of the basolaterally delivered polymeric immunoglobulin receptor. Similarly, the kinetics of apical secretion of a soluble form of -glutamyltranspeptidase were reduced with no effect on the basolaterally secreted fraction. Interestingly, M2 activity had no effect on the rate of secretion of a nonglycosylated protein (human growth hormone [hGH]) that was secreted equally from both surfaces. However, M2 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.

Key Words: acidification • polarity • Madin-Darby • canine kidney • influenza M2 • apical

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AMT, amantadine; AV, adenovirus; AV-TA, adenovirus encoding tTA; BafA₁, bafilomycin A₁; endo H, endoglycosidase H; GT, -glutamyltranspeptidase; ghGH, glycosylated hGH; HA, hemagglutinin; hGH, human growth hormone; pIgR, polymeric Ig receptor; PM, plasma membrane; V-ATPase, vacuolar H⁺-ATPase.

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