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Volume 272, Number 47, Issue of November 21, 1997 pp. 29810-29820
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Phagosomal Acidification
DIFFERENTIAL TARGETING OF Na⁺/H⁺ EXCHANGERS, Na⁺/K⁺-ATPases, AND VACUOLAR-TYPE H⁺-ATPases

(Received for publication, June 19, 1997, and in revised form, August 28, 1997)

David J. Hackam ^§ , Ori D. Rotstein ^§ , Wei-Jian Zhang ^§ , Nicolas Demaurex , Michael Woodside , Olivia Tsai ^§ and Sergio Grinstein

From the  Division of Cell Biology, The Hospital for Sick Children, and the ^§ Department of Surgery, Toronto Hospital, University of Toronto, Toronto M5G 1X8, Canada

Vacuolar-type (V) ATPases are thought to be the main determinant of phagosomal acidification. In phagosomes containing mycobacteria, which ostensibly impair the delivery of V-ATPases to the phagosomal membrane, the pH would be expected to be near neutral. This prediction was tested by microfluorescence ratio imaging using macrophages from mice susceptible to mycobacterial infection. Although less acidic than their counterparts containing dead bacteria, phagosomes containing live Mycobacteria bovis were nearly 1 pH unit more acidic than the cytosol, suggesting the existence of alternate H⁺ transport mechanisms. We therefore investigated whether Na⁺/H⁺ exchange (NHE) contributes to phagosomal acidification. Immunoblotting, reverse transcriptase-polymerase chain reaction, and pharmacological studies indicated that NHE1 is the predominant isoform of the exchanger in macrophages. Fractionation revealed that NHE1 is incorporated into the phagosomal membrane, and measurements of pH indicated that it is functional in this location. Nevertheless, acidification of the lumen of phagosomes containing either latex beads or live M. bovis was insensitive to (3-methylsulfonyl-4-piperidinobenzoyl)-guanidine methanesulfonate, a potent inhibitor of NHE1. This may have been due to the absence of an appropriate lumen to cytosol Na⁺ gradient, because the phagosomal membrane was found to be devoid of Na⁺/K⁺ pumps. Unexpectedly, the acidification of M. bovis phagosomes was fully reversed by specific inhibitors of the vacuolar H⁺-ATPase, suggesting that ATPases are present only transiently or in reduced quantities in the phagosomal membrane. Alternatively, acid equivalents accumulated in endosomes by V-ATPases may be delivered to the mycobacterial phagosome by carrier vesicles devoid of ATPases.

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