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1 Department of Molecular and Cell Biology, University of California - Berkeley, Berkeley, CA, USA
2 Department of Molecular and Cell Biology, University of California - Berkeley, Berkeley, CA, USA; Graduate Group in Bioengineering, University of California-Berkeley, Berkeley, CA, USA
3 Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Toyama City, Toyama, Japan
* To whom correspondence should be addressed. E-mail: machen{at}socrates.berkeley.edu.
The influences of the gastric H+/K+-pump on organelle pH during trafficking to and from the plasma membrane was investigated using HEK293 cells stably expressing the 
and 
subunits of human H+/K+-ATPase (H+/K+-, cells). The pH's of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-, cells with the pH-sensitive GFP pHluorin fused to targeting sequences of either TGN38 or synaptobrevin, respectively. Immunofluorescence showed that H+/K+-ATPase was present in the plasma membrane, TGN and RE. The pHTGN was similar in both H+/K+-, cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-, (pHRE 6.40) and mock cells (pHRE 6.37). SCH28080 (inhibits H+/K+-ATPase) caused TGN to alkalinize by 0.12 pH units; subsequent addition of bafilomycin (inhibits H+ v-ATPase) caused TGN to alkalinize from pHTGN 6.4 up to a new steady state pHTGN 7.0-7.5, close to pHcytosol. Similar results were observed in RE. Thus, H+/K+-ATPases that trafficked to the plasma membrane were active, but had small effects to acidify the TGN and RE compared to H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8,000) active in the TGN to balance a large, passive H+ leak (with PH ~ 10-3 cm/sec) via unidentified pathways out of the TGN. We propose that in the presence of this effective, though inefficient, buffer system in the Golgi and TGN, H+/K+-ATPases (estimated to be approximately 4,000 active in the TGN) and other transporters have little effect on lumenal pH as they traffic to the plasma membrane.
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