title: pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: implications for pH regulation in the secretory pathway.
abstract: The influences of the gastric H+/K+ pump on organelle pH during trafficking to and from the plasma membrane were investigated using HEK-293 cells stably expressing the alpha- and beta-subunits of human H+/K+-ATPase (H+/K+-alpha,beta cells). The pH values of trans-Golgi network (pHTGN) and recycling endosomes (pHRE) were measured by transfecting H+/K+-alpha,beta 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+-alpha,beta cells (pHTGN 6.36) and vector-transfected ("mock") cells (pHTGN 6.34); pHRE was also similar in H+/K+-alpha,beta (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 pH 6.4 up to a new steady-state pHTGN of 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 with H+ v-ATPase. Mathematical modeling predicted a large number of H+ v-ATPases (8000) active in the TGN to balance a large, passive H+ leak (with PH approximately 10-3 cm/s) 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 4000 active in the TGN) and other transporters have little effect on luminal pH as they traffic to the plasma membrane.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA. machen@socrates.berkeley.edu
authors.forename: Terry E
authors.initials: TE
authors.lastname: Machen
authors.forename: Mary Jae
authors.initials: MJ
authors.lastname: Leigh
authors.forename: Carmen
authors.initials: C
authors.lastname: Taylor
authors.forename: Tohru
authors.initials: T
authors.lastname: Kimura
authors.forename: Shinji
authors.initials: S
authors.lastname: Asano
authors.forename: Hsiao-Ping H
authors.initials: HP
authors.lastname: Moore
mesh_terms: D000136:Acid-Base Equilibrium / Q000502:physiology*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000502:physiology; D002478:Cells, Cultured; D011992:Endosomes / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D021381:Protein Transport / Q000502:physiology; D011522:Protons; D011817:Rabbits; D022142:Secretory Vesicles / Q000378:metabolism; D014162:Transfection; D021601:trans-Golgi Network / Q000201:enzymology*
chemical_list: D008164:Luminescent Proteins; D011522:Protons; D017506:H(+)-K(+)-Exchanging ATPase