title: Organelle pH studies using targeted avidin and fluorescein-biotin.
abstract: BACKGROUND
  Mammalian organelles of the secretory pathway are of differing pH. The pH values form a decreasing gradient: the endoplasmic reticulum (ER) is nearly neutral, the Golgi is mildly acidic and the secretory granules are more acidic still ( approximately pH 5). The mechanisms that regulate pH in these organelles are still unknown.
  
  
  RESULTS
  Using a novel method, we tested whether differences in H(+) 'leak' and/or counterion conductances contributed to the pH difference between two secretory pathway organelles. A pH-sensitive, membrane-permeable fluorescein-biotin was targeted to endoplasmic-reticulum- and Golgi-localized avidin-chimera proteins in HeLa cells. In live, intact cells, ER pH (pH(ER)) was 7.2 +/- 0.2 and Golgi pH (pH(G)) was 6.4 +/- 0.3 and was dissipated by bafilomycin. Buffer capacities of the cytosol, ER and Golgi were all similar (6-10 mM/pH). ER membranes had an apparent H(+) permeability three times greater than that of Golgi membranes. Removal of either K(+) or Cl(-) did not affect ER and Golgi H(+) leak rates, or steady-state pH(G) and pH(ER).
  
  
  CONCLUSIONS
  The Golgi is more acidic than the ER because it has an active H(+) pump and fewer or smaller H(+) leaks. Neither buffer capacity nor counterion permeabilities were key determinants of pH(G), pH(ER) or ER/Golgi H(+) leak rates.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Wu
authors.forename: J
authors.initials: J
authors.lastname: Llopis
authors.forename: S
authors.initials: S
authors.lastname: Adams
authors.forename: J M
authors.initials: JM
authors.lastname: McCaffery
authors.forename: M S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: T E
authors.initials: TE
authors.lastname: Machen
authors.forename: H P
authors.initials: HP
authors.lastname: Moore
authors.forename: R Y
authors.initials: RY
authors.lastname: Tsien
mesh_terms: D001360:Avidin / Q000493:pharmacokinetics; D001710:Biotin / Q000493:pharmacokinetics; D002712:Chlorides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D019793:Fluorescein / Q000493:pharmacokinetics; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D017136:Ion Transport; D016253:Microscopy, Immunoelectron; D015388:Organelles / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism
chemical_list: D002712:Chlorides; D005456:Fluorescent Dyes; D017494:Proton Pumps; D001360:Avidin; D001710:Biotin; D011188:Potassium; D019793:Fluorescein