Organelle pH studies using targeted avidin and fluorescein-biotin.
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.
Wu
M M
MM
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Llopis
J
J
Adams
S
S
McCaffery
J M
JM
Kulomaa
M S
MS
Machen
T E
TE
Moore
H P
HP
Tsien
R Y
RY
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
D002712:Chlorides; D005456:Fluorescent Dyes; D017494:Proton Pumps; D001360:Avidin; D001710:Biotin; D011188:Potassium; D019793:Fluorescein