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1 Department of Molecular and Cell Biology and 2 Department of Physics, University of California, Berkeley, California 94720-3200
Work addressing whether cystic fibrosis
transmembrane conductance regulator (CFTR) plays a role in regulating
organelle pH has remained inconclusive. We engineered a pH-sensitive
excitation ratiometric green fluorescent protein (pHERP) and targeted
it to the Golgi with sialyltransferase (ST). As determined by
ratiometric imaging of cells expressing ST-pHERP, Golgi pH
(pHG) of HeLa cells was 6.4, while pHG of
mutant (
F508) and wild-type CFTR-expressing (WT-CFTR) respiratory
epithelia were 6.7-7.0. Comparison of genetically matched F508
and WT-CFTR cells showed that the absence of CFTR statistically
increased Golgi acidity by 0.2 pH units, though this small difference
was unlikely to be physiologically important. Golgi pH was maintained
by a H+ vacuolar (V)-ATPase countered by a H+
leak, which was unaffected by CFTR. To estimate Golgi proton permeability (PH+), we modeled
transient changes in pHG induced by inhibiting the V-ATPase
and by acidifying the cytosol. This analysis required knowing Golgi
buffer capacity, which was pH dependent. Our in vivo estimate is that
Golgi PH+ = 7.5 × 10
4 cm/s when pHG = 6.5, and
surprisingly, PH+ decreased as
pHG decreased.
enhanced yellow fluorescent protein; ultraviolet enhanced green fluorescent protein; trachea; organelle pH; proton permeability; cystic fibrosis
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