1 Department of Molecular and Cell Biology and 2 Department of Physics, University of California, Berkeley, California 94720-3200
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ABSTRACT |
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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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INTRODUCTION |
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CYSTIC FIBROSIS
(CF) is the most common fatal genetic disease among Caucasians. CF is
caused by mutations in the gene encoding the cystic fibrosis
transmembrane conductance regulator (CFTR), which is a cAMP/protein
kinase A-regulated Cl channel that is absent or defective
in CF. CF patients most commonly succumb to respiratory complications
that arise because of colonization of the respiratory tract by
Pseudomonas aeruginosa and other opportunistic bacteria. One
hypothesis to explain how a reduction of Cl
permeability
could lead to colonization of CF lungs by P. aeruginosa is
that pH of the Golgi (pHG), trans-Golgi, and
trans-Golgi network, which are normally acidic relative to
the cytosol, are alkaline in CF. CFTR, which could serve as the
counterion conductance, may be required to prevent generation of large
lumen-positive membrane voltages during pumping by the electrogenic
H+ vacuolar (V)-ATPase (1, 2). The lack of
CFTR would lead to an alkaline pHG, which in turn would
alter the activities of resident enzymes responsible for proper
sialylation, sulfation, and fucosylation [due to sialyltransferase
(ST), sulfotransferase, and fucosyltransferase, respectively] of
secreted and surface membrane components. These alterations could lead
to changes in the chemical properties of membrane and secreted
glycoproteins and glycolipids, such as increased asialo-GM1, a
hypothesized bacterial binding site on epithelia (5, 11, 25,
63) [see also Schroeder et al. (45) for
conflicting opinion].
A number of groups have attempted to test the organelle pH hypothesis. Lukacs et al. (33) demonstrated that CFTR was functional in endosomes of Chinese hamster ovary (CHO) cells heterologously expressing CFTR, but they concluded that factors other than CFTR were the major determinants of endosomal pH. Dunn et al. (12) found that endocytic acidification was independent of CFTR when the CF pancreatic cell line (CFPAC) and CFTR-corrected CFPAC cells were compared. Seksek et al. (48) microinjected liposomes containing pH-sensitive fluid-phase dyes into the Golgi of fibroblasts and epithelial cells and found that pHG was the same in both cell types and also in epithelial cells that normally do (Calu-3) and do not (Madin-Darby canine kidney, SK-MES-1) express CFTR (49).
There were three reasons for performing a rigorous test of the
organelle pH hypothesis. First, human airway epithelial cells may be
different from fibroblasts, lymphocytes, and non-airway epithelial
cells from other species with regard to pHG regulation. Second, none of the previous comparisons of pHG was
performed on cells that were genetically matched F508 (deletion of
Phe-508 in CFTR) and wild-type CFTR-expressing (WT-CFTR) respiratory
cell lines. Third, it was important to measure the pH of Golgi
cisternae where ST and the other critical enzymes reside because pH
regulatory mechanisms may differ in different organelles
(54), and none of the other studies had definitively
measured pH in the ST-containing region of the Golgi.
We developed a ratiometric, green fluorescent protein (GFP)-based pH
sensor that was targeted to the Golgi with ST in genetically matched
F508-CF and CFTR-corrected
F508-CF human airway epithelial cells.
Various pH-sensitive mutants of GFP (pKa
5-7) have been targeted to the Golgi (as well as mitochondria and
endoplasmic reticulum) (28, 32, 55), but artifacts can
arise with the use of the single-wavelength intensity changes of these
GFPs when the apparent or actual fluorophore concentration changes
(e.g., due to bleaching or changes in path length) and pH does not. By fusing ultraviolet enhanced GFP (GFPuv) and enhanced yellow fluorescent protein (EYFP), we created a chimeric protein that has an excitation spectrum with pH-dependent (490 nm) and relatively pH-independent (440 nm) wavelengths. Measurements of pHG in HeLa cells were
used to confirm the method, and measurements in CF (
F508 CFTR)
tracheal (CFT1) and human nasal epithelial (JME) cells and in
CFTR-expressing tracheal (CFT1-CFTR) and human bronchial epithelial
(HBE) cells were used to determine the role of CFTR in the control of
pHG.
It was also expected that pHG would be critically affected
by the magnitude of H+ leaks. Because CFTR may control both
Na+ conductance (29, 39, 50) and anion
(Cl/HCO
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METHODS |
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Materials
All salts, glucose, buffers, dibutyryl-cAMP, adenine, transferrin, insulin, chloroquine, DMSO, triiodothyronine, hydrocortisone, cholera toxin, epithelial growth factor (EGF), epinephrine, endothelial cell growth supplement, DEAE dextran, bafilomycin, nigericin, and monensin were obtained from Sigma (St. Louis, MO); forskolin and sometimes bafilomycin were obtained from Calbiochem (San Diego, CA); solvents were from Fisher Scientific (Pittsburgh, PA); and restriction enzymes were from New England Biolabs (Beverly, MA). Tissue culture reagents were obtained from GIBCO-BRL or Cellgro. Fetal bovine serum (FBS) was obtained from Gemini Bio-Products. BCECF-AM and Pluronic F-127 were from Molecular Probes, (Eugene, OR).Construction of Plasmids
Bacterial expression vector for pHERP. To create a bacterial expression vector for pH-sensitive excitation ratiometric green fluorescent protein (pHERP), EYFP (S65G, S72A, T203Y, H231L) was PCR-amplified with a sense primer (5'-cccaagcttgatggtgagcaagggcgag-3') containing a HindIII restriction site (underlined) and an antisense primer (5'-gacgagctgtacaagggaggaggtctagag-3') that codes for a linker region. An XbaI restriction site (underlined) is present and eliminated the EYFP stop codon. Cloning the product into GFPuv (F99S, M153T, V163A; Clonetech) put EYFP upstream of GFPuv with an intervening linker region having the amino acid sequence GGGLEDPRVPVEK.
ST-pHERP mammalian expression vector. We PCR amplified the ST fragment, amino acids 1-70 containing the cytosolic, transmembrane, and truncated luminal domains, from human 2',6-sialyltransferase (courtesy of Dr. Brian Seed, Harvard Medical School and Massachusetts General Hospital). This portion of ST has been used to target chimeric molecules to the Golgi (60, 61). PCR amplification was performed with primers (5'-cgcgggaagcttgccaccatgattcacaccaacctg-3' and 5'-cgcgggcggatcctgggtgctgcttgagga-3') that allowed cloning of the PCR product into the pcDNA3 vector (Invitrogen, San Diego, CA) with 5' HindIII and 3' BamHI restriction sites. PCR amplification of pHERP was performed with a sense primer (5'-cgcgggagatctagaattcgtgagcaagggcgag-3') that eliminates EYFP's ATG and has a BglII site (underlined) and with an antisense SP6 primer (5'-gatttaggtgacactatag-3'). The EYFP-GFPuv PCR product was subcloned downstream of ST in pcDNA3 with BglII and ApaI. The final construct codes for the chimeric protein with amino acids 1-70 of ST, a 3-amino acid linker (LEF) between ST and EYFP, amino acids 2-239 of EYFP, a 13-amino acid linker (GGGLEDPRVPVEK), and amino acids 1-238 of GFPuv. GT-EGFP [enhanced GFP (EGFP) targeted to Golgi with galactosyltransferase (GT)] was provided by the laboratory of Roger Tsien (Howard Hughes Medical Institute and University of California, San Diego).
In Vitro Spectra of pHERP
Bacteria expressing the various GFPs were grown overnight in liquid cultures and resuspended in one-tenth the volume of a bacterial lysis solution. Bacterial lysis solution contained (in mM) 10 Tris · HCl (pH 7.4), 100 NaCl, 1 MgCl2, 10 dithiothreitol, and protease inhibitors (0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin, and 20 µg/ml phenylmethylsulfonyl fluoride). Lysed bacteria were diluted into buffer containing (in mM) 50 Na-acetate, 50 glycine, and 50 K2HPO4, which were titrated to the various pH values with HCl or KOH. Excitation spectra were obtained with a fluorometer (Spex Flurolog 1681; Spex Industries, Edison, NJ) containing a 150-W xenon arc lamp.Cell Culture
All cells were maintained in a 37°C incubator with 5% CO2. All media were supplemented with penicillin, streptomycin, and glutamine. HeLa cells were maintained in DMEM supplemented with 10% FBS. JMEs (obtained from Dr. Douglas Jefferson, Tufts University) were grown in DMEM/F-12 supplemented with 10% FBS, 180 µM adenine, 5 µg/ml insulin, 5 µg /ml transferrin, 30 nM triiodothyronine, 1.1 µM hydrocortisone, 10 µg/ml EGF, and 5.5 µM epinephrine. HBE, CFT1-C2, and CFT1-CFTR cells were obtained from Dr. James Yankaskas (62) and grown in Ham's F-12 supplemented with 10 µg/ml insulin, 1 µM hydrocortisone, 25 ng/ml endothelial cell growth supplement, 10 ng/ml EGF, 30 nM triiodothyronine, 5 µg /ml transferrin, and 10 ng/ml cholera toxin.Transfections
Cells were transiently transfected with a modified DEAE dextran protocol of Seed and Aruffo (47). Cells were sequentially split first into a tissue culture flask and, on the following day, onto glass coverslips to a confluency of ~30-50%. On the following day, the cells were incubated in a solution containing 5-6 µg/ml plasmid DNA, 100 µg/ml DEAE dextran, and 50 µM chloroquine for 2-4 h. Cells were washed with PBS containing 10% DMSO for 2 min. Medium was then added, and experiments were performed 24-72 h later. Transfection efficiency was low (1-10%) but sufficient for the single-cell experiments. Some transfections were done with a microporator. Cells plated on coverslips were exposed to DNA (2 µg/µl) while three 30-ms pulses of 300 mV/cm were applied (53). This method led to improved transfection efficiency (5-20%).Solutions
Ringer solution contained (in mM) 141 NaCl, 2 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 2 CaCl2, and 10 glucose brought to pH 7.4 with NaOH. Na+-free solutions contained (in mM) 141 N-methyl-D-glucamine (NMDG) base, 2 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 2 CaCl2, and 10 glucose brought to pH 7.4 with HCl. Calibration solutions contained (in mM) 70 NaCl, 70 KCl, 1.5 K2HPO4, 1 MgS04, 10 HEPES, 10 MES, 2 CaCl2, and 10 glucose adjusted to various pH values (5.5, 6.0, 6.5, 7.0, 7.5, or 8.2) with KOH, 0.01 nigericin, and 0.01 monensin. In some experiments in which cells were alkalinized, NH4Cl (30 mM) was substituted for 30 mM NaCl or NMDG where indicated. Buffer capacity experiments were performed in high-K+/0-Na+ solutions with varying amounts of K-acetate or NH4Cl substituting for the KCl. Bafilomycin was used at 100-250 nM. Intracellular cAMP was increased by perfusing cells with solutions containing either 10 µM forskolin alone or 10 µM forskolin plus 500 µM dibutyryl-cAMP.Fluorescence Ratio Imaging of pHC and pHG
Golgi, labeled with pHERP, and cytosol, labeled with 10 µM BCECF-AM, were monitored in separate experiments with the use of digitally processed fluorescence ratio imaging. Dye-loaded cells were placed in an open perfusion chamber on an inverted IM35 Zeiss microscope. A ×40 oil-immersion objective (1.4 NA; Nikon) was used to collect fluorescence from 1 to 30 cells during each experiment. A lens was used to focus the image through a phototube (Diagnostic Instruments) onto a low-light-level DAGE 68 SIT camera. Emission images of the cells were collected through a 530-nm band-pass filter during sequential excitation at 490 and 440 ± 5 nm (Omega Optical, Brattleboro, VT). Filters were changed with a Lambda 10-2 filter wheel (Sutter Instruments, Novato, CA). Separate images for each wavelength were averaged over eight frames by a digital image processor (Axon Image Lightning; Axon Instruments, Foster City, CA) and subsequently converted pixel by pixel to a ratio image. Data collection rate (1 ratio image every 5-60 s), filter wheel position, and shutter opening/closing were controlled by a 133-MHz Pentium computer (Gateway 2000) running the latest update of version 2.x of Imaging Workbench (Axon Instruments). The ratio images were displayed in pseudocolor.Data were collected by electronically selecting regions of the image for quantitation. Cytosolic measurements were made from entire cells. When measurements were made on Golgi, only the brightest perinuclear regions were selected. Like FITC and BCECF, the fluorescence of pHERP excited at 490 nm increases with pH, whereas fluorescence at 440 nm is relatively insensitive to pH. Intensities were balanced with neutral density filters. Photobleaching was negligible during our experiments.
Methods describing calibration of cytosolic and organelle pH measurements have been reported previously (54).
Determination of Golgi Buffer Capacity
We made small, stepwise changes in the extracellular concentration of either NH
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(1) |
Calculation of H+ Permeability of Golgi Membranes
We used the previously described model (18), which accurately describes experimentally determined values of both steady-state and transient acidification of endosomes (56) and Golgi (61), and experimentally determined changes of pHG, pHC (measured in separate but identical experiments), andOne experimental protocol was to add the H+ V-ATPase
inhibitor bafilomycin to cells and model measured rates of alkalization of pHG while pHC remained constant (see Fig.
8A). The second approach was to acidify both cytosol and
Golgi cells with an NH4Cl pulse and then to model changes
of pHG while pHC also changed (see Figs. 6 and
8B). Without loss of generality, the equation that describes transient changes in pHG during the above-mentioned
experimental manipulations is
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(2) |
Using the previously described model of the V-ATPase (19), we found computed values for Jpump to be relatively constant over moderate luminal pH ranges but sensitive to changes in membrane potential (calculated as shown in Eq. 4), consistent with previous current-voltage data (7). In the present work, calculated Jpump changed by only 1-10% over the time course of any experiment.
Although the true nature of the proton leak,
Jleak, is not known, we modeled this transport
as simple, passive diffusion (23)
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(3) |
Because G will affect both Jpump
and Jleak, we included this effect by writing an
explicit form for
G in terms of the excess charge inside
the Golgi, the membrane of which was treated as a parallel plate
capacitor, and by assuming that the dominant counterions were
K+ and Cl
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(4) |
When K+ and Cl permeabilities were assumed to
be 10
5 cm/s (22),
G was
calculated (from Eqs. 2 and 3) to be <10 mV.
This result was consistent with recent experiments showing that the
Golgi and trans-Golgi network had relatively large
conductances to both K+ and Cl
and that
G was likely to be an unimportant determinant of either Jpump or Jleak (43,
61). Schapiro and Grinstein (43) arrived at this
conclusion by finding that [K+]G, measured to
be 107 mM using a null point method, was similar to the cytosolic
[K+] ([K+]C).
Equation 2 together with equations describing the passive
fluxes of K+ and Cl (from Eq. 3)
forms a set of three ordinary differential equations that are coupled
by the algebraic constraint of Eq. 4 and that uniquely
determine the time course of changes of ionic concentrations in the
Golgi given an initial set of conditions. We assumed
[K+]C = 130 mM and
[Cl
]C = 20 mM throughout each
experimental run, and initial [K+]G and
[Cl
]G were chosen to keep the Golgi close
to electroneutral (consistent with
G < 10 mV).
pHC and transient changes of pHG (similar to
those shown in Fig. 8, A or B) were measured in
separate experiments, and then the model was used (Eq. 3) to
predict the changes in Golgi acidification. This was done by varying
PH+, pump, and the
concentration of fixed negative charge in the lumen (B)
until a best fit to pHG was obtained. When results were
modeled from experiments using V-ATPase inhibitors (similar to Fig.
8A),
pump was set to zero and pHC
was held constant to match experimental conditions. The Na+-free, acid-loaded Golgi experiments (see Fig.
8B) were more complex because pHC and
pHG both varied. However, this procedure was advantageous because it allowed us to acidify the Golgi and keep pHC
relatively constant (see Figs. 6 and 8B). For these
experiments, we reported the average of six predicted
PH+ values, for each pHG
experiment, where each pHG run was fit against a different pHC run (see Possible errors in predicting
PH+). All searches were
performed with a Nelder-Mead algorithm, and the ordinary differential
equations were solved with a stiff method in both Matlab and Berkeley
Madonna software (38).
Although Eqs. 2-4 yielded estimates of
PH+, B, and the number of
active H+ V-ATPases (NOP = pump · S), we have reported only
predicted values for PH+. The model
yielded average values of B
140 mM and NOP
2,000. Within a particular data set, these two parameters had
counteracting effects: a decrease in
pump together with
an increase in B sometimes resulted in very similar pH fits.
This made it very difficult for the search algorithm to find a unique,
best fit, and the same data set with different initial search
conditions would yield very different values for B and
pump. In contrast, estimates of
PH+ were more robust to the initial
conditions of the search algorithm, and the same best value for
PH+ was usually found regardless of
the values of B and
pump. As mentioned above,
Jpump changed little for any given run. Thus the
parameter PH+ had the strongest
influence over the shape of the predicted pH curves.
It should be noted that in the limit of = 0 mV, Eqs.
2-4 can be replaced by an intuitive and simple expression for
the PH+ of the Golgi in terms of the
instantaneous rate of change of the Golgi, the H+ gradient,
and a few physical parameters
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(5) |
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Possible Errors in Predicting PH+
There are several uncertain parameters and assumptions that affect our estimates of PH+. We explored how errors in each of the following affected the predicted value of PH+.Surface-to-volume ratio.
We arbitrarily chose the value of 1.33 × 106
cm
1 for the surface-to-volume ratio (S/V) of
the Golgi obtained from rat kidney cells (30). Golgi
S/V in terminal tubule and acinar cells of the rat
submandibular gland (51) is an order of magnitude smaller. If the latter estimates were correct, our
PH+ values would be 10-fold larger;
thus our calculations provide a lower estimate. This S/V
parameter is most likely the largest source of error in our study.
Buffering. If the true buffering capacity is 20% larger or smaller than the value measured, which is consistent with our errors, then the true PH+ will be 20% larger or smaller than predicted.
H+ gradients.
In many of the bafilomycin-induced alkalization experiments,
pHC was assumed to be 7.55 (the average value). Varying the
assumed pHC by ±0.2 pH units for a typical run had a
±25% effect on the estimated PH+.
For the experiments where the Golgi has been acidified, each
pHG run (see examples in Figs. 6 and 8B) was fit
against six pHC runs that represented the entire set of
measured cytoplasmic recovery experiments. Most of these reported PH+ values had SD of 2 × 104, although one had a SD of 2 × 10
3.
Membrane potential.
The movement of counterions influences H+ movement through
effects on G as shown in Eq. 4. When the
Golgi membrane was assumed to have K+ and Cl
permeabilities >5 × 10
9 cm/s,
G was
calculated (from Eqs. 2 and 3) to be <10 mV.
When permeabilities to K+ and Cl
were
<5 × 10
9 cm/s, K+ and Cl
movements became quite slow, and
G began to affect
pHG. The present PH+
predictions remain unchanged for K+ and Cl
permeabilities >5 × 10
9 cm/s. Below this value,
movements of counterions resulted in transient changes of
G that affected calculations of
PH+. When
G was
arbitrarily varied from +7 mV to +50 mV and held constant over the time
course of the simulation, the predicted
PH+ values varied from +50% to
50% of the original value determined with our model of
G. Therefore, the PH+
values determined with Eqs. 2 and 3 were
relatively insensitive to
G.
Obtaining Initial Alkalization Rates and PH+ From the Literature
To compare our data with those from previous work, we scanned published figures as PICT files and used Data Thief (Computer Systems Group of the Nuclear Physics Section at the National Institute for Nuclear Physics and High Energy Physics, Amsterdam, The Netherlands) to extract data points of pHG vs. time. These data points were then used to obtain initial rates or were used with the model (Eqs. 2-4) to determine PH+ (see Table 2).Statistics
Unless otherwise specified, data have been presented as means ± SD. Experimental data were compared using unpaired Student's t-test (two-tailed). Differences were considered significant if P < 0.01.As an objective measure of the quality of our fits, the conventional root mean square (RMS) value was computed. When a particular run did not have an RMS value <0.175 pH units, it was dropped from the analysis. The average RMS value for all runs fit by the model was 0.06 ± 0.03 pH units (mean ± SD).
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RESULTS |
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pH Sensitivity and Golgi Targeting of pHERP
The ST-containing compartment was targeted with pHERP. We first created a chimeric protein of two commercially available mutant GFPs by placing GFPuv NH2-terminal to EYFP. The chimeric protein retains the dominant excitation peaks of the individual molecules at 397 nm (GFPuv) and ~500 nm (EYFP) when emission intensity is measured at 520 nm (Fig. 1). The peak at 500 nm was extremely sensitive to pH, whereas the trough at ~440 nm was relatively insensitive to pH, indicating that this molecule could be used as an excitation ratiometric (490/440) pH indicator. By leaving the luminal and transmembrane amino acids (1-70) of ST intact and replacing its cytosolic domain with pHERP, we were able to target the pH sensor to the Golgi lumen (Figs. 1 and 2). ST was used to target pHERP because it is one of the trans-Golgi enzymes (52) whose activity has been proposed to be altered in CF (2). Cells transiently expressing ST-pHERP displayed the characteristic perinuclear staining pattern typical of Golgi. A typical result for JME cells is shown in Fig. 2, and similar staining patterns were observed in HeLa, HBE, CFT1, and CFT1-CFTR cells (not shown). An in vivo calibration was performed at the end of every experiment (Fig. 1) by perfusing solutions containing nigercin (K+/H+ exchanger) and monensin (Na+/H+ exchanger) with different pH values onto the cells. The pKa of the ratio 490/440 was ~6.5 and thus was optimal for measuring pHG (Fig. 1).
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pHG Measured With pHERP
We performed control experiments on HeLa cells to compare measurements of pHG obtained using ST-pHERP with previous measurements of HeLa cell pHG obtained using other methods. As shown in the typical individual experiment and in the summarized data (Fig. 3), steady-state pHG was 6.4, in good agreement with previous results of 6.4-6.6 obtained using Golgi-targeted fluorescein and GFP-based pH sensors (26, 32, 61). When HeLa cells were pulsed with a solution containing 30 mM NH4Cl, pHG instantly alkalinized because of the entry of the weak base NH3. With removal of the NH4Cl, the cells acidified below basal levels and rapidly recovered (Fig. 3). These results were in good agreement with results obtained in Vero cells using FITC-verotoxin (27, 43) and in CHO cells using a mutant GFP (28).
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ST-pHERP consists of ST fused to the relatively pH-insensitive GFPuv
and pH-sensitive EYFP. EYFP has recently been reported also to be
sensitive to [Cl], with decreases in
[Cl
] causing increases in fluorescence
(57). Although the changes in EYFP fluorescence due to
changes in [Cl
] are smaller than those due to pH
(32, 57), we were concerned that changes in ST-pHERP
fluorescence ratio in vivo would be larger than predicted from
calibrations in which pH was varied but [Cl
] was held
constant. We therefore compared measurements of pHG made
with ST-pHERP with those obtained with GT-EGFP (13, 28, 32). EGFP is insensitive to [Cl
] but sensitive
to pH, with a pKa of 6.4 (32, 57).
When expressed in both CFT1 and CFT1-CFTR cells, GT-EGFP and ST-pHERP
yielded similar results when pHG was perturbed. When cells
expressing either ST-pHERP (see Fig. 6) or GT-EGFP (data not shown)
cells were pulsed with NH4Cl followed by removal and then
Na+-free treatment, pHG acidified and recovered
partially. Full recovery was completed only when Na+ was
added back to the cells. These results support the conclusion that
ST-pHERP was accurately reporting pHG. The fact that
ST-pHERP was more easily calibrated in terms of pHG made it
preferable to GT-EGFP.
CFTR and Steady-State pHG
As summarized in Table 1, pHG in respiratory epithelial cells was more alkaline (6.7-7.0) than in HeLa cells (6.4; see Fig. 3). Airway epithelial cells from
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The activity of CFTR is increased by cellular cAMP concentration, and
the effects of cAMP on pHG have been controversial
(32, 48). We therefore tested the effects of cAMP in CFT1
and CFT1-CFTR cells. In both cell types, there was little change in
pHG in response to cAMP whether experiments were performed
in the presence of 25 mM HCO
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Golgi H+ "Leak" and Potential Effect of CFTR
We determined the role of CFTR in controlling H+ leak across the Golgi membrane into the cytosol in CFT1 and CFT1-CFTR cells that had been treated with bafilomycin. Once the bafilomycin effect had reached a steady state with pHG and pHC approximately equal, cells were treated with an NH4Cl pulse followed by the Na+-free condition. This caused both the Golgi and the cytosol to acidify. We then added back extracellular Na+, which allowed both pHG and pHC to realkalinize. Rates of alkalinization of the Golgi lumen (using ST-pHERP) and cytosol (using BCECF) were measured in separate experiments. It was expected that if the H+ leak in the Golgi were large, removal of H+ from the cytosol by the Na+/H+ exchanger (NHE) in the plasma membrane would cause a similarly rapid alkalinization of pHG because H+ in the Golgi lumen would rapidly leak into the cytosol to be pumped out of the cell across the plasma membrane by the NHE. By comparing recovery rates of pHC and pHG measured using the same experimental protocol, we found that the Golgi in CFT1 cells alkalinized at a rate of 2.2 ± 0.7 × 10Golgi-to-Cytosol [H+] Gradient
We compared pHC and pHG from experiments in which the CFT1 cells were acidified with a 5-min pulse of 30 mM NH4+ followed by Na+-free Ringer. Removal of extracellular Na+ prevented the NHE in the plasma membrane from pumping out the accumulated H+. On average, the steady-state pHC of CFT1 cells was 7.5 ± 0.2 (n = 68), while under the conditions used to acidify the cells, pHC dropped to 6.4 ± 0.2 (n = 55). Under the same conditions, average pHG dropped from 6.7 ± 0.2 (n = 45) to 5.9 ± 0.3 (n = 31) and then partially recovered with a single-exponential time course (see Fig. 6C). The average [H+] gradient (Golgi to cytosol) returned to 2 × 10Golgi H+ Permeability and Buffer Capacity
When the H+ V-ATPase was inhibited by bafilomycin, the Golgi rapidly alkalinized with an initial rate of 8.6 ± 7.8 × 10
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It is well established that the cytosolic buffering capacity varies
with pH (58, 59), which is a function of the
pKa and concentration of titratable groups
(41). Previous measurements of Golgi buffer
capacity have reported only one value (42, 61) or claimed
that G was constant between pH 6 and 7 (13). We felt that it was necessary to measure
G over a wide pH range to calculate
PH+ of the Golgi at the various pH values observed in our experiments. Values for
G were
obtained by first treating the cells with bafilomycin (250 nM) and then titrating in various amounts of weak base (NH3) or weak
acid (HOAc). We measured
G at pH < 7 by following
the bafilomycin treatment with an acidification step (NH4Cl
prepulse followed by incubation in Na+-free solution) and
then adding either NH4Cl or HOAc to the
Na+-free solutions. A typical experiment in which
G was measured is shown in Fig.
7A. Data summarizing
measurements of
G as a function of pH are summarized in
Fig. 7B. Buffer capacities were grouped into 0.2-pH unit
buffering domains for simplicity. At pH 6.9 ± 0.02 (mean ± SE),
G = 17.2 ± 4 mM per pH unit (mean ± SE), in good agreement with previous results of 10-40 mM per pH
unit (13, 42, 61). At higher and lower pH values,
G varied, and the variation was well fit by a single
exponential (Fig. 7B), which allowed us to extrapolate to
acidic values that were attained in some measurements of
PH+.
By using the experimentally determined G and a model of
pHG (Eqs. 2-4), we were able to estimate
the PH+ of bafilomycin-treated cells
when pH > 6.4 (Fig.
8, A and
C). Additionally, the model allowed estimates of
PH+ under various conditions where
organelle and pHC varied over a considerable range:
recovery of both pHG and pHC were measured
after acidification (using an NH
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DISCUSSION |
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ST-pHERP for Measuring pHG
By fusing EYFP and GFPuv, each having different excitation spectra and different pH dependencies, we created the genetically targeted ratiometric pH indicator pHERP (Figs. 1 and 2). A variety of other techniques have been used to measure pHG, but each has limitations that our technique attempted to improve. Isolated Golgi vesicles are rarely pure and can be damaged and lose soluble regulatory factors during preparation. Electron microscopic methods (2) allow only imprecise quantitation at single time points. Microinjection of dye-filled liposomes (48, 49) is laborious and invasive, and since the dyes are delivered to the Golgi relatively slowly and then removed relatively rapidly at 37°C, measurements can be performed only during short time periods. FITC-labeledGFPs have many advantages as organelle pH sensors: any organelle can be targeted, a variety of mutants with a wide range of pKa values (5.5-7.0) is available (28, 32, 55), and changes of pH can be measured with little background signal, no diffusive loss of fluorophore, minimal bleaching, and no requirement for exogenous dyes that require hydrolysis and might lead to cytotoxicity. Despite these many advantages, calibrating the single-wavelength intensity changes of these GFPs is problematic, because artifacts can arise when apparent fluorophore concentration changes and pH does not. Miesenbock et al. (34) overcame this problem by developing a GFP with a spectrum that shifted with pH, thereby providing an excitation ratiometric pH indicator. Our development of pHERP allows an alternative approach that has many of the same advantages as pHlorin for measuring pHG.
CFTR, Counterion Conductance, and Membrane Potential in Determining pHG
As measured with pHERP, HeLa cells had an average pHG of 6.4 (Fig. 3), which was nearly identical to previous measurements obtained using a variety of other methods in HeLa, fibroblast, and Vero cells (27, 32, 42, 43, 61). In contrast, the Golgi of airway epithelial cells was more alkaline (pHG = 6.7-7.0) than in HeLa cells. Although, there was no clear correlation between the presence of CFTR and pHG when all the airway epithelial cells were compared, there was a significant difference when the genetically matched CFT1 cells were compared (Table 1). Cells expressingAl-Awqati and colleagues (1, 2) suggested that the Golgi
Cl permeability was able to limit H+ flux by
dissipating the membrane potential. Subsequent data in the literature
indicated that conductances to both K+ and Cl
in the Golgi and trans-Golgi network are so high that
membrane voltage is small and unimportant in determining organelle pH
(10, 43, 61). This is consistent with observations that
neither ouabain nor its membrane-permeant analog acetyltrophanthidin
have any effect on pHG in HeLa cells, which have no CFTR
(Wu M and Machen T, unpublished observations), implying that the
Na+-K+-ATPase activity was low or
nonexistent. Our model confirmed and extended the conclusions
regarding the small Golgi membrane potential and also allowed us to
estimate the number of counterion channels necessary to achieve this
goal. Our model showed that K+ and Cl
permeability could be decreased to as low as 5 × 10
9 cm/s before membrane potential became a factor in
fitting the pH transients (see METHODS and Fig. 8). For a
Golgi with a surface area of 8×10
6 cm2
(30), these very low permeabilities corresponded to
K+ and Cl
conductances <20 pS for the entire
Golgi, which could be achieved with between one and two K+
or Cl
channels. Calculation of K+ conductance
was performed by using the Goldman-Hodgkin-Katz (GHK) current equation
(23) after compensating for the assumed surface area of
the Golgi. Conductance was calculated as the slope of the current with
respect to voltage at 0 mV, assuming
[K+]G = 110 mM (43) and
[K+]C =130 mM. In this circumstance, it is
likely that eliminating CFTR has no effect on pHG, because
other K+ and Cl
channels can provide
sufficient counterion conductance to ensure that membrane potential in
the Golgi is small and therefore has little effect on H+
pumps or leaks. Two other studies have provided evidence for the
presence of Cl
channels in the Golgi: Nordeen et al.
(35) found an anion channel in enriched Golgi fractions,
and Schwappach et al. (46) found that Gef1p (the only
yeast ClC Cl
channel) localizes to the Golgi.
Additionally, elimination of Gef1p from yeast does result in altered
cation homeostasis (17) but does not effect Kar2p
secretion or glycosylation of invertase, both dependent on an acidic
Golgi (46).
It might be argued that since our experiments were performed on single cells, they are not comparable to those performed on confluent monolayers. We attempted to make pHG measurements on confluent cells grown on filters, but the background fluorescence contributed by the filter exceeded the specific fluorescence of pHERP when excited at 440 and 490 nm under acidic conditions. Therefore, it was impossible to perform experiments when cells were grown on filters. Because of technical difficulties, we were able to obtain only one pHG measurement from a CFT1-CFTR cell in a confluent patch. Results from this experiment clustered with most of the other CFT1-CFTR data (Fig. 8C). We also note that CFTR transits through the Golgi and becomes functional in the plasma membrane of a variety of cells grown on glass either as single cells or in confluent patches (14, 15). We therefore believe, but have not proven, that pHG experiments performed on isolated cells grown on glass reflect those of confluent cells.
Roles of H+ Pump, pH-Dependent Leak, and pHC in Determining pHG
It has been proposed that pH of the Golgi and other organelles in the secretory pathway is determined primarily by a balance between the active accumulation of protons by the H+ V-ATPase and the passive loss of H+ through leaks (10, 61) and that the flux of protons is balanced at pHG (13). The previous work has not accurately distinguished between effects of the pH gradient and the PH+ in determining H+ fluxes across the Golgi. Farinas and Verkman (13) showed that as pHG decreased, the passive H+ flux out of the Golgi increased. Regardless of the true nature of the H+ leak pathway, this is not a surprising finding because as the luminal [H+] is increased, the efflux rate will most probably increase. The present work has extended these conclusions by estimating PH+ and showing that this permeability decreased as pH decreased. We also showed that Golgi buffer capacity was approximately equal to, and showed a pH dependency similar to, that of the cytosol (see Refs. 58 and 59), which supports the assumption thatAs summarized in Fig. 8C,
PH+ in Golgi of CFT1 cells was
~7.5 × 104 cm/s when pHG was 6.5, and
there was little difference to CFTR-corrected CFT1 cells. Similar
values for Golgi PH+ of HeLa, Vero,
and CHO cells were calculated using our model (Eqs.
2-4), and bafilomycin-induced alkalinization data were
obtained from the literature (Table 2). These
PH+ values are large compared with
typical permeability values for Na+, K+, and
Cl
: 10
12 cm/s for a membrane without
channels (37) and up to 10
5 cm/s for a
membrane with channels (22, 24). However, our estimates of
PH+ compare favorably with
PH+ in lipid bilayers and liposomes:
10
7-10
2 cm/s (8, 21, 37, 40).
Similar to the data presented for
PH+ of the Golgi, artificial lipid
bilayers also have pH-dependent PH+
(8, 21). It therefore seems possible that
PH+ of the Golgi might be solely due
to simple H+ diffusion through membranes, and variations in
PH+ could therefore be due to
differences in fatty acids (which act as weak acid shuttles) and/or
lipids (which can form water "wires") (8, 20, 21) in
the Golgi of different cells. In addition, H+ channels may
also provide leak pathways for H+ in the Golgi
(43). However, estimates of
PH+ > 5 × 10
1 cm/s for plasma membranes of lung alveolar cells with
H+ channels [using GHK, reported pH gradients, and
measured current densities (6, 9)] was 100 times larger
than the largest PH+ observed in the
present experiments. Finally, Na+/H+ exchange
in the Golgi might contribute to the H+ permeability.
However, Schapiro and Grinstein (43) showed that NHEs play
no role in the efflux of H+ from the Golgi. Also, we have
found that treatment of CFT1 cells with hexamethylene amiloride
(membrane-permeant analog of amiloride) had no effect on steady-state
pHG (data not shown).
A consequence of the Golgi having a relatively large, pH-dependent
PH+ is that pHG will be
a complicated function of pHC,
PH+, and H+ pumping. The
role of pHC in determining pHG is shown by
experiments in which cells were treated with
NH3/NH
|
The role of H+ pumps in setting pHG was
suggested by the fact that HeLa cells had a more acidic Golgi than
respiratory epithelial cells (Table 1). This was not solely due to the
more acidic pHC in HeLa cells because the Golgi-to-cytosol
[H+] gradient was larger in HeLa (3.6 × 107 M) than in respiratory epithelial cells
(0.75-1.7 × 10
7 M) (Table 1). Given that
PH+ of HeLa and other cells was
similar in the pH 6.4-6.6 range (Fig. 8C), the most likely explanation is a difference in H+-pump activity
between HeLa cells and CFT1 cells. This explanation would also apply to
the difference between pHG of CFT1 and CFT1-CFTR cells,
although why or how various pump activities would arise in different
cells is not clear.
|
|
A potential regulatory role for the H+ pump has been considered previously by Kim et al. (27), who found that pHG was constant in the face of altered pHC. In contrast, the present data show that when pHC was acidified from an average of 7.5 to 6.5, pHG acidified on average from 6.5 to 6.2. These averages predict that the Golgi-to-cytosol [H+] gradient is constant in the face of an apparently decreasing PH+ and constant H+ pump. It will be necessary to measure pHC and pHG in the same cells to determine whether H+ pump and leak may be regulated. It also will be important to determine the roles of pHC, H+ pump, and pH-dependent H+ leak in generating the wide range of pH values found in other acidic organelles [e.g., endosomes (pH 6.0-6.5), lysosomes (pH 4-5), and secretory granules (pH 4-5)] in different cells.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Minnie Wu for useful discussions and for providing measurements of average pHC for HeLa cells. We thank Eric Wunderlich for providing measurements of average pHC for JME cells.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-51799 (to T. Machen), and grants from Cystic Fibrosis Research, Inc. (to G. Chandy and H.-P. H. Moore). M. Grabe was supported by National Science Foundation Grant DMS9220719 (to George Oster, whom we thank for useful discussions).
Present address of G. Chandy: Dept. of Molecular Pharmacology, Stanford, CA 94305-5175.
Address for reprint requests and other correspondence: T. E. Machen, 231 LSA, Univ. of California, Berkeley, CA 94720-3200 (E-mail: machen{at}socrates.berkeley.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 26 October 2000; accepted in final form 16 April 2001.
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