1Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3200; 2Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Toyama City, Toyama 930-0194, Japan; and 3Graduate Group in Bioengineering, University of California, Berkeley, Calilfornia 94720-1762
Submitted 9 January 2003 ; accepted in final form 13 March 2003
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ABSTRACT |
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pHluorin; H+ v-ATPase; trans-Golgi network; organelle pH; H+ permeability
First, the Golgi is more acidic than the ER because the Golgi has an active H+ v-ATPase and a lower passive H+ permeability than the ER. This H+ permeability in the Golgi appears to be blocked by Zn2+ (33), a well-known blocker of H+ channels/conductance in the plasma membranes of multiple cell types (3, 6, 7, 9, 12, 15, 18, 38).
Second, the Golgi and TGN, like the ER, have large apparent permeabilities to both K+ and Cl so that these ions are likely to equilibrate rapidly with ions in the cytosol and membrane potential is likely to be small (<10 mV) (5, 11, 19, 21, 33, 40). Thus membrane potential seems not to play an important role in regulating pH in the Golgi and TGN, and the pH values of these compartments are likely to be maintained primarily by H+ pumping by the H+ v-ATPase balanced by a passive H+ leak driven solely by the differences in pH between the Golgi or TGN lumen and the cytosol.
Third, recycling endosomes appear to be a heterogeneous organelle with variable pH depending on the population in which measurements are made (4, 13, 16, 36, 41): endosomes that contain the transferrin receptor appear to be less acidic than cellubrevin-containing endosomes (pH 6.5 vs. pH 6.0), and this difference may arise because a subpopulation of endosomes (transferrin receptor-positive endosomes) contains the electrogenic Na+/K+-ATPase and generates a lumen-positive membrane voltage that inhibits H+ pumping by the electrogenic H+ v-ATPase (4, 16, 36).
An interesting and important question remains unanswered about pH regulation in these organelles. Different cells have different ion and acid-base transporters to accomplish different physiological functions. Because these transporters likely become active in the ER (28, 29, 31), do they affect the pH-regulatory properties of the Golgi and other organelles of the secretory pathway as the transporters are trafficked to the plasma membrane? A particularly dramatic example of this problem exists in the Golgi of parietal cells of the stomach. These cells express H+/K+-ATPase pumps in the apical plasma membrane. When these pumps and K+ and Cl channels are active in the apical membrane, the H+/K+-ATPases are capable of accumulating isotonic HCl, pH 0.8, in the gastric lumen (10, 26, 32). Because H+/K+-ATPases are continually synthesized to replace degraded pumps and the Golgi has large, inherent permeabilities to both K+ and Cl (thereby providing KCl needed for the operation of the pump), it might be expected that the H+/K+-ATPases present in the Golgi are functionally active. What effect do these pumps have on pHG and other organelles of the secretory pathway in parietal cells? We attempted to answer this question for the TGN and recycling endosomes using the genetically targeted, pH-sensitive green fluorescent protein (GFP) derivatives TGN38-pHluorin and synaptobrevin (SV)-pHlourin (24).
Because parietal cells are difficult to transfect, we used HEK-293 cells
that had been stably transfected with both the - and
-subunits of
human H+/K+-ATPase
(H+/K+-
,
cells; see Ref.
22). These cells express
functional H+/K+-ATPase in the plasma membrane, as shown
by the fact that the cells exhibit 86Rb+ (K+
substitute) uptake and Na+-independent H+ secretion that
is blocked by SCH28080 (specific blocker of the
H+/K+-ATPase). In addition, evidence from membrane
fractionation indicated that a large fraction of the
H+/K+-ATPase was expressed in organelles. Pulsechase
labeling studies showed that the ATPase had a half-life of
12 h, so
steady-state expression in the plasma membrane required continual synthesis
(22). We confirmed the
organelle localization of the pHluorins and the
H+/K+-ATPase using immunofluorescence and then tested
for the activity of the H+/K+-ATPase by measuring pH in
the Golgi and recycling endosomes during treatments with SCH28080 (specific
inhibitor of H+/K+-ATPase). Experiments were performed
with normal Ringer's solutions bathing the cells to assure that conditions in
the TGN would be as close to the in vivo state as possible. We used 50 µM
of this inhibitor to assure that all the H+/K+-ATPase
pumps that were present in the TGN and recycling endosomes would be inhibited.
For comparison, we also tested the effects of bafilomycin, the specific
blocker of the H+ v-ATPase. Results from these experiments
indicated that the H+/K+-ATPases that are trafficked to
the plasma membrane are active in both the TGN and recycling endosomes, but
their activity is much lower than that of endogenous H+ v-ATPase,
so trafficking pumps have little effect on the pH of the organelles they
traverse on their way to the plasma membrane.
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METHODS |
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H/K-,
and mock cells. cDNAs of
- and
-subunits of H/K-ATPase were prepared from rabbit gastric mucosa as
described elsewhere (2). The
- and
-subunit cDNAs were digested with EcoRI and
XhoI. The obtained fragments were each ligated into the pcDNA3 vector
(for the
-subunit cDNA) or pcDNA3.1(+) (Zeo) (for the
-subunit
cDNA) treated with EcoRI and XhoI. DNA sequencing was done
by the dideoxy chain termination method using an ABI Prism 377 DNA sequencer
(Applied Biosystems, Tokyo, Japan).
Stable cell lines expressing - and
-subunits of gastric
H+/K+-ATPase were generated as follows. HEK-293 cells
were cultured as described previously
(1,
2). HEK-293 cells were
transfected with pcDNA3-H+/K+
cDNA by lipofection
using an Effectene transfection reagent, and stable cell lines were selected
in the presence of 1 mg/ml Geneticin (G-418 sulfate). Single colonies were
isolated, expanded, and maintained in the presence of 0.5 mg/ml Geneticin. The
expression of the
-subunit was confirmed by immunofluorescence and
Western blot. The H+/K+-expressing cells
(H+/K+
,
cells) were then generated by
transfecting HEK cells stably expressing the
-subunit with pcDNA3.1(+)
(Zeo)-H/K-
cDNA construct. Stable cell lines were selected in the
presence of 0.2 mg/ml Zeocin plus 0.5 mg/ml Geneticin. Single colonies were
isolated, expanded, and maintained in the presence of 0.5 mg/ml Geneticin and
0.1 mg/ml Zeocin. The expression of the H+/K+ subunits
in H+/K+-
,
cells was confirmed by
immunofluorescence and Western blot. H+/K+-mock cells
were transfected with empty vectors and then treated identically to the
H+/K+-
,
cells.
Cell culture. Cells were plated onto plastic culture dishes or collagen-coated coverslips and maintained in a 37°C incubator with 5% CO2. All media were supplemented with penicillin, streptomycin, and glutamine. All HEK-293 cells were maintained in DMEM supplemented with 10% FBS.
Transfections of
H+/K+-,
and mock
cells with TGN38-pHluorin or synaptobrevin-pHluorin. HEK-293-mock and
H+/K+-
,
cells were transiently transfected
with TGN38-pHluorin or synaptobrevin-pHluorin plasmids using the Effectene
transfection reagent (Qiagen, Germany). Cells were split onto a
collagen-coated 10-cm culture dish at 50% confluency 68 h before
transfection. They were then transfected with 2 µg of DNA complexed with 16
µl of enhancer and 60 µl of Effectene reagent. After 24 h, cells were
split onto collagen-coated glass coverslips and incubated with growth medium
containing 6 mM Na-butyrate for 1316 h to induce gene expression. Cells
were returned to normal growth medium lacking Na-butyrate 1 h before imaging
experiments.
Solutions. Ringer's solution contained (in mM) 141 NaCl, 2 KCl, 1.5 K2HPO4, 1 MgSO4, 10 HEPES, 2 CaCl2, and 10 glucose brought to pH 7.4 with NaOH. Calibration solutions contained (in mM) 70 NaCl, 70 KCl, 1.5 K2HPO4, 1 MgSO4, 10 HEPES, 10 MES, 2 CaCl2, 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 experiments in which cells were acidified using a brief NH4 treatment, 30 mM NH4Cl was substituted for 30 mM NaCl. Bafilomycin was used at 100500 nM (yielding equivalent effects) and SCH28080 at 50 µM.
Fluorescence ratio imaging of pH in the cytosol, TGN, and recycling endosomes of HEK-293 cells. General methods used in this laboratory for measuring organelle pH values have been described previously (5, 39). Briefly, TGN labeled with TGN38-pHluorin, recycling endosomes labeled with SV-pHluorin, and cytosol labeled with 10 µM BCECF-AM (Molecular Probes, Eugene, OR) were monitored in separate experiments using digitally processed fluorescence ratio imaging. Labeled cells were placed in an open perfusion chamber on an inverted microscope (Zeiss IM35 or Nikon Diaphot). The solutions used for perfusing the cells, the chamber holding the cells, and the objective were all heated to 37°C. A x40 oil-immersion objective (1.3 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 silicon-intensified tube camera. Emission images of the cells were collected through a 530-nm long-pass filter during sequential excitation at either 410 and 470 or 380 and 440 ± 10 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 (one ratio image every 5180 s), filter wheel position, and shutter opening/closing were controlled by a 133-MHz Pentium computer (Gateway 2000) running the version 2.x of Axon's Imaging Workbench.
Data were collected by electronically selecting regions of the image for quantitation. Cytosolic measurements were made from entire cells. When making measurements on TGN and recycling endosomes, only the brightest perinuclear regions were selected. Intensities were balanced with neutral density filters. Photobleaching was negligible.
Methods describing calibration of cytosolic and organelle pH measurements have been reported previously (36, 39). Calibrations of TGN- and SV-pHluorin showed pH-ratio relationships that were nearly identical to those in Miesenbock et al. (24).
Immunofluorescence. HEK-mock and
H+/K+-,
cells were transiently transfected
with TGN38-pHluorin or synaptobrevin-pHluorin plasmids as described above. Two
days after transfection, they were fixed with 3% paraformaldehyde for 20 min
and permeabilized with 0.1% Triton X-100 in PBS for 5 min.
H+/K+-ATPase was detected by incubation with monoclonal
antibody 2G11 (courtesy of Dr. John Forte, Univ. of California, Berkeley)
against the cytoplasmic domain of
-subunit (1:1,000 dilution), followed
by rhodamine-conjugated goat anti-mouse antibodies (1:25 dilution). For some
coverslips, the recycling endosomes were stained by incubating cells in
serum-free medium for 30 min at 37°C followed by incubation with 50
µg/ml rhodamine-conjugated human transferrin (Molecular Probes) in the same
medium for1hat 37°C. GFP and rhodamine fluorescence was visualized using a
Zeiss Axiophot fluorescence microscope.
Statistics. Unless otherwise specified, data are presented as means ± SE. Experimental data were compared using unpaired Student's t-test (two-tailed). Differences were considered significant if P < 0.05.
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RESULTS |
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Antibody to the -subunit of the H+/K+-ATPase
was used to test for the subcellular localization of the pump in
H+/K+-
,
and mock cells transiently
transfected with TGN38-pHluorin. In H+/K+-
,
(Fig. 1Ab), but not in
mock cells (Fig. 1Bb),
H+/K+-ATPase staining was observed in both the plasma
membrane and also in intracellular organelles. Some internal ATPase
colocalized to the same compartment where TGN38-pHluorin was localized
(Figs. 1Ab and
1Ac).
Similar experiments were performed on
H+/K+-,
and mock cells that had been
transiently transfected with SV-pHluorin to compare the cellular localization
of SV-pHluorin and that of the TGN (identified using TGN38) and recycling
endosomes (identified using rhodamine-transferrin). SV-pHluorin is a marker of
synaptic vesicles in neuronal cells. It shares similar subcellular trafficking
patterns as cellubrevin (8),
and like cellubrevin it is targeted to endosomes in HEK-293 cells.
Rhodamine-transferrin colocalized to the same compartment as SV-pHluorin
(Fig. 2, AaAc)
but not to the compartment where TGN38-pHluorin was localized
(Fig. 2,
BaBc). In addition, some
H+/K+-ATPase appeared to colocalize with SV-pHluorin in
the H+/K+-
,
cells
(Fig. 3, AaAc),
whereas there was no H+/K+-ATPase staining in the
SV-pHluorin-transfected mock cells (Fig. 3,
BaBc). These data showed that the
H+/K+-ATPase was expressed in the plasma membrane and
also in both the TGN and recycling endosomes. The next set of experiments was
designed to test for H+/K+-ATPase pumping by testing for
SCH28080-induced changes in cytosolic pH and in luminal pH of the TGN and
recycling endosomes.
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H+/K+-ATPase is active in the plasma membrane of
H+/K+-,
cells. Previous work
(22) showed that
H+/K+-
,
cells exhibited Rb+
influx and H+ efflux consistent with the presence of active
H+/K+-ATPase in the plasma membrane. SCH28080-sensitive
Rb+ uptake amounted to
0.3 nmol · 106
cells1 ·
min1. Assuming that the
H+/K+-ATPase is a neutral one-for-one pump with a
turnover rate of 300/s, this rate of Rb+ uptake indicated that
there were 10,000 active H+/K+-ATPase pumps in the
plasma membrane of each cell.
Further measurements of cytosolic pH (pHC) by Kimura et al.
(22) showed
Na+-independent (i.e., occurred in absence of Na+ in the
medium), SCH28080-sensitive pHC recovery from an acid load occurred
at a rate of 0.06 pH/min. We repeated these experiments here using procedures
similar to those of Kimura et al.
(22).
H+/K+-,
cells were acid-loaded by a 5-min
treatment with 30 mM NH4-containing Ringer's solution followed by
incubation in Na+-free Ringer's solution
(N-methyl-D-glucamine replaced Na+). Under
these Na+-free conditions, pHC recovered toward baseline
at rates of 0.05 and 0.07 pH unit/min (n = 2 experiments), and this
recovery was totally blocked by 50 µM SCH28080. This
Na+-independent, SCH28080-blockable pHC recovery was
absent in two similar experiments on mock cells (data not shown). Assuming
that the buffer capacity of H+/K+-
,
cells
was 25 mmol · liter1 · pH
unit1
(35) and cell volume was
1012 liter, the pHC recovery rate of
0.06 pH/min (22) was used to
calculate that each H+/K+-
,
cell expressed
50,000 active pumps in the plasma
membrane.1 Thus these
previous experiments indicated that H+/K+-
,
cells expressed between 10,000 and 50,000 H+/K+-ATPases
in the plasma membrane. Despite the presence of these "extra" acid
extruders in the plasma membranes of
H+/K+-
,
as compared with mock cells, Kimura
et al. (22) found that
H+/K+-
,
cells were not significantly more
alkaline than mock cells, indicating that the activity of other pH-regulatory
mechanisms dominated over the H+/K+-ATPase in
maintenance of steady-state pHC. Similarly, parietal cells that
have been stimulated to secrete HCl also exhibit only small changes in
pHC due to the fact that anion- and cation-coupled acid-base
transporters in the basolateral membrane are able to acidify the cytosol at
the same rate that the H+/K+-ATPase alkalinizes the
cytosol (25,
27,
37).
H+/K+-ATPase in the TGN: pH measurements using
TGN-pHluorin. It was expected that if the
H+/K+-ATPase was active in the TGN, then the TGNs of the
H+/K+-,
cells would be more acidic than the
mock cells. However, similar to the cytosol of
H+/K+-
,
vs mock cells, the TGNs of
H+/K+-
,
cells and mock cells were nearly
identical: average pHTGN was 6.36 in H/K-
,
cells and
6.34 in mock cells (Table 1).
These values of pHTGN were similar to pHTGN 6.21
measured by Miesenbock et al.
(24) and 6.06.7
measured by Poschet et al.
(30) but somewhat more
alkaline than pHTGN 5.9 measured by Kim et al.
(20). We concluded that the
TGN was roughly one pH unit more acidic than the cytosol in both
H+/K+-
,
cells and mock cells, although
pHTGN in individual cells varied between pH 5.8 and pH 6.8.
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One possible explanation for the fact that the TGNs of
H+/K+-,
cells and mock cells had very
similar average pH values was that the H+/K+-ATPase was
not active in the TGN. Alternatively, there could be an SCH28080-inhibitable
acidification mechanism in H+/K+-
,
cells,
but pHTGN was variable enough that small differences could not be
detected using different sets of cells. We therefore tested for the effects of
SCH28080 on steady-state pHTGN. This
H+/K+-pump blocker caused a slow, but consistent
alkalinization of pHTGN in
H+/K+-
,
cells
(Fig. 4A) but not in
mock cells (Fig. 4B).
Subsequent addition of bafilomycin caused a more rapid and larger increase in
pHTGN to pH 7.07.5 (Fig.
4, A and B), similar to values that were
observed in the cytosol in both H+/K+-
,
cells and mock cells (22).
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The effects of these inhibitors on steady-state pHTGN of
H+/K+-,
and mock cells are summarized in
Table 1. SCH28080 caused
pHTGN to increase (at an average rate of 0.20 ± 0.03 x
103 pH/s) to a new steady-state value that was on
average 0.12 pH units more alkaline than the initial pHTGN in
H+/K+-
,
cells, whereas there was no
significant effect of SCH28080 on pHTGN in mock cells. Further
addition of bafilomycin caused pHTGN to increase by
0.6 pH
units (at initial rates of 1.95 ± 0.07 x
103 pH/s and 1.61 ± 0.13 x
103 pH/s, respectively) in
H+/K+-
,
cells and mock cells. Similar
results were obtained when H+/K+-
,
cells
were treated first with bafilomycin followed by SCH28080: initial pH was 6.34
± 0.13 (1,
6); bafilomycin caused
pHTGN to increase by 1.21 ± 0.16
(1,
6) pH units, and subsequent
addition of SCH28080 caused pH to increase a further 0.23 ± 0.08
(1,
6) pH units; final
pHTGN was 7.62 ± 0.08
(1,
6). Thus bafilomycin caused
large, rapid increases and SCH28080 caused smaller and slower increases in
pHTGN. These results were consistent with the idea that the
H+/K+-ATPase was active in the TGN and contributed to a
small but significant acidification of this organelle in the steady state.
H+/K+-ATPase in recycling endosomes:
pHRE measurements using SV-pHluorin. As shown in
Figs. 5, A and
B, experiments were also performed on
SV-pHluorin-transfected H+/K+-,
cells and
mock cells to test for the effects of SCH28080 and bafilomycin. Similar to the
results obtained for the TGN, average pHRE was similar in both
H+/K+-
,
cells (average pHRE
6.47) and mock cells (average pHRE 6.37; see
Table 2). SCH28080 caused
pHRE to alkalinize slowly but consistently in
H+/K+-
,
cells
(Fig. 5A) but not in
mock cells (Fig. 5B).
Subsequent addition of bafilomycin caused a more rapid and larger increase in
pHRE to pH 7.07.5, similar to effects on
pHTGN.
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The effects of these inhibitors on steady-state pHRE of
H+/K+-,
cells and mock cells are summarized
in Table 2. SCH28080 caused
pHRE to increase by an average of 0.22 pH units (at an average rate
of 0.28 ± 0.24 x 103 pH/s) in
H+/K+-
,
cells, and further addition of
bafilomycin caused pHRE to increase by 0.42 pH units (at a rate of
1.61 ± 0.40 x 103 pH/s) in
H+/K+-
,
cells. There were no significant
effects of SCH28080 on pHRE in mock cells, whereas bafilomycin
caused pHRE to alkalinize as expected
(Fig. 5B and
Table 2).
These experiments indicated that the H+/K+-ATPase was also active in recycling endosomes, although, like the situation in the TGN, inhibition of this pump had smaller effects on pHRE compared with the effects of bafilomycin. The H+/K+-ATPase requires the presence of K+ at its lumen-facing aspect to exchange one-for-one with H+ that are transported from the cytosol into the lumen. Because the K+ permeability properties of recycling endosomes have not been tested, it was possible that the recycling endosomes (unlike the TGN, which has large K+ and Cl permeability; see Refs. 11 and 33) may not have had enough K+ in the lumen to permit full activity of the H+/K+-pumps. We therefore tested whether valinomycin treatment would enhance H+/K+-ATPase activity by allowing K+ to permeate from the cytosol into the lumen of the endosomes, thereby increasing availability of K+ at the lumenal aspect of the pump. Cells were treated first with bafilomycin and then valinomycin, and pHRE attained a new steady state of 6.92 ± 0.08 (3, 26). Further addition of SCH28080 caused pHRE to increase (at a rate of 0.25 ± 0.17 x 103 pH/s) up to a new steady state that was more alkaline by 0.23 ± 0.04 pH units (3, 26), similar to the effects observed when SCH28080 was added before bafilomycin (Table 2). There were no significant effects of SCH28080 on pHRE in mock cells. These results indicated that the H+/K+-ATPase that was active in recycling endosomes was not limited in activity by the availability of K+ in the lumen of this organelle.
Relative rates of H+ pumping by H+ v-ATPase and
H+/K+-ATPase in the TGN and recycling endosomes.
Data presented in the previous sections showed that the
H+/K+ ATPase pump was present and active in both the TGN
and recycling endosomes in H+/K+-,
cells,
but the activity of this pump was less than that of the H+ v-ATPase
that was present endogenously in these organelles. The relative pumping
activities due to these two pumps can be estimated from the alkalinization
that occurred after treatment with SCH28080 and bafilomycin because the two
inhibitors were specific to the two H+ pumps. We reasoned that
blocking the H+ v-ATPase with bafilomycin would lead to an
alkalinization due to the loss of activity of this transporter, and similarly
blocking the H+/K+-ATPase with SCH28080 would lead to an
alkalinization due to the loss of activity of this transporter. Furthermore,
because the TGN appears to have large permeabilities to both K+ and
Cl and likely a small membrane potential
(33; also see Ref.
39), the relative numbers of
H+ v-ATPases and H+/K+-ATPases can be
estimated (assuming their turnover rates are approximately equal) from the
relative changes in [H+] that occurred after each treatment, i.e.,
steady-state [H+] will be directly proportional to the number of
H+ ATPases present in the membrane (see Ref.
17). As shown in the last line
of Table 1, when the
[H+] due to bafilomycin treatment was compared with
[H+] due to SCH28080 treatment in the same experiments, the
ratio was 2.1, indicating that there were 2.1 times as many
bafilomycin-sensitive H+ v-ATPases as
H+/K+-ATPases in the TGN of
H+/K+-
,
cells.
Using similar reasoning and the data in
Table 2 for the recycling
endosomes, it can be concluded that the ratio of H+
v-ATPase/H+/K+-ATPase ratio was 0.8, i.e., that there
were approximately equal numbers of H+ v-ATPases and
H+/K+-ATPases in the recycling endosomes of
H+/K+-,
cells. Although this conclusion is
much less secure because the permeability properties for K+ and
Cl of recycling endosomes have not been determined and the
role of membrane potential in this compartment remains unknown, the results
clearly showed that the H+/K+-ATPase contributed to pH
regulation in recycling endosomes.
An "inefficient" H+ pump/leak system
stabilizes the pH of trafficking organelles. It might have been expected
that steady-state pHTGN in H/K ,
cells would have been
much more acidic than the values of pH 6.06.5 observed in mock cells
(Table 1) and in other cell
types (11,
24,
30): the
H+/K+-ATPase pump has the capability to generate a
highly acidic fluid with pH 1.0 or less, the TGN has the required
K+ and Cl conductances required for the
H+/K+-ATPase to transport H+, and this pump
was active in the TGN during its trafficking to the plasma membrane. Instead,
based on the effects of SCH28080 on pHTGN, the
H+/K+-ATPase appeared to acidify the TGN of H/K
,
cells by only 0.12 pH units compared with the TGN in mock cells
(Table 1).
An explanation for the similar values of pHTGN in the
H+/K+ ,
and mock cells comes from a
consideration of the overall H+ pump and leak characteristics of
the TGN and Golgi in mock cells (Fig.
6A) and in H+/K+
,
cells (Fig. 6B).
Previous experiments have shown that the Golgi has large passive H+
permeability equal to
103 cm/s
(5,
39), which assures that
H+ leaks rapidly across the membrane. The rapid alkalinization of
pHTGN when bafilomycin is added to cells shows that the TGN has a
similarly large H+ permeability. Because the H+
permeability is large, the TGN and Golgi require a large number of
H+ v-ATPase pumps to maintain an acidic lumen. Thus, in the steady
state, the Golgi and TGN operate in an inefficient mode in which H+
are constantly being recycled (pumped and leaked) across the membrane. In this
condition, trafficking H+/K+ pumps through the Golgi and
TGN has only a small effect on the steady-state pH of these organelles because
the number of H+/K+ pumps that are trafficking to the
plasma membrane through these organelles is, in the steady state, small
relative to the number of H+ v-ATPase pumps that are already
operating.
|
|
As shown in Fig. 7, we
predict for a Golgi with H+ permeability of
103 cm/s that 8,000 H+ v-ATPase
pumps are required to generate a Golgi pH of 6.4. Because the ratio of
H+ v-ATPases to H+/K+-ATPases in the Golgi of
H+/K+
,
cells was calculated to be 2.1, we
further estimate that there are 8,000/2.1 = 3,800
H+/K+-ATPases in the Golgi of the
H+/K+
,
cells. As can be seen from
Fig. 7, it is predicted that
these 3,800 H+/K+-ATPases in the Golgi would have caused
the Golgi to acidify to about pH 6.2, i.e., by only 0.20 pH units compared
with the value observed in mock cells, similar to the 0.12 pH unit
acidification observed in the present experiments
(Table 1). This small
acidification is predicted, though, to increase the rate of H+ leak
out of the Golgi or TGN in the steady state, as shown in comparing A
and B of Fig. 6. Thus
pH in the Golgi remains stable because there are so many endogenous
H+ v-ATPase pumps and H+ leaks that any trafficking pH
regulators have only small effects on pHG.
Calculations shown in Fig. 7 also show that the H+ permeability of the Golgi cannot be too high; otherwise, an excessive number of H+ v-ATPases would be required to acidify the Golgi to pH 6.3. In contrast, if the Golgi had a H+ permeability of 104 cm/s (i.e., 10 times lower than what has been previously calculated) or lower, then only 2,500 H+ v-ATPases (or fewer) would be required to generate pH 6.4 (Fig. 7). However, in this circumstance, the trafficking of 3,800 H+/K+-ATPases through the Golgi would cause pHG to acidify to pH 5.3.
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DISCUSSION |
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It might be expected that stability of Golgi pH to trafficking of
H+/K+-ATPases would be particularly important in the
parietal cells of the stomach, which have 0.5 million pumps in their plasma
membranes (J. G. Forte, unpublished observations), compared with the
transfected HEK-293 cells used for the present studies in which we estimated
there were only between 10 and 50,000 H+/K+-ATPases
active in the plasma membrane and 3,800
H+/K+-ATPases in the TGN. However, due to the relatively
slow rate of turnover of H+/K+-ATPase in parietal cells
(t1/2
24 h; see Ref.
26; also, J. G. Forte,
personal communication), it is expected (based on approximate residence time
of 15 min in the Golgi) that there would be only
2,600
H+/K+-ATPase pumps in the Golgi of parietal cells. As
shown in Fig. 7, this number of
H+/K+-ATPase pumps would be expected to acidify the
Golgi only to pH 6.3, which is within the range of reported values in
different cells (5,
11,
14,
39,
40). Thus it seems that the
Golgi and TGN (and, using similar reasoning, the recycling endosomes) likely
have similar pH values in all cells (both in vivo and in transfected cells in
culture) despite trafficking different types and numbers of transporters to
the plasma membrane. This is due to the fact that the Golgi (and possibly
endosomes) has relatively large H+ permeabilities that require
large numbers of H+ pumps to keep the organelles even mildly
acidic; the pumps and other transporters trafficked to the plasma membranes,
therefore, have only small effects on pH of organelles along the secretory
pathway. Thus trafficking organelles seem to sacrifice efficiency to generate
pH stability.
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ACKNOWLEDGMENTS |
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This work was supported by grants from National Institute of Diabetes and Digestive and Kidney Diseases (R-01-DK-51799) and National Science Foundation (MCB-9983342).
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FOOTNOTES |
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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.
1 Number of H+/K+-ATPases in the plasma membrane was
calculated as follows: number of pumps = (pH recovery rate) x (cell
buffer capacity) x (cell volume) x (Avogadro's number) x
(pump turnover number)1 = (0.06 pH/min x 1
min/60 s) x (25 x 103 mol·l
cell water1·pH
unit1) x (1012
liter) x (6.02 x 1023 H+/mol) x (300
H+/s)1 = 5.02 x 104
H+/K+ ATPase pumps in the plasma membrane.
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