Selective Perturbation of Early Endosome and/or
trans-Golgi Network pH but Not Lysosome pH by
Dose-dependent Expression of Influenza M2 Protein*
Jennifer R.
Henkel
,
Jamie L.
Popovich
,
Gregory A.
Gibson
,
Simon C.
Watkins§, and
Ora A.
Weisz
¶
From the
Laboratory of Epithelial Cell
Biology, Renal-Electrolyte Division and § Department of Cell
Biology and Physiology, University of Pittsburgh,
Pittsburgh, Pennsylvania 15213
 |
ABSTRACT |
Many sorting stations along the biosynthetic and
endocytic pathways are acidified, suggesting a role for pH regulation
in protein traffic. However, the function of acidification in
individual compartments has been difficult to examine because global pH
perturbants affect all acidified organelles in the cell and also have
numerous side effects. To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments. We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that
dissipates proton gradients across membranes) and measured the effects
on various steps in protein transport. At low multiplicity of infection
(m.o.i.), delivery of influenza hemagglutinin from the
trans-Golgi network to the cell surface was blocked, but
there was almost no effect on the rate of recycling of internalized transferrin. At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes. Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not
compromised by M2 expression. However, delivery of newly synthesized
cathepsin D to lysosomes was slowed in cells expressing active M2,
suggesting that acidification of the TGN and endosomes is important for
efficient delivery of lysosomal hydrolases. Fluorescence labeling using
a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a
subset of acidified compartments in the cell. The ability to dissect
the role of acidification in individual steps of a complex pathway
should be useful for numerous other studies on protein processing and transport.
 |
INTRODUCTION |
Protein sorting along the biosynthetic and endocytic pathways of
cells is a complex event whose specificities are just beginning to be
understood. Intriguingly, some of the major sorting stations in cells,
the trans-Golgi network
(TGN)1 and endosomes, are
known to be acidified, and pH regulation is known to be required for
some functions of these organelles. For example, acidic pH is required
for the proper sorting and processing of regulated secretory proteins
and hormones in the TGN of endocrine cells (1, 2). Furthermore,
acidification of early endosomes is thought to be necessary for the
dissociation of some internalized ligands from their receptors (3).
However, outside of a few specific examples, the role of acidification
in protein trafficking is not well understood. In part, this is due to
the inherent problems presented by the use of global pH perturbants
such as weak bases or the vacuolar H+-ATPase (V-ATPase)
inhibitors bafilomycin A1 (BafA1) and
concanamycins A and B. These inhibitors disrupt the pH of all acidified
compartments in the cell, have dramatic effects on organelle
morphology, and are only slowly reversible. In addition,
BafA1 may differentially affect V-ATPase isoforms in some
organelles, further complicating interpretation of results (4, 5).
Moreover, at least some cells can become rapidly resistant to treatment
with concanamycin (6). Together these complications may account for the
vast discrepancies between different studies on the effects on
biosynthetic and endocytic traffic when acidification is disrupted
(7-15).
We have taken a different approach to dissect the function of
acidification in transport through individual compartments in the cell.
We have expressed the M2 protein of influenza virus in cells and
examined its effects on transport. M2 is an acid-activated proton-selective channel formed by the self-association of 97 amino
acid single membrane-spanning proteins into tetramers (16-20). M2
activity is controlled by the pH-dependent protonation
state of a transmembrane histidine residue (21). Unlike global
perturbants, M2 expression should affect the pH of only those acidified
compartments in which it is present. Moreover, M2 activity and its
effects on transport can be rapidly and reversibly modulated using the specific ion channel blocker BL-1743 (22-24).
Previously, we demonstrated that intracellular M2 is localized to the
TGN and apical recycling endosomes of polarized Madin-Darby canine
kidney cells and selectively affects transport of itinerant proteins
through these but not through other normally acidified compartments
(24).2 Here we have expanded
our studies to examine the effects of M2 on nonpolarized cells.
Interestingly, in these cells we find that the effect of M2 in
different subcellular compartments can be controlled by adjusting the
expression level of M2. Furthermore, we can rapidly and reversibly
inactivate M2 ion channel activity. This flexibility makes M2
expression a useful tool with which to probe the role of acidification
in protein sorting and delivery.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Adenoviral Infection--
HeLa cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal bovine serum. Generation and characterization of
E1-substituted recombinant adenoviruses encoding Rostock M2 in the
correct and reverse orientations (AV-M2 and AV-M2rev, respectively),
influenza hemagglutinin (AV-HA), and the tetracycline transactivator
(AV-TA) are described in Ref. 24. Cells were plated (3 × 105/35-mm dish or 1 × 105/12-mm well) the
day before infection. Cells were washed with calcium-free
phosphate-buffered saline containing 1 mM MgCl2
(PBS-M). After 5 min at room temperature, the PBS-M was removed, and
400 µl of PBS-M (200 µl for 12-mm wells) containing recombinant
adenovirus was added. Cells infected with AV-M2 at a multiplicity of
infection (m.o.i.) of 100 received AV-TA at a m.o.i. of 50, whereas
cells infected with AV-M2 at a m.o.i. of 500 received AV-TA at a m.o.i. of 100. The dishes were rocked briefly by hand, and the cells were
returned to the incubator for 1-2 h. Mock-infected cells were treated
identically except that virus was omitted during the incubation period.
Dishes were then rinsed with PBS-M, and cells were incubated overnight
in growth medium. The M2 ion channel inhibitors amantadine (AMT, Sigma;
5 µM) or BL-1743 (gift of Dr. Mark Krystal, Bristol-Myers
Squibb Pharmaceutical Research Institute, Wallingford, CT; 5 µM) were added as 1000-fold concentrated stocks prepared
in 95% ethanol at this step or in subsequent steps where indicated to
inhibit M2 activity. Experiments were initiated at 18-24 h postinfection.
Cell Surface Delivery of HA--
Surface delivery of newly
synthesized HA was performed as described in (23) with minor
modifications. Cells were coinfected with AV-M2 or AV-M2rev, AV-TA, and
AV-HA (m.o.i. 40) as described above. The following day, cells were
rinsed once with PBS, then starved for 30 min in medium A
(cysteine-free, methionine-free MEM containing 0.35 g/liter
NaHCO3, 10 mM HEPES, and 10 mM MES, pH 7.0; Ref. 25). AMT was added to the indicated samples at the
beginning of the starvation and included during the pulse and chase
periods. Cells were metabolically labeled with 50-100 µCi/ml
EXPRE35S35S (NEN Life Science Products) in the
same medium, then chased for 2 h at 19 °C in the same medium
supplemented with four times the normal amount of cysteine and
methionine (medium B). At various times, dishes were removed, rapidly
chilled to 0 °C by rinsing with ice-cold PBS, and incubated on ice
for 30 min in 1 ml medium B containing 100 µg/ml
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated
trypsin (Sigma) for 30 min. Trypsin cleaves HA into two subunits (HA1
and HA2) that remain associated via disulfide bonds during
immunoprecipitation. Trypsinization was stopped by incubating the cells
twice for 10 min with ice-cold medium B containing 200 µg/ml soybean
trypsin inhibitor (Sigma). Cells were then rinsed with PBS and lysed in
0.5 ml of detergent solution (50 mM Tris-HCl, 2% Nonidet
P-40, 0.4% deoxycholate, 62.5 mM EDTA, pH 8.0) containing 0.13 TIU/ml aprotinin and 20 µg/ml soybean trypsin inhibitor. Lysates
were centrifuged briefly to remove nuclei, and SDS was added to the
supernatant to a final concentration of 0.2%. HA was
immunoprecipitated using monoclonal antibody Fc125 (23), and
antibody-antigen complexes were collected using fixed
Staphylococcus aureus (Calbiochem). Where indicated, samples
were treated with endoglycosidase H (New England Biolabs, Beverly, MA)
as described previously (23). After electrophoresis on 10%
SDS-polyacrylamide gels, the percentage of cleaved HA was quantitated
using a phosphorimager (GS-363 Molecular Imager System, Bio-Rad).
Transferrin Recycling Assays--
HeLa cells were mock-infected
or infected with recombinant adenovirus as described above. The
following day, cells were depleted of intracellular stores of
transferrin (Tf) by incubation for 1 h at 37 °C in MEM/BSA
(MEM, Hanks' balanced salt solution, 0.6% BSA, 20 mM
HEPES, pH 7.4) and 125I-labeled iron-loaded human Tf (~5
µg/ml; ~5 × 106 cpm/µg) was internalized for 45 min at 37 °C. The cells were washed three times rapidly and twice
for 5 min each with ice-cold MEM/BSA, then incubated at 37 °C for
2.5 min to allow receptor internalization. The medium was replaced with
prewarmed MEM/BSA and the cells incubated at 37 °C. At the
designated times, the medium was collected and replaced. After the
final time point, cells were lysed in detergent solution and the amount
of 125I-labeled Tf in all samples was determined using a
counter (Packard Instrument Co., Downers Grove, IL). The kinetics
of transferrin recycling were calculated by determining the cumulative
percentage of preinternalized transferrin released into the medium at
each time point.
EGF Degradation--
HeLa cells were mock-infected or infected
with recombinant adenovirus as described above. AMT was added to the
indicated samples immediately after infection and included in
subsequent steps. The following day, cells were incubated with
125I-labeled EGF (0.5 µM in MEM/BSA; Amersham
Pharmacia Biotech) for 2 h at 0 °C. Cells were washed three
times on ice for five min each with MEM/BSA, rinsed an additional three
times, and then incubated with prewarmed medium. Chloroquine (50 µg/ml) was added to the indicated samples at this time. At various
times, the medium was collected and replaced; at the end of the time
course, the cells were solubilized with 1% Triton X-100 in 50 mM Tris, pH 7.4. trichloroacetic acid (10% final
concentration added as a 100% w/v stock; Sigma) was added, and the
samples were incubated on ice for 10 min. Samples were then centrifuged
at maximum speed in a microcentrifuge for 15 min at 4 °C, and the
radioactivity in the supernatants and pellets counted using a
counter. The rate of EGF degradation was determined by calculating the
cumulative release of trichloroacetic acid-soluble counts into the
medium over time.
Indirect Immunofluorescence--
Indirect immunofluorescence of
HeLa cells was performed as described previously (23). Briefly, cells
were rinsed once with PBS, fixed for 20 min at ambient temperature in
3% paraformaldehyde, then incubated briefly with PBS containing 10 mM glycine and 0.02% sodium azide (PBS-G). Where
indicated, cells were permeabilized for 3 min in 0.5% Triton X-100 in
PBS-G. Nonspecific binding was blocked with 0.25% ovalbumin in PBS-G
prior to incubation with antibodies. M2 was detected using the
monoclonal antibody 5C4 (a generous gift of Dr. Robert Lamb,
Northwestern University) at a dilution of 1:250 followed by
Cy3-conjugated affinity-purified goat anti-mouse IgG (2 mg/ml, 1:1000
dilution; Jackson ImmunoResearch Laboratories, Inc., Avondale, PA).
Cells were viewed using a Nikon Optiphot microscope (Fryer Company,
Inc., Carpentersville, IL) and images were acquired using a Hamamatsu
C5985 chilled CCD camera (8 bit, 756 × 483 pixels; Hamamatsu
Photonics Systems, Bridgewater, NJ).
Fluorescence Imaging in Live Cells--
To qualitatively assess
the effect of M2 expression on organelle pH, we incubated mock- or
AV-M2-infected HeLa cells with 5 µM LysoSensor (Molecular
Probes, Eugene, OR) in MEM/BSA. Acidified compartments were visualized
using an Olympus Provis microscope (Olympus, Tokyo, Japan) using a
triple pass (blue/green/red) cube, which allows excitation at 384 nm
and collection at 540 nm. Images were collected digitally to a Sony 3 chip color CCD camera (Sony 970; Sony Electronics, Tokyo, Japan)
equipped with hardwired on chip integration circuitry. In each
experiment images were collected over 256 grayscales for each color,
for 3 video frames. The effects of chloroquine (200 µg/ml) and AMT
(10 µM) on the fluorescence patterns of mock-infected and
M2-expressing samples were determined after 1-h and 15-min incubations, respectively.
Cathepsin D Processing--
HeLa cells cultured in 35-mm dishes
were infected with adenoviruses as described above. The following day,
cells were starved, radiolabeled for 20 min with 100 µCi/ml
[35S]methionine in medium A, then chased for the
indicated times in medium B. The medium was collected and a 5-fold
concentrated stock of detergent solution supplemented with aprotinin
was added to a final concentration of 1×. Cells were solubilized in
detergent solution as described above and samples were incubated with
anti-cathepsin D antibody (Calbiochem). Antibody-antigen complexes were
collected using fixed S. aureus and analyzed on 12%
SDS-polyacrylamide gel electrophoresis gels.
 |
RESULTS |
M2 Slows TGN-to-Cell Surface Delivery of Influenza
Hemagglutinin--
Our previous results demonstrated that vaccinia
virus mediated overexpression of M2 inhibited TGN to surface delivery
of influenza HA (23). However, at these extremely high expression
levels, M2 also inhibited earlier steps in transport through
nonacidified compartments (23, 25). Moreover, in our hands,
synchronization of proteins along the secretory pathway by temperature
blocks is not effective in vaccinia infected cells. To circumvent these problems we developed replication-defective recombinant adenoviruses expressing M2 (AV-M2) (24). In our system, expression of M2 is driven
by the tetracycline operon and thus requires coinfection with an
adenovirus encoding the tetracycline transactivator chimera (AV-TA;
expression of the TA protein in this virus is constitutively driven by
the constitutive CMV promoter). We infected HeLa cells using AV-M2 and
AV-TA at various m.o.i. and examined the localization of the protein
using indirect immunofluorescence (Fig.
1). Cells infected at low m.o.i. (100)
had very low levels of M2 staining. More staining was visible in cells
infected with AV-M2 at m.o.i. 500. Interestingly, most of the staining
was localized to the plasma membrane, and no distinct organelle
staining was visible at either infection level. However, the nuclei of
many of the cells infected at high m.o.i. had multiple donut-shaped
inclusions that stained brightly for M2. The origin of these inclusions
is not known. Other than this, we did not observe any effect of viral infection on the morphology of the cells during the course of our
experiments. These expression patterns are very different from M2
overexpressed in HeLa cells using recombinant vaccinia, where the
majority of the protein accumulated in the endoplasmic reticulum and
Golgi complex and cells became rounded after several hours of
expression (23).

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Fig. 1.
Distribution of adenovirally expressed M2 in
HeLa cells. HeLa cells were infected with AV-TA and AV-M2 or
AV-M2rev at the indicated m.o.i. The following day, cells were fixed,
permeabilized, and processed for indirect immunofluorescence to
localize M2. Very little M2 expression was detected in cells infected
at low m.o.i., and more was observed at the higher expression
level.
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To test the effect of M2 on biosynthetic traffic, we infected HeLa
cells at various m.o.i. with AV-TA and AV-M2 (or AV-M2rev, a control
virus that encodes M2 in the reverse orientation) and measured the
effects on HA delivery from the TGN to the plasma membrane. Cells were
radiolabeled for 15 min, then chased for 2 h at 19 °C to
accumulate newly synthesized proteins in the TGN. M2 had a moderate
effect on the amount of HA that was endoglycosidase H-resistant after
this chase period, consistent with slower intra-Golgi transport as has
been reported previously (23, 25) (Fig.
2A). Similar effects on early
Golgi transport have been documented using other pH perturbants
(8-10). We have demonstrated previously that the delay in these early
transport steps in M2-expressing cells is most likely an indirect
effect resulting from prolonged accumulation of proteins, lipids, and
possibly transport machinery in the TGN (23). As expected, M2
expression had a much more dramatic effect on the kinetics of HA
delivery from the TGN to the plasma membrane (Fig. 2, B and
C). The effect of M2 on HA delivery was maximal at m.o.i.
100. Subsequent experiments showed similar inhibition at m.o.i. as low
as 50 (data not shown). These results suggest that M2 is active in the
TGN, and that TGN acidification is required for efficient delivery of
HA to the cell surface in these cells.

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Fig. 2.
M2 blocks delivery of HA to the plasma
membrane. A, M2 expression slows arrival of HA to the
medial Golgi. HeLa cells were infected with AV-M2 or
AV-M2rev, AV-TA, and AV-HA as described under "Experimental
Procedures." The following day, cells were metabolically radiolabeled
for 15 min, then chased for 2 h at 19 °C (in the presence or
absence of AMT). Samples were solubilized, immunoprecipitated with
anti-HA antibody, and treated with endoglycosidase H (endo
H) to determine the amount of HA reaching the medial Golgi during
the chase period. B and C, M2 markedly slows
TGN-to-cell surface delivery of HA. HeLa cells infected with AV-M2 or
AV-M2rev and AV-TA were metabolically radiolabeled and chased for
2 h at 19 °C as above to accumulate newly synthesized HA in the
TGN. AMT was included in the indicated dishes throughout the
pulse/chase to block M2 ion channel activity. The cells were then
rapidly warmed to 37 °C and the kinetics of HA delivery to the
plasma membrane determined using cell surface trypsinization as
described under "Experimental Procedures." The positions of intact
HA (HA0) and its trypsin cleavage products (HA1
and HA2) are marked. Quantitation of the gel in C
is shown in B. Similar results were obtained in three
experiments.
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M2 Expression Causes Dose-dependent Inhibition of
Transferrin Recycling--
We then measured the effect of M2
expression on Tf recycling. In polarized Madin-Darby canine kidney
cells, where M2 expression is restricted to apical endosomal
compartments, we observed no effect on Tf recycling (24). However, we
reasoned that M2 might affect Tf recycling if it localized to endosomal
compartments in nonpolarized cells. Thus, we determined the effect of
increasing M2 expression levels on Tf recycling (Fig.
3). Cells infected with M2rev had
identical Tf recycling kinetics compared with mock-infected cells (Fig.
3A). M2 expressed using low m.o.i. (100) had a slight but reproducible effect on Tf recycling (~10% decrease in initial rate). By contrast, Tf recycling was markedly reduced (~30%) in cells expressing higher levels of M2 (m.o.i. 500). Addition of doxycycline to block M2 expression after viral infection, or addition of AMT to the medium shortly before Tf uptake, resulted in normal Tf
recycling kinetics, suggesting that the effect on recycling was due to
M2-mediated ion channel activity. Fig. 3B shows the results
from several experiments in which the dose-dependence of M2's effect
on Tf recycling was compared. The effect of M2 on Tf recycling was
essentially maximal at m.o.i. 500, as increasing the dose to m.o.i.
2500 had no further effect on recycling. Interestingly, the effect of
M2 on recycling never approached that of the V-ATPase inhibitor
BafA1, which inhibited recycling by approximately 60%. Addition of AMT or the reversible M2 inhibitor BL-1743 completely blocked the effect of M2 at all m.o.i. tested.

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Fig. 3.
M2 expression causes
dose-dependent inhibition of transferrin recycling.
A, effect of M2 on transferrin recycling kinetics. HeLa
cells infected with AV-M2 or AV-M2rev at the indicated m.o.i. were
incubated with 125I-labeled Tf for 45 min, then washed
extensively, and the kinetics of transferrin release into the medium
quantitated. AMT or doxycycline (DOX) were included in the
indicated samples to block M2 activity and expression, respectively.
The mean ± S.D. of triplicate samples is plotted. B,
summary of the effects of M2 on transferrin recycling. HeLa cells were
infected with the AV-M2rev (m.o.i. 100-2500) or AV-M2 and transferrin
recycling monitored as described under "Experimental Procedures."
AMT or the reversible M2 inhibitor BL-1743 were included in the
indicated samples to block M2 activity. Some mock-infected cells were
pretreated with 0.5 µM BafA1 for 30 min prior
to transferrin uptake. The amount of transferrin released into the
medium after 15-min chase is plotted as a percentage of release from
mock-infected cells. The number (n) of times an experiment
was performed is shown for each condition, and the mean ± S.E.
(n > 2) or range (n = 2) is
plotted.
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M2 Expression Does Not Affect Lysosomal pH--
To test whether M2
expression affected lysosomal function, we measured its effect on
degradation of radioiodinated EGF. Interestingly, no effect of M2 on
EGF degradation was observed, whereas treatment with chloroquine
drastically inhibited this process (Fig.
4). To further confirm that M2 did not
disrupt lysosomal pH, we compared the effects of M2 and chloroquine on
lysosome morphology using indirect immunofluorescence (Fig.
5). As expected, incubation with
chloroquine caused dramatic swelling of lysosomal compartments as
visualized using an antibody against the membrane protein LAMP-1. By
contrast, M2 expression had no effect on LAMP-1 staining.

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Fig. 4.
M2 expression does not affect EGF
degradation. HeLa cells (mock-infected or infected with AV-TA and
AV-M2rev or AV-M2 at the indicated m.o.i.) were incubated with
125I-labeled EGF on ice for 2 h. The cells were then
washed extensively, then rapidly warmed, and the kinetics of EGF
degradation measured as described under "Experimental Procedures."
Indicated samples were treated with AMT (5 µM) or 50 µg/ml chloroquine (chlor). Similar results were obtained
in three independent experiments.
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Fig. 5.
M2 does not affect the steady state
distribution of lysosomal membrane proteins. HeLa cells were
mock-infected (mock) or infected with AV-TA and AV-M2 at a
m.o.i. of 500 (M2). After infection, one dish of
mock-infected cells was treated with 50 µg/ml chloroquine
(chlor), and an AV-M2-infected dish was incubated with AMT
(M2+AMT). The following day, cells were fixed and processed
for indirect immunofluorescence to localize LAMP-1.
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Although there is considerable indirect evidence to suggest that M2
expression alters organelle pH, this has never been directly measured
(26-30). We took advantage of a relatively new fluorescence pH
indicator to test whether M2 affected the pH of intracellular organelles. We incubated mock-infected or AV-M2-infected HeLa cells
(m.o.i. 500) with LysoSensor yellow/blue, a membrane permeant probe
that fluoresces bright yellow at acidic pH and dim blue at neutral pH.
Although the blue staining was relatively uninformative, we were able
to monitor acidified compartments in each cell by visualizing the
yellow fluorescence. Mock-infected cells had numerous acidic
compartments, as evidenced by the multitude of bright yellow punctae
(Fig. 6A). As expected,
treatment with chloroquine caused all of the punctae to disappear (Fig.
6B). Interestingly, when cells expressing M2 were incubated
with LysoSensor, a much smaller number of yellow compartments were
present (Fig. 6C). Based on the trafficking assays described
above, we hypothesize that these remaining punctae represent late
endosomal and lysosomal compartments, as the function of these
compartments is not compromised by M2 activity. We observed a similar
staining pattern for all of the cells on the coverslip, suggesting that
essentially all of the cells expressed M2. Within 15 min of addition of
AMT to the M2-expressing cells, there was a dramatic increase in the
number of acidic compartments, as evidenced by the appearance of
numerous new yellow punctae (Fig. 6D). Thus M2 expression
alters the pH of a subset of acidified compartments in the cell, and
the effects of M2 can be rapidly reversed.

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Fig. 6.
M2 expression perturbs the pH of a subset of
acidified organelles. HeLa cell grown on coverslips were incubated
with LysoSensor, a pH-sensitive dye that fluoresces yellow in acidic
compartments. Yellow fluorescence was compared in mock infected cells
(A), cells treated with the weak base chloroquine
(B), cells infected with M2 adenovirus at a m.o.i. of 500 (C), and M2-infected cells treated with the M2 ion channel
blocker AMT (D). As expected, chloroquine treatment
neutralizes all intracellular acidified compartments. By contrast, M2
expression causes a decrease in the number of acidic compartments,
consistent with neutralization of the TGN and endosomes but not
lysosomes. Acidification of juxtanuclear compartments is restored upon
addition of AMT for 15 min. Scale bar: 10 µm.
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M2 Expression Inhibits Lysosomal Delivery of Newly Synthesized
Cathepsin D--
Because we were able to selectively alter TGN or
TGN/endosome function by varying M2 expression levels, we tested the
effect of M2 on delivery of the newly synthesized lysosomal hydrolase cathepsin D. This protein is synthesized as a 53-kDa precursor (P) in
the endoplasmic reticulum, clipped in the TGN and/or endosomes to an
slightly smaller intermediate (I) form, then processed to a mature
30-kDa form (M) in lysosomes (31). Conversion to the mature form is not
dependent on prior cleavage to the intermediate form (32). Pertubation
of acidified compartments using various inhibitors blocks cathepsin D
processing to the mature form and in some cases increases secretion of
the precursor (33-38). However, because cathepsin D processing to its
mature form requires acidic pH, it can be difficult to distinguish
missorting of intracellular cathepsin D from the accumulation of
precursor or intermediate forms in lysosomes. Therefore, we compared
the effects of M2 and BafA1 on processing and delivery of
newly synthesized cathepsin D in HeLa cells (Fig.
7). As predicted, BafA1
blocked proteolytic maturation of cathepsin D and slightly increased
secretion of the precursor. Low expression levels of M2 (m.o.i. 100)
slightly delayed processing of cathepsin D to both the intermediate and mature forms. Higher expression of M2 (m.o.i. 500) had a more dramatic
effect on both steps that was almost completely reversed when AMT was
included. These data suggest that at least some cleavage of cathepsin D
to its intermediate form normally occurs in the TGN. Furthermore,
because higher expression levels of M2 had a greater effect on
cathepsin D maturation, a portion of cathepsin D may traffic through
early endosomes in HeLa cells.

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Fig. 7.
Effect of M2 on cathepsin D processing.
HeLa cells were infected with AV-M2 and AV-TA at the indicated m.o.i.
The following day, cells were radiolabeled for 15 min, then chased for
the indicated times. AMT (5 µM) and BafA1 (1 µM) were added to the indicated dishes 30 min prior to
radiolabeling. Cathepsin D was immunoprecipitated from the cells and
medium as described under "Experimental Procedures." The migration
of the precursor (P), intermediate (I), and
mature (M) forms of cathepsin D is noted. On occasion, a
slowly migrating band of unknown origin was observed in some
samples.
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DISCUSSION |
We have developed a new method to selectively elevate the pH of
some intracellular acidified compartments. By varying the expression
level of virally expressed influenza M2 protein, an acid-activated ion
channel, we can reversibly modulate TGN or TGN/endosome pH in HeLa
cells without affecting lysosomal pH or function. At low expression
levels, maximal effects on TGN-to-cell surface delivery of the marker
protein influenza HA were observed. However, higher expression levels
were required to maximally inhibit the recycling of transferrin. Even
at these high expression levels, no effects on lysosomal function were
observed. Fluorescence staining of live cells using a pH-sensitive dye
confirmed the selective effect of M2 on a subset of acidified
compartments. To our knowledge, this is the first direct
demonstration that influenza M2 perturbs organelle pH in mammalian cells.
Although even low expression levels of M2 dramatically inhibited
TGN-to-cell surface delivery of the marker protein influenza HA, M2 did
not completely block exit from the TGN, as secretion of newly
synthesized cathepsin D continued normally in M2-expressing cells.
Moreover, we have found that cell surface delivery of another heterologous membrane protein, the polymeric immunoglobulin receptor, was completely unaffected by even high levels of M2
expression.3 The selective
effect of M2 on HA but not polymeric immunoglobulin receptor delivery
was also observed in polarized Madin-Darby canine kidney
cells.4 Differences in the
marker proteins as well as cell lines used could thus account for some
of the discrepancies between the various studies on the role of
acidification on secretory traffic.
Interestingly, the maximal effect of M2 on Tf recycling never
approached that of the V-ATPase inhibitor BafA1. Because
essentially all cells are infected under our conditions, this
difference does not reflect activity of M2 in only a subset of cells.
Although it is possible that BafA1 has a greater effect on
endosomal pH than M2, increasing M2 m.o.i. 5-fold had no further effect
on Tf recycling. Rather, our results suggest that M2 is present or active in only a subset of endocytic compartments through which receptor-bound Tf passes. Iron is thought to dissociate from Tf primarily in sorting endosomes; subsequently, receptor-bound Tf is
segregated into recycling endosomes and returned to the cell surface.
In the presence of BafA1, iron remains bound to transferrin throughout the pathway and prevents recycled Tf from dissociating from
its receptor upon return to the cell surface (15). In cells expressing
M2, the pH of a subset of acidified compartments along the transferrin
recycling route could be altered, resulting in partial dissociation of
iron from transferrin during recycling. Alternatively, iron
dissociation could proceed normally in M2-expressing cells and the
effects we observed might reflect effects on the rate of receptor
movement along the recycling pathway. Presley et al. (39)
found that BafA1 treatment caused a ~45% decrease in the
rate of transferrin receptor recycling; this is comparable with the
~35% effect on transferrin release we observed at high levels of M2 expression.
Importantly, even at high expression levels, M2 had no effect on the
kinetics of degradation of internalized EGF, suggesting that both
delivery to lysosomes and lysosomal function were unaffected. By
contrast, BafA1 treatment has been variously reported to
block delivery from late endosomes to lysosomes (15) and lysosomal degradation but not delivery to lysosomes (7). In addition, lysosome morphology and the steady state distribution of lysosomal enzymes were unaffected in M2-expressing cells. This is in sharp contrast to all other pH perturbants, which have dramatic effects on
lysosome pH and function. The ability to alter the pH of a subset of
intracellular compartments using M2 should thus help us to dissect the
role of acidification in individual steps along pathways that involve
transport through multiple acidified compartments.
To test the utility of our system, we examined the effect of M2 on
delivery of newly synthesized cathepsin D to lysosomes. Because many
steps in the sorting of lysosomal hydrolases are pH-sensitive, the role
of acidification in individual compartments remains relatively poorly
understood despite considerable study. Cathepsin D delivery to
lysosomes first involves binding to mannose 6-phosphate receptors
(MPRs) in the TGN. This step is complicated by the differential pH
binding profiles of the two MPRs: the cation-dependent MPR
binds many substrates weakly at neutral pH, while the
cation-independent MPR is pH-insensitive within a relatively broad
range (40). While the two MPRs have overlapping substrate
specificities, they cannot completely substitute for each other when
one is nonfunctional (41, 42). The MPR-hydrolase complexes are
transported to late endosomes where pH-dependent
dissociation of the hydrolases occurs. While a direct route from the
TGN to late endosomes has been proposed, in some cell types cathepsin D
may transit through early endosomes en route to lysosomes
(43, 44). In addition to this pathway, a significant fraction of
cathepsin D delivery in some cells also occurs via MPR-independent
mechanisms (45, 46). BafA1 treatment likely inhibits the
pH-dependent steps; however, since cathepsin D maturation
in lysosomes is also inhibited, it is difficult to determine the effect
of BafA1 on individual steps in transport or on
MPR-independent delivery. Furthermore, neutralized lysosomes might not
be recognized as an appropriate target for fusion by compartments
carrying newly synthesized cargo; for example, inactivation of
lysosomes using HRP blocks the lysosomal delivery of internalized EGF-EGF receptor complexes (47). Processing of newly synthesized cathepsin D to its mature form (a lysosomal event) was progressively inhibited by increased expression of M2 and completely inhibited (as
expected) by BafA1. The progressive effect of increasing M2 expression on cathepsin D processing and delivery supports the idea
that some cathepsin D traffics through early endosomes in these cells.
In neither case was cathepsin D delivery to lysosomes completely
inhibited. The properly targeted fraction could represent cathepsin D
transport via the pH-independent pathway or cathepsin D binding to the
cation-independent MPR, which may still occur at neutral pH.
Importantly, our data also demonstrate that inefficient delivery of
newly synthesized hydrolases in pH-perturbed cells is not simply due to
the loss of recognition of the lysosome as an appropriate target.
In summary, influenza M2 expression provides a novel method to
selectively alter the pH of a subset of acidified organelles. Further
characterization of the effects of M2 on pH in individual compartments
should help us dissect the role of acidification in numerous processing
and transport steps along the biosynthetic and endocytic pathways. In
addition, the possibility of altering M2 localization or proton channel
activity via site-directed mutagenesis may add a new level of
flexibility in manipulating organelle pH.
 |
ACKNOWLEDGEMENTS |
We thank Mark Krystal for his gift of
BL-1743, Robert Lamb for the anti-M2 antibody, Greg Conner for
anti-cathepsin antibody used in unpublished studies, Gerard Apodaca for
critical review of this manuscript, and Doug Fambrough and Nancy Gough
for helpful suggestions.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant R01DK54407 and by grants from the Cystic Fibrosis Foundation, the
Competitive Medical Research Fund of the University of Pittsburgh, and
the Emma and Samuel Winters Foundation (to O. A. W.). The Laboratory of Epithelial Cell Biology is supported in part by Dialysis
Clinic Inc.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.
¶
To whom correspondence should be addressed: Renal-Electrolyte
Division, University of Pittsburgh, 978 Scaife Hall, 3550 Terrace St.,
Pittsburgh, PA 15213. Tel.: 412-383-8891; Fax: 412-383-8956; E-mail:
weisz{at}med1.dept-med.pitt.edu.
2
J. R. Henkel and O. A. Weisz,
unpublished observations.
3
G. A. Gibson and O. A. Weisz,
unpublished observations.
4
J. R. Henkel, G. A. Gibson, and
O. A. Weisz, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
TGN, trans-Golgi network;
AMT, amantadine;
AV, adenovirus;
BafA1, bafilomycin A1;
EGF, epidermal growth
factor;
HA, influenza hemagglutinin;
MPR, mannose 6-phosphate receptor;
PBS, phosphate-buffered saline;
Tf, transferrin;
V-ATPase, vacuolar
H+-ATPase;
m.o.i., multiplicity of infection;
BSA, bovine
serum albumin;
MEM, minimal essential medium.
 |
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