Yeast V-ATPase Complexes Containing Different Isoforms of the
100-kDa a-subunit Differ in Coupling Efficiency and in Vivo
Dissociation*
Shoko
Kawasaki-Nishi
,
Tsuyoshi
Nishi
, and
Michael
Forgac§
From the Department of Physiology, Tufts University School of
Medicine, Boston, Massachusetts 02111
Received for publication, November 29, 2000, and in revised form, February 28, 2001
 |
ABSTRACT |
The 100 kDa a-subunit of the yeast vacuolar
(H+)-ATPase (V-ATPase) is encoded by two genes,
VPH1 and STV1. These genes encode unique
isoforms of the a-subunit that have previously been shown to reside in
different intracellular compartments in yeast. Vph1p localizes to the
central vacuole, whereas Stv1p is present in some other compartment,
possibly the Golgi or endosomes. To compare the properties of V-ATPases
containing Vph1p or Stv1p, Stv1p was expressed at higher than normal
levels in a strain disrupted in both genes, under which conditions
V-ATPase complexes containing Stv1p appear in the vacuole. Complexes
containing Stv1p showed lower assembly with the peripheral
V1 domain than did complexes containing Vph1p. When
corrected for this lower degree of assembly, however, V-ATPase
complexes containing Vph1p and Stv1p had similar kinetic properties.
Both exhibited a Km for ATP of about 250 µM, and both showed resistance to sodium azide and
vanadate and sensitivity to nanomolar concentrations of concanamycin A. Stv1p-containing complexes, however, showed a 4-5-fold lower ratio of
proton transport to ATP hydrolysis than Vph1p-containing complexes. We
also compared the ability of V-ATPase complexes containing Vph1p or
Stv1p to undergo in vivo dissociation in response to glucose depletion. Vph1p-containing complexes present in the vacuole showed dissociation in response to glucose depletion, whereas Stv1p-containing complexes present in their normal intracellular location (Golgi/endosomes) did not. Upon overexpression of Stv1p, Stv1p-containing complexes present in the vacuole showed
glucose-dependent dissociation. Blocking delivery of
Vph1p-containing complexes to the vacuole in vps21
and
vps27
strains caused partial inhibition of
glucose-dependent dissociation. These results suggest that dissociation of the V-ATPase complex in vivo is controlled
both by the cellular environment and by the 100-kDa a-subunit isoform present in the complex.
 |
INTRODUCTION |
The vacuolar (H+)-ATPases (or
V-ATPases)1 are a family of
ATP-dependent proton pumps found in a variety of
intracellular compartments that function in both endocytic and
secretory pathways (1-8). Acidification of these compartments is
essential for many cellular processes, including receptor-mediated
endocytosis, intracellular targeting, protein processing and
degradation, and coupled transport. V-ATPases are also present in the
plasma membrane of certain specialized cells, including osteoclasts
(9), renal intercalated cells (10), and neutrophils (11), where they
function in such processes as bone resorption, renal acidification, and
pH homeostasis, respectively.
The V-ATPases from fungi, plants, and animals are structurally very
similar and are composed of two domains (1-8). The V1 domain is a peripheral complex of molecular mass 570 kDa composed of
eight different subunits of molecular mass 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain
is a 260-kDa integral complex composed of five subunits of molecular mass 100-17 kDa (subunits a, d, c, c', and c'') that is responsible for proton translocation. This structure is similar to that of the ATP
synthases (or F- ATPases) that function in ATP synthesis in
mitochondria, chloroplasts, and bacteria (12-17). Sequence homology between these classes of ATPase has been identified for both the nucleotide binding subunits (18, 19) and the proteolipid subunits (subunits c, c', and c'') (20, 21). Subunit G has also been shown to
have some homology to subunit b of the F-ATPases (22).
The 100-kDa a-subunit of the V-ATPase is an integral membrane protein
possessing an amino-terminal hydrophilic domain and a carboxyl-terminal
hydrophobic domain containing multiple putative membrane-spanning
segments (23-25). In yeast, the 100-kDa subunit is encoded by two
genes, VPH1 and STV1. Vph1p and Stv1p are
homologous proteins displaying 54% identity and 71% similarity (23,
24). These proteins show distinct intracellular localization, with Vph1p localized to the vacuole and Stv1p normally localized to some
other intracellular compartment, possibly Golgi or endosomes (24).
These results suggest that the 100-kDa a-subunit contains information
necessary to target the V-ATPase to the appropriate intracellular site.
To compare the properties of V-ATPase complexes containing Vph1p and
Stv1p, we have taken advantage of the observation that overexpression
of Stv1p in a yeast strain deleted in both VPH1 and
STV1 results in mislocalization of Stv1p to the vacuole (24). This allowed us to compare the properties of V-ATPase complexes
present in the same intracellular compartment (the vacuole) that
differed only in the a-subunit isoform present.
 |
EXPERIMENTAL PROCEDURES |
Materials and Strains--
Zymolyase 100T was obtained from
Seikagaku America, Inc. Concanamycin A was purchased from Fluka
Chemical Corp. Protease inhibitors were from Roche Molecular
Biochemicals. The monoclonal antibody 3F10 (directed against the HA
antigen), which is conjugated with horseradish peroxidase, was also
from Roche Molecular Biochemicals. The monoclonal antibody 8B1-F3
against the yeast V-ATPase A-subunit (26) and the monoclonal antibody
10D7 against the 100-kDa a-subunit (27) were from Molecular Probes.
Escherichia coli and yeast culture media were purchased from
Difco Laboratories. Restriction endonucleases, T4 DNA ligase, and other
molecular biology reagents were from Life Technologies, Inc., Promega,
and New England Biolabs. ATP, phenylmethylsulfonyl fluoride, and most
other chemicals were purchased from Sigma.
Yeast strain MM112 (MAT
vph1::LEU2
stv1::LYS2 his3-(
200 leu2
lys2 ura3-52) and plasmid MM322 (VPH1 in pRS316) (24)
were used to generate and study the HA-tagged Vph1p. Yeast cells were grown in yeast extract-peptone-dextrose (YPD) medium or synthetic dropout (SD) medium (28).
Cloning of the STV1 Gene--
The STV1 gene was
amplified from genomic DNA isolated from yeast strain YPH500 using
primers containing the restriction enzyme sites XbaI and
BamHI and then cloned into pRS316 using the XbaI and BamHI sites (pRS316-STV1). The sequence of
the cloned STV1 gene was confirmed by DNA sequencing using
an automated sequencer from Applied Biosystems. The oligonucleotides
used for amplification of the STV1 gene were as follows,
with the restriction enzyme sites underlined: STV1 forward
(XbaI),
5'-GGCCTCTAGATTAGAGGAAATGCAATTTTCTATCATC-3'; STV1 reverse
(BamHI),
5'-GGCCGGATCCTATTGGTAGACCACTTGACCGAC-3'.
Introducing HA Tags into Vph1p and Stv1p--
An HpaI
site was introduced into VPH1 using polymerase chain
reaction, and then the two tandem repeats of the nine-amino acid HA
epitope (YPYDVPDYA) were ligated into the HpaI site of
pRS316-VPH1 (pRS316-VPH1::HA) and the
BglII site of pRS316-STV1
(pRS316-STV1::HA). This resulted in
insertion of the tandem HA tags after amino acid residue Asn-185 of
Vph1p and the corresponding residue (Leu-227) of Stv1p. A
XbaI-BamHI fragment of
pRS316-STV1::HA was ligated into
XbaI-BamHI sites of the 2-µm plasmid, YEp352
(YEp352-STV1::HA).
Transformation and Selection--
Yeast cells (MM112)
lacking functional endogenous Vph1p and Stv1p were transformed using
the lithium acetate method (29) with the wild-type
pRS316-VPH1 as a positive control, pRS316 vector alone as a
negative control, pRS316-VPH1::HA,
pRS316-STV1::HA, and
YEp352-STV1::HA. The transformants were
selected on uracil minus (Ura
) plates as described
previously (30). Growth phenotypes of the mutants were assessed
on YPD plates buffered with 50 mM
KH2PO4 or 50 mM succinic acid to
either pH 7.5 or pH 5.5.
Isolation of Vacuolar Membrane Vesicles--
Vacuolar membrane
vesicles were isolated using a modification of the protocol described
by Uchida et al. (31). Yeast were grown overnight at
30 °C to 1 × 107 cells/ml in 1 liter of selective
medium. Cells were pelleted, washed once with water, and resuspended in
100 ml of 10 mM dithiothreitol and 100 mM
Tris-HCl, pH 9.4. After incubation at 30 °C for 15 min, cells were
pelleted again, washed once with 100 ml of YPD medium containing
0.7 M sorbitol, 2 mM dithiothreitol, and 100 mM MES-Tris, pH 7.5, resuspended in 100 ml of YPD medium
containing 0.7 M sorbitol, 2 mM dithiothreitol,
100 mM MES-Tris, pH 7.5, and 2 mg of zymolase 100T and
incubated at 30 °C with gentle shaking for 60 min. The resulting
spheroplasts were osmotically lysed, and the vacuoles were isolated by
flotation on two consecutive Ficoll gradients. Protein concentrations
were measured by the BCA protein assay (Pierce).
Analysis of the 100-kDa Subunit Expression and V-ATPase
Assembly--
Yeast were grown to log phase at 30 °C in selective
medium, whole cell lysates were prepared as described previously (26), and the proteins were separated by SDS-PAGE on 8% acrylamide gels. The
expression of the 100-kDa subunit was detected by Western blotting
using the horseradish peroxidase-conjugated monoclonal antibody 3F10
against HA, whereas subunit A was detected by monoclonal antibody
8B1-F3 directed against subunit A followed by a horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Assembly of the
V-ATPase was assessed by measurement of the amount of subunit A present
on isolated vacuolar membranes (27, 32). Vacuolar membrane proteins
were separated by SDS-PAGE on 8% acrylamide gels, and the 100-kDa
subunit, subunit A, and subunit d were detected by Western blotting as
described above. Blots were developed using a chemiluminescent
detection method obtained from Kirkegaard and Perry Laboratories
(Gaithersburg, MD).
Measurement of ATPase Activity and Examination of Effects of
Inhibitors on ATPase Activity--
ATPase activity was measured using
a coupled spectrophotomeric assay as described previously (33) with
some modification. To determine the
KmATP and the
Vmax for V-ATPase complexes containing Vph1p or
Stv1p, ATPase activity was measured over a range of ATP concentrations from 0.1-2 mM ATP, whereas MgSO4 was
maintained at 4 mM. Vacuoles isolated from the
vph1
stv1
strain expressing Vph1p (3 µg of protein) or Stv1p (10 µg of protein) were incubated in ATPase assay
buffer (50 mM NaCl, 30 mM KCl, 20 mM HEPES-NaOH, pH 7.0, 0.2 mM EGTA, 10%
glycerol, 1 mM MgCl2, 1.5 mM
phosphoenolpyruvate, 0.35 mM NADH, 20 units/ml pyruvate
kinase, and 10 units/ml lactate dehydrogenase) with 0.1%
Me2SO or 1 µM concanamycin A at room temperature for 10 min. The assay was then started by the addition ATP
at the indicated concentrations, and the absorbance at 341 nm was
measured continuously using a Kontron UV-visible spectrophotometer. V-ATPase activity was defined as that fraction of the ATPase activity inhibited by 1 µM concanamycin A (typically 90% for
Vph1p-containing vacuoles and 50% for Stv1p-containing vacuoles).
KmATP and Vmax
were calculated from double-reciprocal plots of ATP concentration
versus V-ATPase activity expressed in µmol ATP/min/mg of
protein. To examine the effect of inhibitors on ATPase activity, isolated vacuolar membranes were incubated in ATPase buffer containing 0.1% Me2SO, 1 µM concanamycin A, 0.1 mM sodium vanadate, or 0.5 mM sodium azide at
room temperature for 10 min followed by initiation of the assay by the
addition of 0.5 mM ATP. ATP-dependent proton transport activity was measured by quenching of ACMA fluorescence using
a PerkinElmer LS50B spectrofluorometer as previously described (34).
In Vivo Dissociation of the V-ATPase in Response to Glucose
Deprivation--
Dissociation of the V-ATPase in response to glucose
depletion was measured as described previously (35) with some
modifications. The vph1
stv1
strain MM112
expressing Vph1p or Stv1p from the single copy plasmid pRS316 or Stv1p
using the high copy plasmid YEp352 was grown in selective medium
overnight to an absorbance at 600 nm of <1.0. The yeast strains
SF838-1D (MAT
leu2-3, 112 ura3-52 his4-519 ade6
gal2 pep4-3), SGY79 (MAT
leu2-3, 112 ura3-52 his4-519 ade6 gal2 pep4-3
vps21::Kanr), and SGY73
(MAT
leu2-3, 112 ura3-52 his4-519 ade6 gal2
pep4-3 vps27::LEU2) (36) were grown under
the same conditions except using YPD media. The cells were
converted to spheroplasts by treatment with zymolase 100T and incubated
in YEP media with or without 2% glucose for 40 min at 30 °C.
Spheroplasts were pelleted and lysed in phosphate-buffered saline
containing 1% C12E9, protease inhibitors, and 1 mM dithiobis(succinimidyl propionate). An
aliquot (corresponding to ~6 × 105 cells) was
removed to allow analysis of proteins present in the whole cell lysate.
The V-ATPase complexes were immunoprecipitated from the remainder of
the lysate (corresponding to ~3 × 106 cells) using
8B1-F3 against the A-subunit and protein G-agarose followed by
separation on 8% acrylamide gels and transfer to nitrocellulose. Western blotting was then performed separately using the horseradish peroxidase (HRP)-conjugated monoclonal antibodies 3F10 against HA or
10D7-A7 against Vph1p to detect the V0 domain or antibody 8B1-F3 against the A-subunit to detect the V1 domain
followed by an HRP-conjugated secondary antibody. Dissociation of the
V-ATPase complex is reflected as a reduction in the amount of the
100-kDa subunit immunoprecipitated using the antibody directed against subunit A (located in the V1 domain). Blots were developed
using the chemiluminescent detection system from Kirkegaard and Perry Laboratories.
 |
RESULTS |
We wished to compare the properties of V-ATPases containing Vph1p
and Stv1p, the two isoforms of the yeast 100-kDa a-subunit. Because
these isoforms normally reside in different intracellular compartments,
it was necessary to devise a strategy that would allow complexes
containing these two isoforms to be targeted to the same intracellular
membrane. It has previously been shown that overexpression of Stv1p in
a strain disrupted in both VPH1 and STV1 causes a
significant amount of Stv1p to appear in the vacuolar membrane (24).
Because Vph1p normally localizes to the vacuole, this finding allowed
us to directly compare the properties of V-ATPase complexes present in
the same intracellular compartment that differed only in the a-subunit
isoform present. To detect the expression level of each a-subunit
isoform, tandem HA epitope tags were inserted in the amino-terminal
region of both Vph1p and Stv1p. Expression of the HA-tagged form of
either Vph1p or Stv1p using the low copy plasmid pRS316 in the
vph1
stv1
double-deletion strain MM112 led
to a wild-type growth phenotype. That is, cells showed normal growth at
both pH 7.5 and 5.5. This was also true using expression of Stv1p from
the high copy plasmid YEp352. Western blotting of whole cell lysates
(Fig. 1) indicated that Stv1p is expressed at a lower level from the single copy plasmid pRS316 than is
Vph1p. Expression of Stv1p using the high copy plasmid YEp352 led to a
significant increase in expression levels of Stv1p.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 1.
Western blot analysis of expression levels of
HA-tagged 100-kDa a-subunits expressed using low and high copy
plasmids. Whole cell lysates prepared from the
vph1 stv1 strain MM112 transformed with the
single copy plasmid pRS316 alone, HA-tagged Vph1p in pRS316, HA-tagged
Stv1p in pRS316, or HA-tagged Stv1p in the high copy plasmid YEp352
were subjected to SDS-PAGE on an 8% acrylamide gel followed by
transfer to nitrocellulose and Western blot analysis using the
HRP-conjugated monoclonal antibody 3F10 against the HA epitope or the
monoclonal antibody 8B1-F3 against the 69-kDa A-subunit followed by an
HRP-conjugated secondary antibody. Also shown are the growth phenotypes
of cells transformed with each of the indicated plasmids on YPD plates
buffered to pH 7.5.
|
|
To confirm previous reports that overexpression of Stv1p led to its
presence in the vacuolar membrane, vacuoles were isolated from each of
the strains shown in Fig. 1, separated by SDS-PAGE, and analyzed by
Western blotting using anti-HA antibodies. As shown in Fig.
2, although Vph1p was detectable in the
vacuolar membrane using the single copy plasmid, Stv1p was not. By
contrast, when Stv1p was expressed at higher levels using the multicopy plasmid, it was detectable in the vacuolar membrane at levels comparable with Vph1p. Overexposure of the gel shown in Fig. 2 revealed
very low levels of Stv1p in the vacuolar membrane when expressed using
pRS316. Quantitation using densitometric analysis indicated that
although the ratio of Vph1p to Stv1p using pRS316 in whole cell lysates
was 3:1 (Fig. 1, second and third lanes from the
left), the ratio in the vacuolar membrane was 30:1 (Fig. 2, longer
exposure, not shown). These results confirmed that although Stv1p is
normally not targeted to the vacuole, overexpression results in the
appearance of a significant amount of Stv1p in the vacuole.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 2.
Western blot analysis of levels of HA-tagged
Vph1p and Stv1p and the 69-kDa A-subunit on purified vacuolar
membranes. Vacuolar membranes were isolated from the
vph1 stv1 strain MM112 transformed with
pRS316 alone, HA-tagged Vph1p in pRS316, HA-tagged Stv1p in pRS316, or
HA-tagged Stv1p in Yep352, an aliquot (0.5 µg protein) was subjected
to SDS-PAGE on an 8% acrylamide gel, and the levels of the HA-tagged
Vph1p, HA-tagged Stv1p, and the 69 kDa A-subunit were determined by
Western blot analysis as described in the legend to Fig. 1. The amount
of A-subunit present on the vacuolar membrane is one measure of
assembly of the V-ATPase complex.
|
|
To assess the degree of assembly of V-ATPase complexes containing Vph1p
and Stv1p, vacuoles isolated from each strain were also probed using an
antibody directed against the 70-kDa A-subunit. It has previously been
shown that the A-subunit (and other V1 subunits) only
associates with the vacuolar membrane if assembly of the V-ATPase
complex is normal (27, 32). As shown in Fig. 2, although the level of
Stv1p in the vacuolar membrane using the high copy plasmid is
comparable with that of Vph1p using pRS316, the level of A-subunit
associated with Stv1p is much lower than that associated with Vph1p.
Quantitation by densitometry indicates an 8-fold reduction in the level
of A-subunit associated with vacuoles containing Stv1p relative to
vacuoles containing Vph1p. This result indicates that, at least when
present in the environment of the central vacuole, V-ATPase complexes
containing Stv1p are less assembled than V-ATPase complexes containing Vph1p.
To determine whether introduction of the HA epitope tags into Vph1p and
Stv1p altered assembly of the V-ATPase complex, vacuolar membranes from
strains expressing either tagged or untagged versions of Vph1p and
Stv1p were probed by Western blot using antibodies against HA, Vph1p,
subunit A, and subunit d (a V0 subunit). No antibodies
specific for the untagged version of Stv1p are currently available. As
can be seen from Fig. 3, the presence of
the HA tag had virtually no effect on the amount of Vph1p, subunit A, or subunit d present in the vacuolar membrane, indicating that introduction of the tag did not disrupt assembly of complexes containing Vph1p. Similarly, for Stv1p-containing complexes, the amounts of subunit A and subunit d present in the vacuoles was unchanged by the introduction of the epitope tag. Thus, the presence of
the HA tag does not alter the assembly of V-ATPase complexes. Interestingly, the ratio of subunit A to subunit d staining in the
vacuoles is almost the same for Vph1p and Stv1p (Fig. 3) despite the
much lower ratio of subunit A to 100-kDa subunit for Stv1p than for
Vph1p (Fig. 2). This result suggests that the lower assembly of
V1 and V0 observed for Stv1p-containing
complexes may be the result of reduced association of Stv1p with
subunit d, at least in the environment of the vacuolar membrane.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 3.
Comparison of assembly competence of
HA-tagged and untagged versions of Vph1p and Stv1p by Western blot
analysis of vacuolar membranes. Vacuolar membranes were isolated
from the vph1 stv1 strain MM112 transformed
with Vph1p in pRS316, HA-tagged Vph1p in pRS316, Stv1p in YEp352, or
HA-tagged Stv1p in YEp352, an aliquot (20 µg protein) was subjected
to SDS-PAGE on an 8% acrylamide gel, and the levels of the HA-tagged
Vph1p, HA-tagged Stv1p, Vph1p, subunit A, and subunit d were determined
by Western blot analysis as described in the legend to Fig. 1.
|
|
To compare the kinetic properties of V-ATPase complexes containing
Vph1p and Stv1p, vacuoles were isolated from the
vph1
stv1
strain expressing either Vph1p
using pRS316 or Stv1p using the high copy plasmid. In this case, the
untagged versions of Vph1p and Stv1p were expressed. As indicated in
Table I, the Km for
ATP was ~250 µM for complexes containing both Vph1p and
Stv1p. Although the Vmax for ATP hydrolysis for
Stv1p-containing complexes was lower than for Vph1p-containing
complexes, the 8.6-fold reduction in activity could be entirely
accounted for by the lower degree of assembly of V-ATPases containing
Stv1p (Fig. 2). Measurement of proton transport using ACMA fluorescence
quenching (Table I) indicated an even lower level of proton transport
for Stv1p-containing vacuoles relative to Vph1p-containing vacuoles
(the ratio of Vph1p:Stv1p was 55:1). This result indicates that the
ratio of proton transport to ATP hydrolysis is ~4-5-fold lower for
Stv1p-containing complexes compared with Vph1p-containing
complexes.
The inhibitor sensitivity of V-ATPase complexes containing Vph1p and
Stv1p was also compared. As shown in Table
II, ATPase activity for Vph1p-containing
vacuoles was 90% inhibited by 1 µM concanamycin A but
was only 8% inhibited by 0.1 mM vanadate and was not
affected by 0.5 mM azide. By contrast, ATPase activity in
Stv1p-containing vacuoles was only 44% inhibited by concanamycin A and
25% inhibited by vanadate. This apparent discrepancy between Vph1p and
Stv1p may be partially accounted for by the presence of one or more
concanamycin-resistant ATPase activities in the vacuolar membrane, one
of which is vanadate-sensitive. Under normal conditions
(i.e. with vacuoles containing Vph1p), these other activities are quite minor since the bulk of ATPase activity in the
vacuolar membrane corresponds to the V-ATPase. By contrast, because of
the much lower levels of V-ATPase activity observed with Stv1p (Table
I), the percentage of the total ATPase activity in the vacuolar
membrane that corresponds to these "other" ATPases is significantly
larger.
View this table:
[in this window]
[in a new window]
|
Table II
Effects of inhibitors on ATPase activity of isolated vacuoles
containing Vph1p and Stv1p
ATPase assays were carried out on isolated vacuolar membranes at 0.5 mM ATP in the presence or absence of the indicated
inhibitors as described under "Experimental Procedures." Activities
are expressed relative to the control sample, which received 0.1%
Me2SO. Both Vph1p and Stv1p were untagged.
|
|
To compare the affinity of V-ATPase complexes containing Vph1p and
Stv1p for concanamycin A, ATP-dependent proton pumping was
measured in vacuoles using ACMA fluorescence quenching. As shown in
Fig. 4, the concentration of concanamycin
A required for inhibition of 50% of proton transport for both Vph1p
and Stv1p-containing complexes was ~0.1 nM. A small
residual concanamycin-resistant component of ATP-dependent
fluorescence quenching in Stv1p-containing vacuoles was again observed.
Nevertheless, the results suggest that Vph1p- and Stv1p-containing
complexes have nearly the same affinity for concanamycin A.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibitory effect of concanamycin A on
ATP-dependent proton transport in vacuoles containing Vph1p
or Stv1p. Vacuolar membranes were isolated from the
vph1 stv1 strain MM112 transformed with
Vph1p in pRS316 or Stv1p in YEp352 (both untagged), and
ATP-dependent proton transport was measured at the
indicated concentrations of concanamycin A by fluorescence
(F) quenching using the fluorescence dye ACMA as described
under "Experimental Procedures." For Vph1p-containing membranes, 1 µg of protein was employed, whereas for Stv1p-containing membranes, 5 µg of protein was used. Activities are expressed as the initial rate
of fluorescence change after the addition of ATP.
|
|
Reversible dissociation of the V-ATPase complex has been shown to occur
in response to glucose depletion in yeast and has been proposed to
represent an important mechanism of regulating V-ATPase activity
in vivo (35). To compare glucose-dependent dissociation of V-ATPase complexes containing Vph1p and Stv1p, spheroplasts were prepared from the Vph1p- and Stv1p-expressing strains, incubated in the presence or absence glucose, and solubilized with C12E9, and immunoprecipitation was carried
out using the antibody 8B1-F3 directed against the A-subunit of the
V1 domain. After SDS-PAGE, Western blotting was performed
using either 8B1-F3 to detect the A-subunit or the anti-HA antibody to
detect Vph1p or Stv1p in the V0 domain. Dissociation of the
V-ATPase is reflected as a decrease in the amount of Vph1p or Stv1p
immunoprecipitated using the antibody against subunit A. As shown in
Fig. 5a, glucose depletion led
to a decrease in the amount of Vph1p immunoprecipitated using the
anti-A-subunit antibody (first and second lanes), indicating glucose-dependent dissociation of Vph1p-containing
complexes. Because of the lower level of Stv1p expressed using the
pRS316 vector, assembled V-ATPase complexes could not be detected at this exposure (Fig. 5, panel a, third and fourth
lanes). A longer exposure, however, revealed that
Stv1p-containing complexes do not show dissociation on removal of
glucose but instead show some increase in assembly (Fig. 5, panel
b, third and fourth lanes). By contrast, when Stv1p was
expressed at higher levels (under which conditions a significant amount
of Stv1p appears in the vacuole), V-ATPase complexes containing Stv1p
showed glucose-dependent dissociation (Fig. 5, panel
a, fifth and sixth lanes). By comparing the total
expression levels of Vph1p and Stv1p in whole cell lysates (Fig. 5,
panel c) with the amounts of Vph1p and Stv1p
immunoprecipitated with the anti-A-subunit antibody (Fig. 5,
panel b), it is clear that a smaller fraction of Stv1p is
assembled with V1, even when expressed at low levels and in
the presence of glucose than for Vph1p. This result is consistent with
the data shown in Fig. 2. Quantitation of the results from five
independent experiments by densitometry (Fig. 5d) indicates
that glucose removal leads to 65% dissociation of complexes containing
Stv1p expressed at high levels as compared with 45% dissociation of
complexes containing Vph1p. By contrast, V-ATPase complexes formed from
Stv1p expressed at low levels show no significant dissociation upon
removal of glucose. These results suggest that the membrane environment
plays a crucial role in determining whether the V-ATPase undergoes
dissociation in response to glucose depletion, with V-ATPase present in
the central vacuole undergoing dissociation and V-ATPase present in the
Golgi/endosomes remaining assembled.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 5.
In vivo dissociation of V-ATPase
complexes containing Vph1p or Stv1p after glucose depletion.
a, The vph1 stv1 strain MM112
transformed with HA-tagged Vph1p in pRS316, HA-tagged Stv1p in pRS316,
or HA-tagged Stv1p in YEp352 were grown in selective media overnight
and converted to spheroplasts. The spheroplasts were incubated for 40 min in YEP media with or without 2% glucose. The V-ATPase was then
solubilized with C12E9 and immunoprecipitated
using the monoclonal antibody 8B1-F3 against the A-subunit. Samples
were then subjected to SDS-PAGE on 8% acrylamide gels followed by
transfer to nitrocellulose and Western blot analysis using the
HRP-conjugated monoclonal antibody 3F10 against HA or the monoclonal
antibody 8B1-F3 against the 69-kDa A-subunit followed by an
HRP-conjugated secondary antibody. Dissociation of the V-ATPase complex
is reflected as a decrease in the amount of Vph1p or Stv1p
immunoprecipitated using the antibody against subunit A. b,
a longer exposure of the film shown in panel a showing the
degree of glucose-dependent dissociation for Stv1p
expressed at lower levels. c, Western blot analysis was
performed on an aliquot of the whole cell lysate derived from each of
the strains described in part a using antibodies against HA or subunit
A. d, quantitation of the data from five independent
determinations was performed by densitometric analysis, and the results
are expressed as the amount of assembled V-ATPase observed upon glucose
depletion relative to that observed in the presence of glucose.
|
|
Dissociation of Vph1p-containing complexes in response to glucose
depletion has previously been shown to be reversible upon the
readdition of glucose to the media (35). To test whether dissociation
of Stv1p-containing complexes that have been targeted to the vacuole is
also reversible, spheroplasts were prepared from cells expressing Stv1p
from the high copy plasmid, incubated in the presence or absence of
glucose for 20 min, or incubated in the absence of glucose for 20 min
followed by the addition of glucose and incubation for an additional 20 min. After these incubations, spheroplasts were solubilized with
C12E9, and immunoprecipitation was carried out
using the anti-A-subunit antibody followed by SDS-PAGE and Western
blotting using antibodies against both HA and subunit A, as described
above. As can be seen from the data in Fig.
6, dissociation of Stv1p-containing
complexes targeted to the vacuole in response to glucose depletion is
also reversible upon the readdition of glucose to the media.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 6.
Reversal of dissociation of Stv1p-containing
complexes present in the vacuolar membrane upon the readdition of
glucose to the medium. The vph1 stv1
strain MM112 transformed with HA-tagged Stv1p in YEp352 was grown in
selective media overnight and converted to spheroplasts. The
spheroplasts were incubated for 20 min in YEP media with 2% glucose
(+), for 20 min in YEP media without glucose ( ), or for 20 min in YEP
media without glucose followed by the addition of 2% glucose and
incubation for an additional 20 min ( +).
The V-ATPase was then solubilized with C12E9
and immunoprecipitated using the anti-A-subunit antibody followed by
SDS-PAGE and Western blot analysis using the anti-HA and anti-A-subunit
antibodies as described in the legend to Fig. 5.
|
|
To further test the importance of the membrane environment in affecting
in vivo dissociation of the V-ATPase complex,
glucose-dependent dissociation was examined in two yeast
strains that are disrupted in different steps of the membrane traffic
pathway from the Golgi to the vacuole. In vps21
mutants,
Vph1p is found in Golgi-derived vesicles that accumulate as a result of
a block in fusion with the prevacuolar compartment, whereas in the
vps27
mutants, Vph1p appears in an exaggerated form of
the prevacuolar compartment (36). Glucose-dependent
dissociation of Vph1p-containing complexes was examined in these
strains relative to the parent strain using an antibody specific for
Vph1p. As shown in Fig. 7, glucose
depletion appeared to lead to more complete dissociation of
Vph1p-containing complexes in the wild-type strain (87%) than was
observed for Vph1p-containing complexes in the
vph1
stv1p
strain described in Fig. 5. When
the experiment in Fig. 5 was repeated using the antibody against Vph1p,
approximately the same level of dissociation was observed upon glucose
depletion (90%) as was seen in Fig. 7. This indicates that the
apparent difference in dissociation observed between Fig. 5 and Fig. 7
is due to differences in the ability of the anti-HA and anti-Vph1p
antibodies to detect low levels of the 100-kDa proteins. However, it
was necessary to use the anti-HA antibody for the experiments described
in Fig. 5 because of the absence of antibodies specific for Stv1p.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 7.
In vivo dissociation of V-ATPase
complexes containing Vph1p in the wild-type strain and
vps21 and vps27
strains disrupted in vacuolar targeting. a, yeast
strain SF838-1D (wild type), SGY79 (vps21 ), and SGY73
(vps27 ) were grown in selective media overnight and
converted to spheroplasts. The spheroplasts were incubated for 40 min
in YEP media with or without 2% glucose. The V-ATPase was then
solubilized with C12E9 and immunoprecipitated
using the monoclonal antibody 8B1-F3 against the A-subunit. Samples
were then subjected to SDS-PAGE on 8% acrylamide gels followed by
transfer to nitrocellulose, and Western blot analysis was performed
using the monoclonal antibody 10D7-A7 against Vph1p or the antibody
8B1-F3 against subunit A. Also shown are the growth phenotypes of each
strain on YPD plates buffered to pH 7.5. b, Western blot
analysis was performed on an aliquot of the whole cell lysate derived
from each of the strains described in part a using antibodies against
Vph1p or subunit A. c, quantitation of the data from three
independent determinations was performed by densitometric analysis, and
the results are expressed as the amount of assembled V-ATPase observed
upon glucose depletion relative to that observed in the presence of
glucose.
|
|
In both the vps21
and vps27
strains,
dissociation of the V-ATPase in response to glucose depletion was
observed but was less complete than for the wild-type strain.
Quantitation of three independent experiments (Fig. 7c)
revealed dissociation levels of 63 and 76% in the vps21
and vps27
strains, respectively, as compared with the
87% dissociation observed in the parental wild-type strain. Western
blot analysis of whole cell lysates indicated a slight reduction in the
level of Vph1p in the vps21
strain (Fig. 7b).
Glucose-dependent dissociation of the V-ATPase was shown to
be reversible upon the readdition of glucose to the media for the
wild-type, vps21
, and vps27
strains (Fig.
8). Because Vph1p-containing complexes
are prevented from reaching the vacuole in the vps21
and
vps27
strains, the results indicate that complexes
containing Vph1p that are present in other intracellular compartments
are still able to undergo glucose-dependent dissociation, although to different degrees. It should be noted that because the
compartments that accumulate in the vps21
and
vps27
strains are aberrant (36), it is possible that the
reduction in dissociation observed in these strains may be due to some
altered property of these compartments. For example, the multilamellar
structure of the prevacuolar compartment that accumulates in the
vps27
strain may prevent some essential cytoplasmic
signal from reaching a significant fraction of the V-ATPase present in
this compartment. Nevertheless, together with the experiments described
above, these results suggest that in vivo dissociation of
the V-ATPase complex is affected by both the a-subunit isoform present
and the intracellular environment in which the complex resides.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 8.
Reversal of dissociation of V-ATPase in wild
type and vps21 and
vps27 strains upon the readdition of
glucose to the medium. Yeast strain SF838-1D (wild type), SGY79
(vps21 ), and SGY73 (vps27 ) were grown in
selective media overnight and converted to spheroplasts. The
spheroplasts were incubated for 20 min in YEP media with 2% glucose
(+), for 20 min in YEP media without glucose ( ), or for 20 min in YEP
media without glucose followed by the addition of 2% glucose and
incubation for an additional 20 min ( +).
The V-ATPase was then solubilized with C12E9
and immunoprecipitated using the anti-A-subunit antibody followed by
SDS-PAGE and Western blot analysis using the anti-Vph1p and
anti-A-subunit antibodies as described in the legend to Fig. 7.
|
|
 |
DISCUSSION |
V-ATPases have been shown to reside in a large number of different
intracellular compartments in eukaryotic cells (1-8). These include
clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles
and secretory vesicles such as chromaffin granules and synaptic
vesicles. V-ATPases have also been identified in the plasma membrane of
numerous animal cell types, including osteoclasts, renal intercalated
cells, macrophages, neutrophils, insect goblet cells, and certain tumor
cells (9-11, 37, 38). An important question is how V-ATPases are
targeted to their correct cellular destination. Recent results suggest
that the 100-kDa a-subunit may play a crucial role in this targeting
process. Thus, three unique isoforms of the a-subunit have been
identified in mouse (a1, a2, and a3) (39, 40). In osteoclasts, the a1
isoform has been shown to localize to intracellular compartments,
whereas the a3 isoform localizes to the plasma membrane (40).
Consistent with this observation, it has been shown that disruption of
the a3 gene in mouse leads to defective bone resorption (41). In yeast,
the a-subunit is also encoded by multiple genes whose protein products
show distinct cellular localization. Vph1p is localized to the central
vacuole, whereas Stv1p is localized to some other intracellular
compartment, possibly Golgi or endosomes (24). An important but
unanswered question is how the properties of V-ATPases containing
unique isoforms of the 100-kDa a-subunit compare.
To address this question, we have overexpressed Stv1p in a strain
disrupted in both VPH1 and STV1, under which circumstances a
significant amount of Stv1p becomes localized to the vacuole (24). This
allowed a direct comparison of the properties of V-ATPase complexes
present in the vacuolar membrane that differed only in the a-subunit
isoform present. The results suggest that many of the kinetic
properties of V-ATPases containing Vph1p or Stv1p are very similar.
Thus, these enzymes exhibit the same affinity for ATP and the same (or
very similar) inhibitor sensitivity. By contrast, V-ATPases containing
the two a-subunit isoforms differ markedly in three properties. First,
they show a significant (4-5-fold) difference in the ratio of proton
transport to ATP hydrolysis, with Stv1p-containing complexes exhibiting
a much lower efficiency of coupling. Changes in coupling efficiency
have been proposed to play a role in controlling V-ATPase activity
in vivo (42, 43), but this is the first comparison of
coupling for two V-ATPases expressed in the same cell. This result also
suggests that the V1 and V0 domains may be more
loosely connected for Stv1p-containing complexes than for those
containing Vph1p.
Consistent with this idea is the second major difference between the
two isoforms, namely the degree of assembly of V1 and V0. Thus, V0 complexes containing Stv1p showed
a lower degree of assembly with V1 than did V0
complexes containing Vph1p. This may be related to the observation that
Stv1p present in the vacuole also shows a lower association with
subunit d, which is normally an essential component of the
V0 domain (44). Thus, the ratio of subunit d to subunit A
in vacuoles derived from the strain overexpressing Stv1p is the same as
that observed for vacuoles derived from the strain expressing Vph1p
(Fig. 3). The absence of subunit d has been shown to lead to the loss
of assembly of V1 subunits with the vacuolar membrane (44),
and this may account for the reduced assembly of Stv1p-containing
complexes. Nevertheless, it should be noted that Stv1p expressed at
high levels appears to be stable in the vacuolar membrane (Figs. 2 and
3), suggesting that assembly of the entire V0 domain is not defective.
It is possible that these differences between Vph1p and Stv1p may in
part be due to the presence of Stv1p in an intracellular compartment in
which it does not normally reside. However, it is also possible that
V-ATPase complexes in the Golgi (which normally contain Stv1p) may not
need to be as active or as tightly coupled as V-ATPases in the central
vacuole (which normally contains Vph1p). In support of this idea is the
observation that in animal cells the pH of the Golgi is generally
maintained in the range of 6.0-6.5 (45, 46), whereas the pH of
lysosomes (the compartment analogous to the vacuole in higher
eukaryotes) is typically 4.0-5.0 (47). Moreover, the lower degree of
assembly of V1 and V0 was also observed for
Stv1p present in its normal cellular location (Fig. 5).
The third property in which V-ATPase complexes containing Vph1p and
Stv1p differ is their degree of dissociation in response to glucose
deprivation. The Kane laboratory (35) demonstrates that the V-ATPase in
yeast undergoes rapid and reversible dissociation in response to
removal of glucose from the medium and has proposed that this process
represents an important mechanism of regulating V-ATPase activity
in vivo. They have shown that many of the signal transduction pathways activated by glucose starvation, including the
Ras-cyclic AMP and protein kinase C-dependent pathways, are not involved in this response (48). Dissociation of the V1
and V0 domains has also been shown to occur in insects
during molting, under which conditions the need for an active V-ATPase
at the apical membrane of goblet cells lining the midgut may be
dramatically reduced (49). Pools of free V1 and
V0 domains have also been identified in Madin-Darby bovine
kidney cells (50), but rapid and reversible dissociation of the
V-ATPase in mammalian cells has not yet been reported.
In the present report it is shown that dissociation of V-ATPase
complexes in response to glucose depletion appears to be controlled by
both the a-subunit isoform present and the intracellular membrane in
which the complex resides. Thus, Stv1p-containing complexes show no
glucose-dependent dissociation when localized to their normal intracellular site (Golgi/endosomes) but do dissociate when
targeted to the vacuole. By contrast, Vph1p-containing complexes appear
to undergo dissociation both in their normal cellular environment (the
vacuole) and when retained in Golgi or prevacuolar compartments. Nevertheless, dissociation of Vph1p-containing complexes is at least
partly affected by the cellular environment in which these complexes
reside, although altered properties of the compartments that accumulate
in the vps mutants may be partly responsible for the reduced
glucose-dependent dissociation. These results suggest that
there are signals present both in the sequence of the a-subunit and in
distinct intracellular sites that control dissociation of the V-ATPase
complex in vivo. One possible reason that V-ATPase complexes
present in the vacuole show more complete dissociation in response to
glucose depletion than complexes present in other intracellular
compartments (such as the Golgi) is that there may be a larger reserve
of active V-ATPase in the central vacuole that the cell is able to
survive without. Thus, the activity remaining after dissociation may be
sufficient to keep the central vacuole relatively acidic while
conserving significant amounts of cellular ATP. By contrast, V-ATPases
present in the Golgi (or other prevacuolar compartments) appear to be
less abundant but may therefore be less dispensable for cell viability.
It should be noted, however, that because Stv1p-containing complexes
show a lower degree of assembly with V1 than
Vph1p-containing complexes, even in the presence of glucose, glucose
withdrawal may not be able to affect any greater degree of dissociation
of these complexes.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Tom Stevens, Institute of
Molecular Biology, University of Oregon, for the gift of antibody
against subunit d and yeast strains SGY73 and SGY79 disrupted in
VPS27 and VPS21, respectively.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM34478 (to M. F.) and a Charles A. King Trust Fellowship Award (to T. N.).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.
These authors contributed equally to this work.
§
To whom correspondence should be addressed: Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston, MA
02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail address: michael.forgac@tufts.edu.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M010790200
 |
ABBREVIATIONS |
The abbreviations used are:
V-ATPase, vacuolar
proton-translocating adenosine triphosphatase;
ACMA, 9-amino-6-chloro-2-methoxyacridine;
HA, influenza hemagglutinin;
MES, 2-(N-morpholino)ethanesulfonic acid;
PAGE, polyacrylamide
gel electrophoresis;
SD medium, synthetic dropout medium;
YPD medium, yeast extract-peptone-dextrose medium;
YEP medium, yeast
extract-peptone medium;
HRP, horseradish peroxidase,
C12E9, polyoxyethylene-9-lauryl ether.
 |
REFERENCES |
1.
|
Forgac, M.
(1999)
J. Biol. Chem.
274,
12951-12954[Free Full Text]
|
2.
|
Stevens, T. H.,
and Forgac, M.
(1997)
Annu. Rev. Cell Dev. Biol.
13,
779-808[CrossRef][Medline]
[Order article via Infotrieve]
|
3.
|
Margolles-Clark, E.,
Tenney, K.,
Bowman, E. J.,
and Bowman, B. J.
(1999)
J. Bioenerg. Biomembr.
31,
29-38[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Kane, P. M.
(1999)
J. Bioenerg. Biomembr.
31,
49-56[CrossRef][Medline]
[Order article via Infotrieve]
|
5.
|
Nelson, N.,
and Harvey, W. R.
(1999)
Physiol. Rev.
79,
361-385[Abstract/Free Full Text]
|
6.
|
Moriyama, Y.,
Yamamoto, A.,
Yamada, H.,
Tashiro, Y.,
and Futai, M.
(1996)
Biol. Chem. Hoppe-Seyler
377,
155-165[Medline]
[Order article via Infotrieve]
|
7.
|
Anraku, Y.,
Umemoto, N.,
Hirata, R.,
and Ohya, Y.
(1992)
J. Bioenerg. Biomembr.
24,
395-405[Medline]
[Order article via Infotrieve]
|
8.
|
Sze, H.,
Ward, J. M.,
and Lai, S.
(1992)
J. Bioenerg. Biomembr.
21,
371-382
|
9.
|
Chatterjee, D.,
Chakraborty, M.,
Leit, M.,
Neff, L.,
Jamsa-Kellokumpu, S.,
Fuchs, R.,
and Baron, R.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6257-6261[Abstract]
|
10.
|
Gluck, S. L.
(1992)
J. Bioenerg. Biomembr.
21,
351-360
|
11.
|
Nanda, A.,
Brumell, J. H.,
Nordstrom, T.,
Kjeldsen, L.,
Sengelov, H.,
Borregaard, N.,
Rotstein, O. D.,
and Grinstein, S.
(1996)
J. Biol. Chem.
271,
15963-15970[Abstract/Free Full Text]
|
12.
|
Weber, J.,
and Senior, A.
(1997)
Biochim. Biophys. Acta
1319,
19-58[Medline]
[Order article via Infotrieve]
|
13.
|
Fillingame, R. H.
(2000)
J. Exp. Biol.
203,
9-17[Abstract]
|
14.
|
Cross, R. L.,
and Duncan, T. M.
(1996)
J. Bioenerg. Biomembr.
28,
403-408[Medline]
[Order article via Infotrieve]
|
15.
|
Capaldi, R. A.,
Schulenberg, B.,
Murray, J.,
and Aggler, R.
(2000)
J. Exp. Biol.
203,
29-33[Abstract]
|
16.
|
Pedersen, P. L.
(1996)
J. Bioenerg. Biomembr.
28,
389-395[Medline]
[Order article via Infotrieve]
|
17.
|
Futai, M.,
and Omote, H.
(1996)
J. Bioenerg. Biomembr.
28,
409-414[Medline]
[Order article via Infotrieve]
|
18.
|
Zimniak, L.,
Dittrich, P.,
Gogarten, J. P.,
Kibak, H.,
and Taiz, L.
(1988)
J. Biol. Chem.
263,
9102-9112[Abstract/Free Full Text]
|
19.
|
Bowman, B. J.,
Allen, R.,
Wechser, M. A.,
and Bowman, E. J.
(1988)
J. Biol. Chem.
263,
14002-14007[Abstract/Free Full Text]
|
20.
|
Mandel, M.,
Moriyama, Y.,
Hulmes, J. D.,
Pan, Y. C.,
Nelson, H.,
and Nelson, N.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
5521-5524[Abstract]
|
21.
|
Hirata, R.,
Graham, L. A.,
Takatsuki, A.,
Stevens, T. H.,
and Anraku, Y.
(1997)
J. Biol. Chem.
272,
4795-4803[Abstract/Free Full Text]
|
22.
|
Hunt, I. E.,
and Bowman, B. J.
(1997)
J. Bioenerg. Biomembr.
29,
533-540[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Manolson, M. F.,
Proteau, D.,
Preston, R. A.,
Stenbit, A.,
Roberts, B. T.,
Hoyt, M.,
Preuss, D.,
Mulholland, J.,
Botstein, D.,
and Jones, E. W.
(1992)
J. Biol. Chem.
267,
14294-14303[Abstract/Free Full Text]
|
24.
|
Manolson, M. F.,
Wu, B.,
Proteau, D.,
Taillon, B. E.,
Roberts, B. T.,
Hoyt, M. A.,
and Jones, E. W.
(1994)
J. Biol. Chem.
269,
14064-14074[Abstract/Free Full Text]
|
25.
|
Perin, M. S.,
Fried, V. A.,
Stone, D. K.,
Xie, X. S.,
and Sudhof, T. C.
(1991)
J. Biol. Chem.
266,
3877-3881[Abstract/Free Full Text]
|
26.
|
Kane, P. M.,
Yamashiro, C. T.,
and Stevens, T. H.
(1989)
J. Biol. Chem.
264,
19236-19244[Abstract/Free Full Text]
|
27.
|
Kane, P. M.,
Kuehn, M. C.,
Howald-Stevenson, I.,
and Stevens, T.
(1992)
J. Biol. Chem.
267,
447-454[Abstract/Free Full Text]
|
28.
|
Sherman, F.
(1991)
Methods Enzymol.
194,
3-21[Medline]
[Order article via Infotrieve]
|
29.
|
Gietz, D.,
St. Jean, A.,
Woods, R. A.,
and Schiestl, R. H.
(1992)
Nucleic Acids Res.
20,
1425[Medline]
[Order article via Infotrieve]
|
30.
|
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K.
(eds)
(1992)
Short Protocols in Molecular Biology
, John Wiley & Sons, Inc., New York
|
31.
|
Uchida, E.,
Ohsumi, Y.,
and Anraku, Y.
(1985)
J. Biol. Chem.
260,
1090-1095[Abstract/Free Full Text]
|
32.
|
Leng, X. H.,
Manolson, M.,
Liu, Q.,
and Forgac, M.
(1996)
J. Biol. Chem.
271,
22487-22493[Abstract/Free Full Text]
|
33.
|
Forgac, M.,
Cantley, L.,
Wiedenmann, B.,
Altstiel, L.,
and Branton, D.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
1300-1303[Abstract]
|
34.
|
Feng, Y.,
and Forgac, M.
(1992)
J. Biol. Chem.
267,
5817-5822[Abstract/Free Full Text]
|
35.
|
Kane, P. M.
(1995)
J. Biol. Chem.
270,
17025-17032[Abstract/Free Full Text]
|
36.
|
Gerard, S. R.,
Bryant, N. J.,
and Stevens, T. H.
(2000)
Mol. Biol. Cell
11,
613-626[Abstract/Free Full Text]
|
37.
|
Wieczorek, H.,
Putzenlechner, M.,
Zeiske, W.,
and Klein, U.
(1991)
J. Biol. Chem.
266,
15340-15347[Abstract/Free Full Text]
|
38.
|
Martinez-Zaguilan, R.,
Lynch, R.,
Martinez, G.,
and Gillies, R.
(1993)
Am. J. Physiol.
265,
C1015-C1029[Abstract/Free Full Text]
|
39.
|
Nishi, T.,
and Forgac, M.
(2000)
J. Biol. Chem.
275,
6824-6830[Abstract/Free Full Text]
|
40.
|
Toyomura, T.,
Oka, T.,
Yamaguchi, C.,
Wada, Y.,
and Futai, M.
(2000)
J. Biol. Chem.
275,
8760-8765[Abstract/Free Full Text]
|
41.
|
Li, Y. P.,
Chen, W.,
Liang, Y.,
Li, E.,
and Stashenko, P.
(1999)
Nat. Genet.
23,
447-451[CrossRef][Medline]
[Order article via Infotrieve]
|
42.
|
Nelson, N.
(1992)
J. Bioenerg. Biomembr.
24,
407-414[Medline]
[Order article via Infotrieve]
|
43.
|
Arai, H.,
Pink, S.,
and Forgac, M.
(1989)
Biochemistry
28,
3075-3082[Medline]
[Order article via Infotrieve]
|
44.
|
Bauerle, C.,
Ho, M. N.,
Lindorfer, M. A.,
and Stevens, T. F.
(1993)
J. Biol. Chem.
268,
12749-12757[Abstract/Free Full Text]
|
45.
|
Kim, J. H.,
Lingwood, C. A.,
Williams, D. B.,
Furuya, W.,
Manolson, M. F.,
and Grinstein, S.
(1996)
J. Cell Biol.
134,
1387-1399[Abstract]
|
46.
|
Seksek, O.,
Biwersi, J.,
and Verkman, A. S.
(1995)
J. Biol. Chem.
270,
4967-4970[Abstract/Free Full Text]
|
47.
|
Ohkuma, S.,
and Poole, B.
(1978)
Proc. Natl. Acad. Sci. U. S. A.
75,
3327-3331[Abstract]
|
48.
|
Parra, K. J.,
and Kane, P. M.
(1998)
Mol. Cell. Biol.
18,
7064-7074[Abstract/Free Full Text]
|
49.
|
Sumner, J. P.,
Dow, J. A.,
Early, F. G.,
Klein, U.,
Jager, D.,
and Wieczorek, H.
(1995)
J. Biol. Chem.
270,
5649-5653[Abstract/Free Full Text]
|
50.
|
Myers, M.,
and Forgac, M.
(1993)
J. Cell. Physiol.
156,
35-42[Medline]
[Order article via Infotrieve]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.