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J Biol Chem, Vol. 275, Issue 20, 15449-15457, May 19, 2000
From the The vacuolar-type H+-ATPase
(V-ATPase) is composed of a peripherally bound (V1) and a
membrane-associated (V0) complex. V1 ATP
hydrolysis is thought to rotate a central stalk, which in turn, is
hypothesized to drive V0 proton translocation. Transduction of torque exerted by the rotating stalk on V0 requires a
fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V1 relative to
V0; this work sought to identify stator components. The
95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH2
terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid
assays demonstrated that Nt-Vph1p interacts with the catalytic
V1 subunit, Vma1p. Co-immunoprecipitation of Vma1p with
Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a
Vacuolar-type H+-ATPases
(V-ATPases)1 are evolutionary
conserved multimeric complexes that mediate the lumenal acidification of various organelles (yeast and plant vacuoles, endosomes, lysosomes, and clathrin-coated vesicles). Organellar acidification is essential for a variety of cellular processes such as receptor-mediated endocytosis, processing of proteins, intracellular degradation of
ingested pathogens, and proton-coupled transport of small molecules (reviewed in Ref. 1).
V-ATPases are evolutionarily related to the mitochondrial
F1F0 H+-ATPases (F-ATPases) (2).
Thus, the 2.8-Å resolution of the catalytic complex of the F-ATPase
(3) and experiments describing how intersubunit rotation leads to
energy transduction in F-ATPases (4, 5) have provided significant
insight into the structure and function of V-ATPases. V-ATPases have a
similar bipartite structure to F-ATPases, consisting of a membrane
bound proton channel (V0) and a peripherally bound
catalytic core (V1). Electron microscopy reveals the
V1 complex as a lollipop-like structure with a 6-nm long
and 4-nm wide "central stalk" rising out of the membrane (6, 7) and
a 12 × 14-nm diameter ball symmetrically situated on top of the
stalk (7, 8). The ball is a hexamer composed of alternating 69- (Vma1p)
and 60-kDa (Vma2p) subunits symmetrically arranged to form a hollow
core through the middle of the sphere. Vma1p and Vma2p both contain
consensus sequences for ATP-binding domains (9-11); Vma1p has been
ascribed a catalytic function (12) while Vma2p is thought to play a
regulatory role (13). The hollow core formed by the ATP-binding
subunits contains a central stalk, the composition of which is still
debated. Xu et al. (14) propose that the central stalk is
composed of just Vma7p (14 kDa) and Vma8p (32 kDa) while Tomashek
et al. (15) hypothesize that the structure also includes
Vma4p (27 kDa) and Vma10p (16 kDa). The membrane-bound V0
complex contains six copies of the 17-kDa proteolipid, Vma3p (16), one
copy of Vma6p (36 kDa), and at least one (17), but possibly up to three
copies (18), of Vph1p (95 kDa). Vma11p (19) and Vma16p (20) are 17- and
23-kDa subunits with 56 and 35% amino acid identity to Vma3p,
respectively. They are indispensable for V-ATPase activity, yet their
role and stoichiometry in the V0 complex is unknown (21).
In F-ATPases, ATP hydrolysis rotates a stalk structure composed of the
To date, the structure of the V-ATPase remains less well defined,
however, the extensive homology between V- and F-ATPases suggests that
the mechanical coupling of the V1 and V0
complexes likely occurs by a method analogous to that described for
F-ATPase. Visualization of bacterial and bovine V-ATPases by electron
microscopy supports both the presence of a central shaft linking
V1 to V0 and a peripheral stalk (13 nm long and
6.5 nm off-center from the central stalk) that appears to originate
from the V0 complex and extend to the top of the
V1 complex (6, 7, 25). As there are no clear V-ATPase
homologues for the F-ATPase a, b, or The 95-kDa Vph1p subunit is unique to vacuolar H+-ATPases
with no homologue found in any F-ATPase. Hydropathy analysis of Vph1p predicts two distinct structural domains; a hydrophilic
NH2-terminal domain (~45 kDa) and a hydrophobic
COOH-terminal domain (~50 kDa) containing up to 9 putative
membrane-spanning regions (26). We have unambiguously localized the
NH2 terminus of Vph1p to the cytosol, making this domain an
ideal candidate to contribute to the formation of a peripheral stalk
(27). Here we demonstrate that the NH2 terminus of Vph1p
interacts with Vma1p and Vma13p, and present proteolytic evidence that
suggests Vph1p is involved in coupling V1 ATP hydrolysis to
V0 proton translocation.
Materials and Strains
Zymolase was purchased from ICN Biochemicals and bafilomycin
A1 from Kamiya Biomedical Co. The strains of
Saccharomyces cerevisiae utilized were the
protease-deficient wild type BJ926 (a/ Plasmids
pRS316-VPH1 (MM322)--
A 4.2-kilobase
SalI-ScaI fragment of pVIP1-82 containing the
entire open reading frame of VPH1 (28) cloned into
SalI-SmaI cut pRS316. Expresses Vph1p at wild
type levels in a pRS316-Nt-Vph1p (MM501)--
Identical to pRS316-VPH1
except that an in-frame stop codon (and a HpaI restriction
site) was inserted just before the first putative transmembrane region
of Vph1p through site-directed mutagenesis with oligonucleotide MO29
(5'-ctgtgactgttaacgtattgctcag-3'). It does not complement a
pRS426-Nt-Vph1p (MM506)--
A 4.15-kilobase
XhoI-NotI fragment of pRS316-Nt-Vph1p cloned into
XhoI-NotI cut pRS426. Despite the high copy
plasmid, levels of the expected 55-kDa protein were still greatly
reduced as compared with wild type Vph1p.
pET-Nt-Vph1p (MM658)--
PCR was performed on pVIP1-82 (28)
with oligonucleotide MO33 (5'-caaggaaaaccatggcagag-3': introduces
NcoI restriction site at first methionine of Vph1p) and
oligonucleotide MO74 (5'-gcgaattccttaattgatttctctgtactgagc-3': introduces an EcoRI restriction site and a stop codon just
before the first putative transmembrane region of Vph1p). The PCR
product was cut with NcoI and EcoRI and cloned
into the NcoI-EcoRI restriction sites of pET32b
(Novagen, Madison, WI). Sequencing confirmed fidelity of the resulting plasmid.
pAS2-1-Nt-Vph1p (MM570)--
PCR was performed on pVIP1-82 with
oligonucleotide MO33 (5'-caaggaaaaccatggcagag-3': introduces
NcoI restriction site at first methionine of Vph1p) and
oligonucleotide MO19 (5'-agcattgatagatctgtactgagc-3': introduces
BglII restriction site just before the first putative transmembrane region of Vph1p). The PCR product was cut with
NcoI and BglII and cloned into the
NcoI-BamHI sites of pAS1-CYH2
(CLONTECH Laboratories Inc., Palo Alto, CA).
Sequencing confirmed fidelity of the resulting plasmid.
pACT2-Nt-Vph1p (MM606)--
A 1.2-kilobase
NcoI-SalI fragment of pAS2-1-Nt-Vph1p was cloned
into the NcoI-XhoI sites of pACT2
(CLONTECH Laboratories Inc., Palo Alto, CA).
pAS2-1-Nt-Stv1p (MM546)--
PCR was performed on
pKS-STV1 (33) with oligonucleotide MO38
(5'-tatgaatacatatgaatcaagaagagg-3': introduces NdeI
restriction site at first methionine of Stv1p) and oligonucleotide MO39
(5'-ccagcattggatcctttatatgttgcg-3': introduces BamHI
restriction site just before the first putative transmembrane region of
Stv1p). The PCR product was cut with NdeI and
BamHI and cloned into the NdeI-BamHI
sites of pAS1-CYH2. pACT1-CT-Vma1p (MM582), original plasmid
obtained through a two-hybrid screen using pAS2-1-Nt-Vph1p as bait.
Contains 955 base pairs of VMA1 (encoding the last 285 amino
acid residues of Vma1p) cloned into the XhoI site of pACT.
pAS2-1-CT-Vma1p (MM610)--
A 975-base pair PaeR7I
fragment of pACT1-CT-Vma1p was cloned into the SalI site of
pAS2-1.
pBS-KS pRS316-VPH1-R53S (MM636)--
A 730-base pair
NheI-BstEII fragment of
pBS-KS pRS306-VPH1-R53S (MM637)--
A 4.2-kilobase
XhoI-SacI fragment of pRS316-VPH1-R53S was cloned
into XhoI-SacI cut pRS306. The unique
PstI restriction site in the URA3 gene was used
to integrate the plasmid into the ura3-52 locus of the
yeast genome.
MM638--
Antibodies
ATPase subunits were detected or immunoprecipitated with one of
the following antibodies. 1) A monoclonal antibody directed against
Vma1p, the 69-kDa catalytic V-ATPase subunit (clone 8B1-F3, Molecular
Probes). 2) A rabbit polyclonal antibody raised against the plant
homologue of Vma1p, purified from Beta vulagris L.
(described in Ref. 30). 3) A rabbit polyclonal antibody directed
against full-length Vph1p (described in Ref. 28). 4) A rabbit
polyclonal antibody directed against the NH2 terminus of
yeast Vph1p (27). 5) A polyclonal antibody directed against Vma13p, the
54-kDa V-ATPase subunit (kind gift from Dr. T. Stevens, Oregon State University).
Two-hybrid Screening
The two-hybrid library, a kind gift from Dr. Stephen J. Elledge,
was created from S. cerevisiae cDNA (size selected to be >600 base pairs) cloned into the XhoI site of Lambda ACT as
described in Ref. 31. All components were obtained from
CLONTECH and all assays were carried out as
suggested by the manufacturer. The bait, pAS2-1-Nt-Vph1p, was created
as described above. The pACT yeast cDNA library was introduced the
yeast reporter strain, Y190, and 5 × 106
transformants were selected for tryptophan, leucine, and histidine prototrophy. Isolated colonies (100) were subsequently tested for
Transformed cells were streaked onto sterile Whatman No. 1 filters and grown on selective media plates. Following colony growth, the filters were assayed for Immunoprecipitation
Yeast cells were grown overnight (to a concentration no greater
than 1.4 × 107 cells/ml in selective medium), spun
down, and resuspended at 3 × 107 cell/ml in 2 mM dithiothreitol, 50 mM glycine-NaOH, pH 10, for 10 min at 30 °C. Cells were pelleted and resuspended at 6 × 107 cells/ml in 1.2 M sorbitol, 2 mM dithiothreitol, 10 mM Tris-HCl, pH 7.5, with
4 units/ml Zymolyase 100T (ICN) for 30 min at 30 °C. Following
confirmation of cell wall digestion, the spheroplasts were gently
washed twice in 1.2 M sorbitol and resuspended at 1 × 108 cells/ml in solubilization buffer (phosphate-buffered
saline (137 mM NaCl, 2.7 mM KCl, 5.4 mM Na2HPO4, 1.8 mM
KH2PO4, pH 7.4), 1%
N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate
(ZW3-14), 2 mM dithiothreitol, 5 mM ATP, 5 mM MgCl2, 2 mM sodium fluoride, 10 mM benzamidine, 20 µg/ml aprotinin, 20 µg/ml antipain,
20 µg/ml chymostatin, 20 µg/ml leupeptin, 20 µg/ml pepstatin, 0.5 mg/ml bovine serum albumin, and 2 mM PMSF). After 1 h
on ice, samples were diluted to 1.5-2 mg/ml in 50 mM
Tris-HCl, pH 7.5, 10% (w/v) glycerol, 0.8% phosphate-buffered saline,
0.8% ZW3-14, 2 mM dithiothreitol, 5 mM ATP, 5 mM MgCl2, 2 mM sodium fluoride, 10 mM benzamidine, 20 µg/ml aprotinin, 20 µg/ml antipain,
20 µg/ml chymostatin, 20 µg/ml leupeptin, 20 µg/ml pepstatin, 0.5 mg/ml bovine serum albumin, and 2 mM PMSF. To remove
nonspecific protein A-binding proteins, 70 µl of a 50% (v/v) protein
A-Sepharose CL-4B (Amersham Pharmacia Biotech) were incubated with 5 ml
of the solubilized proteins prepared above for 30 min at 4 °C, after
which the beads were removed by centrifugation. Primary antibodies (2.5 µg of Protein Expression and Purification
BL21(DE3) E. coli were transformed with unmodified
pET-32b (Novagen, Madison, WI), pET-Nt-Vph1p (described above), or
pMH21. pMH21 is a pEXP-based protein expression vector encoding the
COOH-terminal fragment (aa160-478) of S. cerevisiae Vma13p
(32). Bacterial cultures containing one of each of the three plasmids
were grown overnight (37 °C at 300 rpm) in LB with 100 µg/ml
ampicillin. The cultures were diluted 1:20 in LB/ampicillin and grown
(37 °C at 300 rpm) to A600 = 0.6. Protein
expression was induced with 1 mM
isopropyl- Thawed pellets were resuspended in 50× in lysis buffer (50 mM Tris-Cl, pH 8.0, 300 mM NaCl, 1% Triton
X-100, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin,
0.1 mM PMSF, 1 mg/ml chicken egg white lysozyme (Sigma)),
incubated for 30 min at 24 °C, sonicated on ice, and centrifuged
(2540 × g at 4 °C for 30 min). The supernatant resulting from the induction of pMN21 was aliquoted and stored at
The supernatants resulting from the induction of the pET-based vectors
were incubated (30 min at 24 °C) with Ni-NTA-agarose (Qiagen GmbH,
Hilden, Germany). The resin was pelleted (45 × g) and
washed (5 × 4 volumes) with wash buffer (50 mM
Tris-Cl, pH 8.0, 300 mM NaCl, 20 mM imidazole).
Resin-bound proteins were suspended (75% slurry) in binding buffer (50 mM Tris-Cl, pH 8.0, 120 mM NaCl, 0.5% Triton
X-100), aliquoted, and stored at Vma13p Binding Experiment
Ten µg of Ni-NTA-bound pET-32b and pET-Nt-Vph1p (as estimated
by Coomassie staining) were each incubated for 30 min with 2 µl of
induced supernatant containing Vma13p (described above) in a final
volume of 75 µl of binding buffer. The resin was then pelleted
(45 × g), the supernatant collected (unbound protein fraction), and the resin washed (5 × 4 volumes binding buffer). The bound proteins were then eluted from the Ni-NTA resin by
boiling the resin in SDS sample buffer.
The bound and unbound proteins from the pET-32b and pET-Nt-Vph1p
affinity matrices and a sample of the induced supernatant containing
Vma13p were resolved by SDS-PAGE and transferred to nitrocellulose. The
membrane was cut at 30 and 46 kDa and assessed with the following
primary antibodies, <30 kDa 1:500 Purification of Vacuolar Membranes
Isolation of vacuolar membranes through flotation of intact
vacuoles on Ficoll gradients was performed as described in Ref. 33 with
modifications detailed in Ref. 28. Protein concentrations of vacuolar
vesicles were estimated as described in Ref. 34. Purified vacuolar
membranes were subjected to controlled trypsin proteolysis in the
following manner: 20 µg of purified vacuolar membranes in 25 mM MES/Tris, pH 7.8, 25 mM KCl, 5 mM ATP were incubated with 0.1 µg/ml trypsin at 37 °C
for 30 min in a final reaction volume of 100 µl. Trypsin proteolysis
was inhibited by the addition of PMSF to a final concentration of 1 mM and
N Proteolysis of Purified Vacuolar Membranes
Purified vacuolar membranes were subjected to controlled trypsin
proteolysis in the following manner: 20 µg of purified vacuolar membranes in 5 mM ATP, 25 mM MES/Tris, pH 7.8, and 25 mM KCl were incubated with either 0.1 or 1.0 µg/ml
trypsin at 37 °C for 30 min in a final reaction volume of 100 µl.
Trypsin proteolysis was inhibited as described above. Samples were
denatured, assayed by SDS-PAGE, and immunoblotted as described above.
ATPase Activity and Proton Translocating Activity
ATPase assays and proton pumping activity were assayed on
freshly prepared vacuolar membrane vesicles. Vesicles were trypzinised with 0.1 µg/ml in 25 mM MES/Tris, pH 7.8, 25 mM KCl, 5 mM ATP for 15 or 30 min at 37 °C.
The final protein concentration was 0.2 µg/ml. Control samples were
similarly treated excluding the addition of proteases. Trypsin activity
was inhibited by the addition of PMSF (1 mM final
concentration) and TLCK (5 mM, final concentration). Samples were incubated for 10 min on ice and trypsin-treated and control samples were assayed for ATPase and proton pumping activity. Proton pumping activity was measured by tracking the
ATP-dependent quenching of acridine orange using a
Perkin-Elmer 650-40 fluorescence spectrophotometer with emission at 545 nm and excitation at 493 nm. Assays were performed in a 1-ml volume of
25 mM MES/Tris, pH 7.2, 5 mM ATP, 25 mM KCl, 5 mM acridine orange, and 5 mM MgCl2. Acidification rate was defined as the
slope of the quench during the first 10 s immediately after the
addition of MgCl2; in all cases the first 10 s gave a
linear response and was not limited by the formation of a proton-motive
force. Bafilomycin A1-sensitive ATP hydrolysis of purified
vacuolar membranes was assayed by measuring the production of inorganic
phosphate utilizing the Ames assay as detailed in Ref. 28. Assays were
performed in the presence of 0.5 mM azide and 0.05 mM vanadate to inhibit mitochondrial and plasmalemmal
ATPase activity, respectively. Inhibitors were added to the assay
mixture 10 min before initiating the reaction. Final assay mixtures
contained 5 µg of protein in a final volume of 250 µl plus 1 µl
of 50 µM bafilomycin A1 or 1 µl of dimethyl sulfoxide.
Preparation of Sample for NH2-terminal Sequencing
Large scale trypsin digests of vacuolar membranes were performed
in order to purify sufficient quantities of the 80-kDa tryptic fragment
for NH2-terminal sequencing. Briefly, 10 mg of purified vacuolar membranes were treated with 0.5 µg/ml trypsin in 25 mM MES/Tris, pH 7.8, 25 mM KCl, 5 mM ATP, and 5 mM MgCl2 at 37 °C for 2 h. Trypsin proteolysis was inhibited by the addition of PMSF
to a final concentration of 1 mM, TLCK to a final
concentration of 5 mM, and incubation on ice for 15 min.
Subsequently, peripheral membrane proteins were removed by incubation
with 100 mM NaCO2, pH 11, for 20 min on ice.
Vacuolar membranes were washed twice with phosphate-buffered saline, pH
7.4, and solubilized in an appropriate volume of 1 × Laemmli
sample buffer. Samples were resolved by standard SDS-PAGE, transferred
to PVDF, and submitted for microsequencing analysis. Microsequencing
was performed by Harvard Microsequencing Facilities.
The NH2 Terminus of Vph1p Interacts with the COOH
Terminus of Vma1p--
The 95-kDa subunit (Vph1p) is unique to the
V-ATPase, having no homologous counterpart in the F-ATPase complex.
Vph1p is defined by two structurally distinct domains: an
NH2-terminal hydrophilic domain (~45 kDa) and a
COOH-terminal hydrophobic domain containing up to 9 putative
transmembrane segments (21). The NH2 terminus of Vph1p has
been unambiguously localized to the cytosol (27), where it is available
to associate with cytosolic proteins. Current evidence suggests that
the cytosolic domain of Vph1p interacts with the catalytic sector. The
peripheral complex blocks binding of an anti-NH2 terminus
Vph1p antibody to vacuolar membranes containing an assembled
V0 complex (27, 37). As well, substrate binding to the
catalytic sector induces a conformational change within the 95-kDa
subunit (27). A yeast two-hybrid system was utilized to identify
cytosolic proteins that potentially interact with the NH2
terminus of Vph1p.
The 45-kDa NH2-terminal domain of Vph1p (amino acid
residues 1-406) was utilized as bait (pAS2-1-Nt-Vph1p) to
screen 5 × 106 prey clones, yielding 100 clones
prototrophic for tryptophan, leucine, and histidine. One of these
positive clones encoded the COOH-terminal portion (residues 332 to 617)
of Vma1p, the catalytic 69-kDa V-ATPase subunit. Co-expression of the
NH2 terminus of the 95-kDa subunit (Nt-Vph1p) and the COOH
terminus of Vma1p (Ct-Vma1p) resulted in activation of
The NH2 Terminus of Vph1p Interacts with the Soluble
V1 Complex in Vivo--
The interaction between Vph1p and
Vma1p was confirmed in vivo by co-immunoprecipitation of the
two proteins. Co-immunoprecipitation in a wild type strain would be
uninformative, as antibodies to any V-ATPase subunit will
immunoprecipitate the holoenzyme. Thus, Nt-Vph1p was expressed in a
Incubation of the yeast lysate (MM53 + pRS426-Nt-Vph1p) with
Efforts to refine the essential domains required for association
between Vph1p and Vma1p yielded inconclusive results. Co-expression of
truncated domains of the interactive regions of Vph1p and Vma1p did not
result in reproducible interactions. The failure to identify discreet
regions required for Vma1p-Vph1p interaction may indicate that the
complete domains identified are essential to establish stable
protein-protein contacts. Alternatively, the inability to demonstrate
interaction between truncated forms of these proteins may indicate that
the interaction between Vma1p and Vph1p is weak and requires additional
components of the V1 complex to stabilize the association.
The NH2 Terminus of Vph1p Is Required for Vma13p
Association with the V1 Sector--
Given that Vph1p is
part of the V0 complex, and that Nt-Vph1p interacts
directly with the catalytic subunit of the V1 complex, it
is reasonable to suggest that the 95-kDa subunit serves as a fixed
structural link between the two V-ATPase sectors. If this is true,
Vph1p is likely to interact with additional components of the
V1 complex. Of the remaining subunits of the catalytic sector, we focused on Vma13p for a number of reasons. Dissociation of
the peripheral sector from the vacuolar membrane yields an inactive
complex containing the majority of V1 components (69, 60, 32, and 27 kDa) and free forms of Vma5p (42 kDa) and possibly Vma13p
(54 kDa) (37, 41). Sector re-association results in recruitment of
Vma13p and Vma5p to the V1 sector with concomitant restoration of V-ATPase activity (41). Given that Vph1p is essential for V1V0 association and that Vma13p only binds
to a fully assembled complex, we hypothesized that Nt-Vph1p may be
required for recruiting the 54-kDa subunit to the catalytic complex.
To determine if this was the case, yeast cell lysates were prepared
from a The NH2 Terminus of Vph1p Interacts with the COOH
Terminus of Vma13p in Vitro--
To determine if Nt-Vph1p recruits
Vma13p to the V1 complex by direct interaction or by
inducing a conformational change within the catalytic sector, an
in vitro Nt-Vph1p-Vma13p binding assay was designed.
Briefly, the sequence encoding Nt-Vph1p was cloned into the pET32b
expression plasmid and was used to produce histidine-tagged (His-tagged) Nt-Vph1p (pET32b-Nt-Vph1p, see "Experimental
Procedures"). Bacterial lysates were also prepared from a strain
expressing a truncated form (aa 160-478) of Vma13p (Ct-Vma13p), a kind
gift from Dr. Tom Stevens as described in Ref. 32. This truncated form
of Vma13p fully complements a Dissociation of ATPase and Proton Pumping Activity--
Both
in vivo (37, 42) and in vitro (43-47),
V1 ATP hydrolytic activity is tightly coupled to
V0 proton translocation. V1V0 dissociation renders the V1 hydrolytically inactive
(43-46) and the V0 proton impermeant (47), with both
activities restored by complex reassociation (37, 42). Chemical
inhibition of the V1 (by N-ethylmaleimide) or
the V0 (by bafilomycin A1) prevents both proton
translocation and ATP hydrolysis, reiterating the functional dependence
of the two sectors. Surprisingly, limited proteolysis of the V-ATPase
in clathrin-coated vesicles partially dissociated these two activities
(48). Trypzinization completely abolished proton translocation with
only a 50% reduction in ATP hydrolysis leading to the conclusion that
proteolytic cleavage of one or more of the mammalian homologues of
Vph1p, Vma1p, Vma2p, Vma6p, and/or Vma4p was responsible for uncoupling
of the two activities. To elucidate whether cleavage of one specific
subunit was responsible for the dissociation of hydrolysis from proton pumping, intact vacuolar membranes were treated with 0.1 µg/ml trypsin as described under "Experimental Procedures." These
conditions are gentler than those previously employed, yielding a more
discrete pattern of proteolytic cleavage. Only Vph1p, Vma2p, and Vma13p were cleaved under the conditions utilized (Fig.
3, panels 1, 3, and
4). The remaining subunits (Vma1p, Vma5p, Vma6p, and Vma4p) remain intact (Fig. 3, panels 2, 5, 6, and 7),
with a 10-fold increase in the amount of trypsin not cleaving Vma1p,
Vma4p, or Vma6p. The possibility that Vma7p, Vma8p, or Vma10p are
cleaved and thus contribute to uncoupling cannot be excluded, as
antibodies to these subunits were unavailable.
To determine if cleavage of Vph1p, Vma2p, and/or Vma13p subunits was
sufficient to uncouple hydrolysis from proton translocation, vacuolar
membranes were trypsinized under identical conditions described above
and assayed for proton translocating and ATP hydrolytic activity.
Trypsin treatment did not have a significant effect on ATP hydrolysis
(Fig. 4, panel A). The minimal
reduction in ATPase activity with or without trypsin treatment is
likely a consequence of thermal denaturation. In contrast, the same
trypsin treatment resulted in a significant decrease in proton
translocation. Trypsin-treated samples showed a 22 and 67% reduction
in proton pumping at 15 and 30 min, respectively, as compared with
control (Fig. 4, panel B). These data suggest that
proteolytic cleavage of Vph1p, Vma2p, or Vma13p, alone or in
combination, results in the functional uncoupling of the two V-ATPase
complexes.
Due to its unique bipartite structure and its interactions with
components of the V1 complex, Vph1p is ideally suited to
serve as a structural link between the V1 and
V0 complexes. To test whether the proteolytic cleavage of
Vph1p was responsible for the dissociation of hydrolysis from proton
translocation, the trypsin-sensitive site on Vph1p was identified and
eliminated by site-directed mutagenesis. We have previously
demonstrated that the limited trypsinization of intact vacuolar
membranes results in the cleavage of 6 kDa from the NH2
terminus of Vph1p (27). The remaining 90-kDa COOH-terminal fragment was
isolated by SDS-PAGE, transferred to PVDF membrane, and subjected to
NH2-terminal sequencing as described under "Experimental
Procedures." The NH2-terminal 7-amino acid residues of
the 90-kDa fragment are T54FVNEIR indicating that the
preceding arginine (Arg53) residue is the site of trypsin
cleavage. Given that intact vacuoles were subjected to limited
proteolysis, identification of arginine 53 as the trypsin cleavage site
reconfirms that the hydrophilic NH2 terminus of Vph1p is
disposed toward the cytosolic space. To remove the trypsin cleavage
site within the NH2 terminus of Vph1p, arginine 53 was
mutated to a serine residue as described under "Experimental
Procedures" (MM635). The single copy CEN-based plasmid
pRS316-VPH1-R53S (MM636) was able to complement a
The V-ATPases and F-ATPases share a common architecture with
V-ATPase subunits having structural counterparts within F-ATPases. An
exception to this generalization is the 95-kDa V0 subunit
which is unique to V-ATPases. This molecule is divided into two
distinct structural domains, the NH2-terminal hydrophilic
domain (~45 kDa) and the COOH-terminal membrane-associated region. To
date, the function of this subunit has not been elucidated. The initial observations that Vph1p isoforms were localized to specific organelles (38) and were differentially expressed in mammalian tissues (49-51)
suggested that Vph1p was involved in the targeting or regulation of
single copy V-ATPase subunits in various cellular locations (52).
Recent experimental evidence points to a more direct role in V-ATPase
activity. Point mutations within the transmembrane domain of Vph1p do
not impair V-ATPase assembly or targeting yet eliminate enzyme activity
(53, 54). Moreover, substrate binding to the catalytic domain induces
conformational changes within Vph1p (27). Given the localization of the
NH2 terminus of Vph1p to the cytoplasm and the subunit's
conformational responsiveness to substrate binding, it was proposed
that this domain interacts directly with the V1 sector
(27). In vitro and in vivo binding studies
demonstrate that the NH2 terminus of Vph1p interacts with the catalytic subunit, Vma1p, and Vma13p (Table I and Figs. 1 and 2).
Although Vma13p is a component of the soluble sector (55), recruitment
of the 54-kDa subunit to V1 requires the expression of
Nt-Vph1p (Fig. 1, panel C).
As with Vph1p, the function of the 54-kDa V1 subunit
(Vma13p) has not been defined, although it is essential for V-ATPase activity (55). Genetic (55) or biochemical (56, 57) removal of Vma13p
from the holoenzyme activity results in complex inactivation. Although
Vma13p is not required for the assembly of either complex, or
V1/V0 association, it stabilizes binding of the
catalytic sector to the vacuole (55). Dissociation/re-association and
subsequent inactivation/re-activation of V-ATPase activity has also
been observed for the bovine homologue of Vma13p, The only other subunit with similar properties to Vma13p is the 42-kDa
V1 subunit, Vma5p. With the exception of Vma13p and Vma5p,
disruption of an individual V-ATPase subunit prevents assembly of its
parent complex (39). Vma13p and Vma5p readily dissociate from the
holoezyme, inactivating the V-ATPase complex (32, 55). To reproducibly
co-immunoprecipitate the two subunits with the holoenzyme, the complex
must be chemically cross-linked prior to solubilization (39).
Furthermore, although both Vma13p and Vma5p are considered
V1 subunits, neither subunit is part of the catalytic core
composed of Vma1p and Vma2p, or the central stalk, thought to be
composed of Vma4p, Vma7p, Vma8p, and Vma10p (15, 40, 58). Taken
together these data suggest that Vma13p and Vma5p bind at the periphery
of a fully assembled V1 complex. Moreover, data presented
here suggest that, in the case of Vma13p, Vph1p is essential for the
recruitment of the subunit to the V1 sector.
The NH2 terminus of Vph1p, through its interaction with
Vma1p, also associates with the V1 complex. Since
V1 and V0 complexes can disassemble/re-assemble
in vivo (37, 42), wild type Vph1p must also readily
dissociate/re-associate from the V1 complex. Given that
Vph1p, Vma13p, and Vma5p all readily dissociate/re-associate with
assembled V1 complexes, and that they are not part of the catalytic core or central stalk, it is reasonable to suggest that these
three subunits are asymmetrically attached to the periphery of the
V1 complex. This has led us to hypothesize that Nt-Vph1p, Vma13p, and possibly Vma5p compose the peripheral stalk visualized by
electron microscopy (6, 25) and likely represents a fixed structural
link between V1 and V0. This asymmetric
peripheral structure is analogous to the one identified in the closely
related F-ATPases. In this complex, subunits a, (b)2, and
Given this functional model, proteolytic cleavage of the stator would
likely result in uncoupling of ATP hydrolysis from proton translocation. Trypsin treatment of purified vacuoles results in
cleavage of both Vph1p and Vma13p with the resultant loss of proton
translocation and preservation of ATP hydrolysis (Figs. 3 and 4).
Removal of the trypsin-sensitive site on Vph1p partially prevents this
phenomenon, suggesting the 95-kDa subunit plays a key role in coupling
the two V-ATPase complexes. Consistent with our model of Vma13p forming
part of the stator, cleavage of Vma13p would also contribute to the
dissociation of V-ATPase activity.
It is of note that the uncoupled ATP hydrolysis resulting from trypsin
proteolysis remained sensitive to bafilomycin A1. This V-ATPase-specific inhibitor binds tightly but noncovalently to the
hydrophobic V0 complex, blocking passive proton conductance (16, 59, 60). Evidence suggests that the binding site is located on
either the proteolipid Vma3p (61) or Vph1p (16), presumably on its
hydrophobic COOH-terminal domain. Given that bafilomycin A1
binds to the V0 complex, one might expect trypsin-uncoupled V1 ATP hydrolysis to be bafilomycin insensitive. Our
results to the contrary indicate that the inhibitory effect of
bafilomycin A1 is not through disruption of the
V1-Nt-Vph1p interaction but rather through another
V1V0 interaction. Models of V-ATPases suggest two separate links between the V1 and V0
complex: the peripheral stator (that we propose is partly composed of
Nt-Vph1p) and the central rotor. Bafilomycin A1 could
inhibit ATP hydrolysis by preventing rotation of the central rotor.
Indeed, one would expect a potent inhibitor to affect a dynamic rather
than a static interaction within an enzyme complex. The fact that the
uncoupled ATP hydrolysis remains bafilomycin-sensitive fits well with
our model of the Nt-Vph1p-V1 interaction composing the
static link and the central rotor as the dynamic link between the
V1 and V0 complexes.
The structural model presented in Fig. 5
reconciles data presented in this paper with previously published
observations. The V-ATPase can be defined by a number of regions; the
catalytic core composed of Vma1p and Vma2p (40); the central stalk,
suggested to be composed of Vma4p, Vma7p, Vma8p, and Vma10p (15); the stator likely composed of NT-Vph1p, Vma13p, and Vma5p; and the proton-translocating domain composed of Ct-Vph1p, Vma3p, Vma11p, Vma16p, and Vma6p (21, 53, 54, 62-64).
The stoichiometry of Vph1p is still uncertain as quantitative amino
acid analysis has calculated from one (17), to three (18) Vph1p
subunits per complex. We show here that Nt-Vph1p interacts with Vma1p.
As Vma1p is present in three copies/complex, there are three binding
sites available for Nt-Vph1p. Although there is nothing to suggest that
all available binding sites are occupied, more than one Vph1p subunit
per complex can be theoretically accommodated. We hypothesize here that
Nt-Vph1p is part of the peripheral stalk structure visualized by
electron microscopy (6, 7). As recent electron microscopy by Boekema
et al. (25) reveal that there are at least two, and possibly
three peripheral stalks, we have drawn Fig. 5 to depict multiple copies
of Vph1p.
Recent work (14) has identified a potential contact between Vph1p and
the stalk subunit Vma4p. Based on our model, this interaction would be
predicted to be transient, allowing Vma4p to rotate with respect to Vph1p.
We are extremely appreciative of Tom Stevens
for the plasmid expressing the truncated form of Vma13p and the
*
This work was support by Medical Research Council of Canada
Operating Grant MT12053 and a Medical Research Council of Canada scholarship (to M. F. M.).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.
Published, JBC Papers in Press, March 9, 2000, DOI 10.1074/jbcM000207200
The abbreviations used are:
V-ATPase, vacuolar
proton-translocating adenosine triphosphatase;
Ct-Vph1p, carboxyl-terminal 50-kDa hydrophobic domain of Vph1p;
Nt-Vph1p, amino-terminal 45-kDa hydrophilic domain of Vph1p;
PAGE, polyacrylamide
gel electrophoresis;
PCR, polymerase chain reaction;
PMSF, phenylmethylsulfonyl fluoride;
SFD, sub-58-kDa dimer;
aa, amino acid;
MES, 4-morpholineethanesulfonic acid;
TLCK, N
Evidence That the NH2 Terminus of Vph1p, an Integral
Subunit of the V0 Sector of the Yeast V-ATPase, Interacts
Directly with the Vma1p and Vma13p Subunits of the V1
Sector*
§,,, and¶
Hospital for Sick Children, Toronto, Ontario
M5G 1X8, Canada, the ¶ Faculty of Dentistry, University of
Toronto, Toronto, Ontario M5G 1G6, Canada, and the
§ Department of Medicine, University of Toronto, Toronto,
Ontario M5G 2C4, Canada
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
vph1 mutant was necessary to recruit Vma13p to
V1. Vma13p bound to Nt-Vph1p in vitro
demonstrating direct interaction. Limited trypsin digests cleaves both
Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of
proton translocation while not reducing bafilomycin
A1-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at
arginine 53. Elimination of the tryptic cleavage site by substitution
of arginine 53 to serine partially protected vacuolar acidification
from trypsin digestion. These results suggest that Vph1p may function
as a component of a fixed structural link, or stator, coupling
V1 ATP hydrolysis to V0 proton translocation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit in a counterclockwise direction within the core of the
catalytic hexamer (5). As energy coupling is thought to occur from the
physical rotation of the stalk connecting the F1 and
F0 complexes, a "stator"-like structure must exist to
prevent the rotation of the catalytic hexamer with respect to the
F0 complex. A stator (defined as the stationary portion of
a motor, dynamo, or turbine) would have to be anchored within the
membrane and extend up to the catalytic hexamer without interfering
with the rotation of the stalk. Structural evidence supports the
presence of this second structural link between the two sectors.
Electron microscopy of Escherichia coli F-ATPases revealed
the presence of two stalks, a central shaft as previously discussed,
and a second peripheral stalk that extends from the side of the
F1 complex down to the F0 complex (22). This
second physical link between the two complexes is ideally suited to
serve as a stator. Cross-linking studies suggest that the peripheral
stalk structure is likely composed of the a, (b)2, and 
subunits (23, 24).
subunits, the components of
the V-ATPase peripheral stalk are unknown. The function of the
catalytic (Vma1p), regulatory (Vma2p), and proteolipid (Vma3p) subunits
are reasonably established. The composition of the central stalk is
suggested to be either Vma7p and Vma8p (14), or Vma7p and Vma8p
together with Vma4p and Vma10p (15). Thus, four to six candidate
subunits with unassigned function remain to form the second peripheral
stalk or stator: Vma4p, Vma5p, Vma6p, Vma10p, Vma13p, and/or
hydrophilic portions of Vph1p.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, trp1/+, +/his1,
prc1-126/prc1-126, pep4-3/pep4-3, prb1-1122/prb1-1122, can1/can1,
gal2/gal2), Y190 (a gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, 112 URA3::GAL:LacZ::GAL:HIS3
cyhr) for two-hybrid assays, and MM53 ( ura3-52 vph1::LEU2), which bears a disruption of the
95-kDa V-ATPase subunit, Vph1p (28). Additional transformed strains are
described below. Wild type yeast cells were grown overnight in 1%
yeast extract (Difco), 2% Bacto-peptone (Difco), and 2% dextrose
(YEPD medium). The vma1p strains were grown in YEPD
adjusted to pH 5.0 with HCl, because cells lacking vacuolar ATPase
activity grow optimally in acidified medium (29).
vph1 strain.
vph1 mutation and the expected 55-kDa protein was barely
detectable as assayed by immunoblotting.
-VPH1-R53S (MM635)--
PCR was performed on
pVIP1-82 (28) with oligonucleotide MO66 (5'-ccccagtactttcgtgaacg-3')
and oligonucleotide MO67 (5'-cgccagtactttgaaacgcacgc-3'). These
oligonucleotides changed arginine 53 to a serine residue while
introducing a ScaI site. The PCR product was cut with
ScaI and re-circularized through ligation.
-VPH1-R53S replaced the corresponding
NheI-BstEII fragment of pRS316-VPH1.
The plasmid was able to complement a vph1 disruption.
Sequencing confirmed that the only change in the plasmid was the
desired R53S mutation.
ura3-52::URA3::VPH1-R53S
vph1::LEU2.MM53 ( ura3-52
vph1::LEU2) was transformed with PstI cut
pRS306-VPH1-R53S. Immunoblots performed on Ura+
Vma+ transformants confirmed Vph1p expression.
-galactosidase activity.
-Galatosidase Assay
-galactosidase activity. The cells were
permeabilized by a freeze-thaw cycle. Filters were floated on liquid
nitrogen for 20 s and allowed to thaw at room temperature. Each
filter was soaked with 2 ml of Z-buffer (CLONTECH
MatchmakerTM protocol manual) containing
5-bromo-4-chloro-3-indoyl--D-galactosidase. The filters
were placed in a covered plastic container and incubated at 37 °C
for a maximum of 3 h.
Vma1p or 20 µl of affinity purified Vph1p (28) were
added for 1 h at 4 °C with gently rocking followed by 70 µl
of 50% (v/v) protein A-Sepharose CL-4B for an additional hour at
4 °C. Sepharose beads were washed 3 times in 10 mM
Tris-HCl, pH 7.5, 0.1% Triton X-100, resuspended in 80 µl of 10%
glycerol, 1% SDS, 20% -mercaptoethanol, 62.5 mM
Tris-HCl, pH 6.8, and then heated for 10 min at 60 °C. Solubilized
proteins were subjected to SDS-PAGE and immunoblotting.
-D-thiogalactopyranoside (Life Technologies,
Inc., Gaithersburg, MD) for 4 h at 24 °C at 300 rpm. Cultures
were cooled to 4 °C, the cells were pelleted (2540 × g at 4 °C), washed with ice-cold phosphate-buffered
saline, and stored at 
20 °C.
20 °C.
20 °C.
-6xHis
(CLONTECH, Palo Alto, CA), 30-46 kDa 1:500
-Vma13p (32), >46 kDa 1:5000 -Vph1p, each followed by 1:7500
horseradish peroxidase-conjugated protein A.
-p-tosyl-L-lysine
chloromethyl ketone (TLCK) to a final concentration of 5 mM
followed by incubation on ice for 15 min. Samples were utilized for
activity assays as detailed below. Remaining samples were denatured by
the addition of 5 × SDS sample buffer and assayed by SDS-PAGE
(35) and immunoblotting as described (36).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase transcription (Table I). Activation was independent of whether
Nt-Vph1p or Ct-Vma1p were in either pACT or pAS2-1 vectors, but neither
Nt-Vph1p nor Ct-Vma1p activated -galactosidase transcription with
the counter empty vector, confirming that protein-protein interaction
between the V-ATPase subunits were required for -galactosidase
activation. The amino terminus of Stv1p, the functional homologue of
Vph1p (38), was equally effective in activating -galactosidase
activity in the presence of Ct-Vma1p, further demonstrating the
specificity of the interaction between the V-ATPase subunits.
The hydrophilic amino terminus of Vph1p and Stv1p, isoforms of the
95-105-kDa subunit of the V-ATPase, interacts with the COOH terminus
of the 69-kDa catalytic subunit, Vma1p
-galactosidase activity
as described under "Experimental Procedures."
vph1 yeast mutant lacking the V0 sector, yet
expressing fully assembled and functional V1 complexes (39). Yeast whole cell lysates were prepared from the
vph1 mutant strain MM53 transformed with the supporting
vector alone (MM53 + pRS426) or with a vector expressing the
NH2 terminus of Vph1p (MM53 + pRS426-Nt-Vph1p). As
expected, immunoprecipitation with an -Vma1p antibody recovered the
catalytic subunit from the vector alone (V) and Nt-Vph1p (I) expressing
lysates (Fig. 1, lanes 1 and
2). The 69-kDa doublet seen in lane 2 likely
reflects post-translational modification of Vma1p; size heterogeneity
of the V-ATPase catalytic subunit has been noted in several other species (30). Immunoprecipitation of Vma1p from lysates expressing Nt-Vph1p recovered both the catalytic subunit and the 55-kDa Nt-Vph1p (Fig. 1, lane 2). This observation demonstrates that
Nt-Vph1p interacts with the soluble V1 domain in
vivo, confirming the results obtained from the original two-hybrid
screen. Binding of Nt-Vph1p likely occurs on the external surface of
the catalytic hexamer given that assembly of the V1 complex
(catalytic core (34, 40) and stalk (15)) is independent of Vph1p and
that Nt-Vph1p binds to a fully assembled complex.
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Fig. 1.
The NH2 terminus of Vph1p and
Vma13p are co-immunoprecipitated with the catalytic subunit (Vma1p) of
V-ATPase. Yeast cells bearing a vph1 mutation (MM
53) were transformed with a high copy plasmid expressing the
NH2 terminus of Vph1p (MM 506) or an empty carrier vector
(pRS426). Despite the high copy plasmid, levels of the expected 55-kDa
protein (NH2 terminus Vph1p) were greatly reduced as
compared with wild type Vph1p. Immunoprecipitations were performed as
described under "Experimental Procedures." Yeast lysates were
immunoprecipitated with the antibodies listed on the upper
legend and subsequently assayed by immunoblotting with the
antibodies listed on the left side. Upper legend,
IP, immunoprecipitated; V, carrier vector;
I, vector with insert.
-Vph1p
antibodies successfully immunoprecipitated the NH2 terminus of Vph1p (Fig. 1, lane 3). Unfortunately, only trace levels
of Vma1p were recovered under these experimental conditions. The majority of Nt-Vph1p precipitated likely represents free protein, with
only a minimal component associated with the V1 complex. The failure to immunoprecipitate bound Nt-Vph1p suggests that association of the truncated 95-kDa subunit to the catalytic complex occludes available epitopes. Immunofluorescence microscopy with -Nt-Vph1p antibodies is only successful when the V0
complex is dissociated from the catalytic sector (27, 37),
demonstrating that association of the V1 complex to the
membrane sector blocks access to Nt-Vph1p epitopes. Conversely,
immunoprecipitation of the catalytic subunit would not be impeded by
Nt-Vph1p binding if there is unequal stoichiometry between the two
subunits. Even with Nt-Vph1p binding to one or two Vma1p subunits, the
third Vma1p subunit would remain accessible to antibodies. The levels of immunoprecipitated Nt-Vph1p in lanes 2 and 3 do not reflect levels of bound versus unbound protein as
immunoprecipitations were performed with an excess of cell lysate
compared with antibodies.
vph1 mutant strain transformed with a construct expressing Nt-Vph1p or the carrier vector alone (as described above).
Immunoprecipitation with -Vma1p sera was unable to co-precipitate Vma13p in the absence of Nt-Vph1p (Fig. 1, lane 1),
suggesting that the 54-kDa subunit is not a component of the isolated
V1 complex. Immunoprecipitation of the catalytic subunit in
the presence of Nt-Vph1p did result in the co-precipitation of Vma13p
(Fig. 1, lane 2, bottom panel). Protein extracted from a
vma13 mutant demonstrated that the 65-kDa polypeptide
seen in lanes 1 and 2 of the blots probed with
-Vma13p sera is not Vma13p (data not shown). These results suggest
that Nt-Vph1p is essential for the interaction of Vma13p with the
catalytic sector, recruiting the 54-kDa subunit from its free cytosolic
state to the fully assembled V-ATPase. If Nt-Vph1p interacts directly
with Vma13p, one might expect co-immunoprecipitation of Nt-Vph1p and
Vma13p, even in the absence of the V1 complex.
Surprisingly, immunoprecipitation with -Vph1p sera was unable to
co-precipitate Vma13p (Fig. 1, lane 3). One possibility to
explain why a V1-free Vph1p-Vma13p interaction was not
detected by immunoprecipitation could be that bound Vma13p occludes
Vph1p epitopes. Alternatively, the NH2 terminus of Vph1p
may not interact with Vma13p alone, but could require additional
components of the V1 complex to mediate the interaction. A
third possibility is that binding of Nt-Vph1p to the V1
complex could induce a conformational change within the catalytic
sector, allowing for Vma13p binding. Thus, the ability of the
NH2 terminus of Vph1p to recruit Vma13p to the catalytic
sector may be an indirect effect rather than through direct binding.
Either of the latter interpretations would be consistent with the
failure of the original two-hybrid screen to select for a Vma13p clone.
To distinguish between these possibilities, we asked whether Vma13p
could interact directly with Vph1p in an in vitro assay.
vma13 disruption indicating that removal of the 159 N2H-terminal region is not required
for V-ATPase assembly or activity (32). Ni-NTA bound proteins produced from pET-32b-Nt-Vph1p or the pET-32b vector alone were incubated with
equal amounts of bacterial lysates containing Ct-Vma13p. The Ni-NTA
bound and unbound fractions were assayed by immunoblotting as described
under "Experimental Procedures." Ct-Vma13p did not bind to the
protein purified from the unmodified pET32b vector, and was completely
restricted to the unbound fraction (Fig.
2, panels B and C, lanes
2 and 3). In the presence of Nt-Vph1p, Ct-Vma13p was
retained in the bound fraction (Fig. 2, lane 4)
demonstrating that Vma13p and Nt-Vph1p interact directly. Despite the
overabundance of Nt-Vph1p, approximately 50% of the available Vma13p
remained in the unbound fraction (Fig. 2, lane 5). This
population of unbound Vma13p may indicate unequal stoichiometry of
these subunits in vivo or that the bacterially expressed
constructs are not assuming native conformations in solution. Dynamic
light-scattering experiments with heterologously expressed Nt-Vph1p
revealed that the protein is polydisperse (data not shown) indicating
aggregation in solution. Aggregation of Nt-Vph1p could occlude
Vma13p-binding sites.
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Fig. 2.
NH2 terminus of Vph1p interacts
with the COOH terminus of Vma13p in vitro. Ten µg of
Ni-NTA-bound protein lysates from pET-32b and pET-Nt-Vph1p (as
estimated by Coomassie staining) were each incubated with 2 µl of
induced supernatant containing Ct-Vma13p as described under
"Experimental Procedures." The resin was pelleted, and the
supernatant/unbound protein fraction (UB) was collected. The
resin was washed multiple times and bound proteins (B) were
eluted in SDS sample buffer. Proteins were resolved by SDS-PAGE,
transferred to nitrocellulose, and the membrane was cut at 30 and 46 kDa and then subjected to immunoblotting with the following primary
antibodies: <30 kDa; 1:500 -6xHis (CLONTECH,
Palo Alto, CA), 30-46 kDa; 1:500 -Vma13p (32), > 46 kDa; 1:5000
-Vph1p. Lane 1 contains 2 µl of the induced supernatant
containing Ct-Vma13p. Lanes 2 and 3 contain
20 µl of bound (B) or unbound (UB) fractions
from the pET-32b affinity resin (pET). Lanes 4 and 5 contain 20 µl of bound or unbound fractions from the
pET-Nt-Vph1p-affinity resin (pET-Nt-Vph1p).
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Fig. 3.
Trypsin (0.1 µg/ml)
cleaves Vph1p, Vma2p, and Vma13p. Tryptic digests of purified
vacuolar membranes were performed at 37 °C for 30 min in 5 mM ATP, 25 mM MES/Tris, pH 7.8, and 25 mM KCl. Final trypsin concentrations were 0.1 or 1.0 µg/ml, as indicated above the panels. All lanes
contain 2 µg of purified total vacuolar proteins. Panels
1-7, immunoblots were probed with the following antibodies:
1, 1:5000 -Vph1p; 2, 1:5000 -Vma1p;
3, 1:10,000 -Vma2p; 4, 1:1000 -Vma13p;
5, 1:500 -Vma5p; 6, 1:1000 -36 kDa;
7, 1:500 -27 kDa. Size of the full-length protein for
each of the V-ATPase subunits assayed is noted on the left
side of the corresponding panel.
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Fig. 4.
Trypsinization of the yeast V-ATPase leads to
the dissociation of ATPase catalytic activity and proton
translocation. Panel A, bafilomycin
A1-sensitive ATP hydrolysis of vacuolar membranes
pretreated with trypsin (0.1 µg/ml) as described for pumping assays.
Hydrolytic activity was assayed for 30 min at 30 °C in 25 mM MES-Tris, pH 6.9, 5 mM MgCl2, 5 mM Na2ATP, and 25 mM KCl with
released inorganic phosphate determined by the Ames method.
Bafilomycin-treated samples were preincubated with a final inhibitor
concentration of 1 µM for 10 min at room temperature
prior to the assays. 6 µg of total protein were utilized per assay
(n = 4). All assays were performed in the presence of
0.05 mM vanadate and 0.5 mM azide.
Hatched bars correspond to control and shaded
bars correspond to trypsinized samples. Panel B,
V-ATPase-mediated acidification of purified intact vacuoles pretreated
with trypsin. Intact vacuoles were pretreated with 0.1 µg/ml trypsin
for 0, 15, or 30 min in the presence of 5 mM ATP, 25 mM KCl, MES-Tris, pH 7.8. Control samples were prepared
under identical conditions. Reactions were stopped by the addition of
final concentrations of 1 mM PMSF and 5 mM
TLCK. Luminal acidification was detected by following the rate of
fluorescence quenching of acridine orange in buffer conditions
identical to those described for the ATPase activity assays. 20 µg of
total protein was utilized per assay. Hatched bars
correspond to control and shaded bars correspond to
trypsinized samples. Relative activities were calculated based on the
maximal extent of acidification. Panels C and D,
a plasmid coding for Vph1p lacking the trypsin-sensitive site (R53S)
was expressed in a vph1p yeast strain. Vacuolar membranes
purified from either this strain or a wild type strain were used to
perform the experiments detailed in panels C and D. Panel C, bafilomycin-sensitive ATP hydrolysis of vacuolar
membranes prepared from wild type and (R53S) yeast strains were
pretreated with trypsin (0.1 µg/ml) as described in panel
A. White bars correspond to wild type vacuolar
membranes and shaded bars correspond to (R53S) vacuolar
membranes. Panel D, V-ATPase mediated acidification of
vacuoles prepared from wild type and (R53S) yeast strains pretreated
with trypsin (0.1 µg/ml) was performed as described in panel B. White bars correspond to wild type vacuolar membranes and
shaded bars correspond to (R53S) vacuolar membranes.
vph1 mutation (MM53) and rendered Vph1p insensitive to
0.1 µg/ml trypsin demonstrating that the R53S mutation eliminated the
NH2-terminal proteolytic cleavage site while not
interfering with the function of Vph1p (data not shown). In order to
purify vacuoles from rich non-selective media, the VPH1 R53S
mutation was integrated into the yeast genome as described under
"Experimental Procedures" (MM638: ura3-52::URA3::VPH1-R53S
vph1::LEU2); MM638 regained bafilomycin
A1-sensitive ATPase activity and vacuolar acidification. Vacuolar membranes purified from MM638 and BJ926 (wild type control) were subjected to limited proteolysis for 15 and 30 min as described above. As previously noted, bafilomycin A1-sensitive ATPase
activity was equally affected with or without proteolysis with 0.1 µg/ml trypsin in vacuoles purified from either strain (Fig. 4,
panel C). In contrast, the R53S mutation partially protects
the MM638 vacuoles from loss of proton conductance when compared with
wild type vacuoles (Fig. 4, panel D). That the R53S mutation
only afforded limited protection was anticipated as 0.1 µg/ml trypsin
did not achieve 100% proteolysis of Vph1p (Fig. 3, panel
1), while proton translocation was effectively abolished (Fig. 4,
panel B). This suggests that cleavage of additional
components of the V-ATPase also contribute to the loss of proton
pumping activity. It is tempting to speculate that proteolysis of
Vma13p may be involved in uncoupling ATP hydrolysis from proton
translocation given that Vma13p was also cleaved by limited proteolysis
(Fig. 3, panel 4), that it does not have an established role
in ATP hydrolysis, and that it interacts with Vph1p (Figs. 1 and 2).
However, it cannot be concluded that cleavage of Vma13p is responsible
for the remaining 38% of proton-pump inhibition not eliminated by the
R53S mutation without controls similar to those performed on Vph1p.
Nevertheless, these results do show that the proteolytic cleavage of
the NH2 terminus of Vph1p is, in part, responsible for
dissociating V1 ATP hydrolysis from V0 proton translocation.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SFD, in
clathrin-coated V-ATPases (56, 57). This dynamic process necessitates
that Vma13p be peripherally located with respect to the catalytic core, allowing for its release without disruption of the Vma1p/Vma2p hexameric center. The observations that Vma13p interacts with the
NH2 terminus of Vph1p and that it stabilizes the
association of the two V-ATPase sectors suggest that the 54-kDa subunit
forms part of a fixed structural link between V1 and
V0.
form a stator which prevents loss of energy through the futile
rotation of F1 relative to F0. A V-ATPase
stator would serve a similar role by preventing rotation of
V1 relative to V0, permitting the efficient transduction of torque exerted by the rotating stalk on
V0.
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[in a new window]
Fig. 5.
Model of V1 and V0
dissociation and reassociation. The NH2 terminus of
Vph1p (Nt-Vph1p) interacts with the catalytic subunit of the
V1 complex. This region of Vph1p is also required to
recruit Vma13p to the V1 complex. Dissociation of the
V1 complex releases both Vma13p and Vma5p into the cytosol
with complex reassociation resulting in their recruitment. In mammalian
V-ATPases, there are two isoforms of the Vma13p ortholog, SFD and
SFD, each present in one copy per complex (14, 57) and one copy of
the Vma5p ortholog, subunit C, per complex (14). The question of
whether there is one or more copies of Vph1p/complex is still under
debate (17, 18). We hypothesize the Vph1p is part of the peripheral
stalk visualized by electron microscopy, and have thus drawn two
subunits of Vph1p/complex to reflect the two peripheral stalks/complex
resolved by Boekema et al. (25).
ACKNOWLEDGEMENTS
-Vma13p sera and Patricia Kane for supplying the -Vma5p
monoclonal antibody and critical review of the manuscript. As well,
M. F. M. also thanks Petra Kuehl for moral support and patience.
FOOTNOTES
To whom correspondence should be addressed: Faculty of
Dentistry Research Institute, 124 Edward St., Rm. 430, Faculty of
Dentistry, University of Toronto, Toronto, Ontario M5G 1G6, Canada.
Tel.: 416-979-4900 (ext. 4392); Fax: 416-979-4936; E-mail:
m.manolson@utoronto.ca.
ABBREVIATIONS
-p-tosyl-L-lysine
chloromethyl ketone.
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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K. Keenan Curtis and P. M. Kane Novel Vacuolar H+-ATPase Complexes Resulting from Overproduction of Vma5p and Vma13p J. Biol. Chem., January 18, 2002; 277(4): 2716 - 2724. [Abstract] [Full Text] [PDF]
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Y. Arata, J. D. Baleja, and M. Forgac Cysteine-directed Cross-linking to Subunit B Suggests That Subunit E Forms Part of the Peripheral Stalk of the Vacuolar H+-ATPase J. Biol. Chem., January 25, 2002; 277(5): 3357 - 3363. [Abstract] [Full Text] [PDF]
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G. Borrelly, J.-C. Boyer, B. Touraine, W. Szponarski, M. Rambier, and R. Gibrat The yeast mutant vps5Delta affected in the recycling of Golgi membrane proteins displays an enhanced vacuolar Mg2+/H+ exchange activity PNAS, August 14, 2001; 98(17): 9660 - 9665. [Abstract] [Full Text] [PDF]
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