From the Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235.
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ABSTRACT |
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The vacuolar proton pump of clathrin-coated
vesicles is composed of two general sectors, a cytosolic, ATP
hydrolytic domain (V1) and an intramembranous proton
channel, V0. V1 is comprised of 8-9 subunits
including polypeptides of 50 and 57 kDa, termed SFD (Sub
Fifty-eight-kDa Doublet). Although SFD is
essential to the activation of ATPase and proton pumping activities
catalyzed by holoenzyme, its constituent polypeptides have not been
separated to determine their respective roles in ATPase functions.
Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and
Stone, D.K. (1998) J. Biol. Chem. 273, 5878-5884). To determine the functional characteristics of the
57-kDa (SFD Vacuolar, or V-type proton pumps acidify a wide array of
intracellular compartments and are essential to functions of
constitutive endocytotic and regulated secretory pathways. These
H+ pumps are also found in the plasma membrane of polarized
cells such as osteoclasts and renal tubular epithelial cells, where they function to acidify discrete extracellular compartments. In fact,
V-pumps have been localized to virtually all intracellular compartments, except the nucleus and mitochondria, and their functions are equally diverse, ranging from promoting receptor-ligand
dissociation in clathrin-coated vesicles and endosomes, to energizing
neurotransmitter and catecholamine storage in synaptic vesicles and
adrenal chromaffin granules (1-3).
Key questions thus arise regarding the mechanisms by which these pumps
are targeted to their cellular sites and how differential regulation of
the enzymes is achieved in these disparate locales. Several regulatory
elements of V-pumps have been described, including activator proteins
(4, 5) and chloride channels that operate in parallel with V-pump to
dissipate the charge generated by these electrogenic pumps and thereby
facilitate pH gradient formation (6, 7).
Recently we provided biochemical (8) and molecular (9) evidence for the
roles of a 50- and 57-kDa polypeptide doublet, termed SFD
(Sub Fifty-eight-kDa Dimer, or
Doublet), in the activation of the V-pump of
clathrin-coated vesicles (CCV) of bovine brain. These proteins were
discovered in the course of our attempts to achieve biochemical
resolution of components of the V-pump of CCV. This enzyme, like all
V-pumps, is comprised of two general sectors, a multisubunit, cytosolic
ATP hydrolytic domain (V1), and a multisubunit proton
channel (V0). Biochemical and genetic studies have revealed
that the V1 domain in eukaryotic organisms is composed of 7 core subunits (A-G), some of which are present in multiple copies (10).
Although less well characterized, the V0 domain of the
V-pump of CCV contains between 3-6 different subunits, ranging from a
116-kDa polypeptide (subunit a) to a small proteolipid (subunit
c) (11).
Attempts at defining the components of V1 (subunits A-G)
and their functions revealed that an additional factor(s) was required for pump function, namely the polypeptides of SFD. When selectively depleted of these proteins, the V-pump of CCV, though assembled as a
V1V0 complex, cannot support ATP hydrolysis or
proton pumping. Purified SFD, when added to SFD-depleted pump, was
shown to restore these functions (8).
From a molecular standpoint, we recently determined the 57- and 50-kDa
polypeptides of SFD, termed SFD Of note, the activation properties of SFD had been previously ascribed
by others (13, 14) to AP50, a component of the AP2 complex which is
responsible for the assembly of the clathrin coats of coated pits and
vesicles (15-17). Our more recent work demonstrated that SFD, and in
particular its 50-kDa component (SFD The current studies add final proof to the molecular identity of the
SFD isoforms. Purified, recombinant SFD Preparations and Materials--
Isolation of clathrin-coated
vesicles from bovine brains (18) and preparations of liposomes from
purified lipids (19) were performed as reported. Purification of the
proton-translocating ATPase of clathrin-coated vesicles was performed
by sequential solubilization with C12E9,
hydroxylapatite chromatography,
(NH4)2SO4 fractionation, and
glycerol gradient centrifugations (20); purified H+-ATPase
had a specific activity of 14-16 µmol of Pi·mg of
protein
Restriction enzymes, T4 DNA sequencing ligase, and a nick translation
kit were obtained from Roche Molecular Biochemicals; DNA sequencing
reagents and enzymes and GeneAmp PCR reagent kit were from
Perkin-Elmer-Cetus; E. coli strains DH5 Expression and Purification of Recombinant SFD Proteins--
The
coding region for bovine SFD Northern Blot Analysis--
An 856-bp PCR product, originally
used to isolate the cDNAs encoding SFD RT-PCR--
RT-PCR was performed using a GeneAmp RNA PCR kit
from Perkin-Elmer. Bovine poly(A)+ RNA (0.1 µg) was
reverse-transcribed using poly(dT)16 as primer, and PCR amplification
was subsequently performed using Primer I (5'-CTGAGGAGAAGCAGGAGATG-3')
and Primer II (5'-GCATCAGCTGCAGACACCCG-3'). These primers were designed
to yield a PCR product that included the 54-bp insertion site that
distinguishes SFD Purification of Recombinant SFD Assembly of SFD-depleted Proton Pump with Recombinant SFD Measurement of ATPase and Proton Pumping--
Measurement of
ATPase activity was assessed by the liberation of
32Pi from [
For assessment of ATP-driven proton pumping, samples of reassembled
proton pump (0.5 pmol/assay) were reconstituted into liposomes (300 µg/assay) prepared from purified lipids by the cholate-dilution, freeze-thaw method, as described (19). Proteoliposomes were diluted
into 1.6 ml of pumping assay buffer consisting of 150 mM
KCl, 10 mM Tricine (pH 7.5), 2.5 mM
MgCl2, 0.15 mM Na3VO4, and 7 µM acridine orange. Proton pumping was assessed by
ATP-dependent quenching of the absorbance of acridine
orange and was measured in Amino DW2C dual wavelength spectrophotometer
as Western Blot Analysis--
The preparation of antibodies Q50
(specific to SFD Our previous attempts to resolve the 50- and 57-kDa polypeptides
of SFD by conventional biochemical methods failed (8). While this may
have owed to the fact that SFD existed as a heterodimer, it was also
possible that their copurification owed to the fact that the two
proteins differed by only 18 amino acids (9). Moreover, results of
molecular mass determination by gel filtration chromatography were
equivocal, perhaps because of the presence of residual amounts of
detergent used for release of the polypeptide pair from holoenzyme. Key
issues regarding the 50- and 57-kDa components of SFD are thus whether
both components are required for pump functions, and if not, do
differences exist in the functional properties of the two proteins.
To explore these points, we expressed and purified recombinant forms of
the two polypeptides, as described under "Experimental Procedures."
Shown in panel A of Fig. 1 are
the purified recombinant )1 and 50-kDa
(SFD
) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFD
and rSFD
, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of
chromaffin granules has only the SFD
isoform in its native state and
that rSFD
and rSFD
are equally effective in restoring ATPase and
proton pumping activities to SFD-depleted enzyme. Finally, we
found that SFD
and SFD
structurally interact not only with V1, but also withV0, indicating that these
activator subunits may play both structural and functional roles in
coupling ATP hydrolysis to proton flow.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and SFD
, respectively, arise from
a single gene by an alternative splicing mechanism and that the SFD
isoform has a smaller molecular mass because of an 18-amino acid
deletion (9). Further characterization revealed that the SFD proteins
have sequence homology to the VMA13 product of Saccharomyces
cerevisiae. As is the case with SFD, loss of the VMA13 gene
product yielded a yeast vacuolar proton pump that was assembled but
inactive (12).
), is molecularly distinct from
AP50 and that removal of AP50 from impure pump preparations had no
effect on enzyme activity, whereas removal of SFD
and SFD
accounted for the deactivation we had previously observed (9).
and SFD
are shown to
restore ATPase and proton pumping activity to V-pump depleted of SFD.
Moreover, we have identified a source of V-pump (chromaffin granules)
that in its native form has only SFD
. When depleted of SFD, the
chromaffin granule pump loses ATPase and proton pumping activities, and
both are restored by addition of either SFD
or SFD
. Finally, we
have determined that SFD binds to both isolated V1
andV0, thus providing evidence that it may function in a
structural role of coupling ATP hydrolysis to proton pumping.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1·min
1. V0 was
isolated from purified V-pump (21), and recombinant subunit B (22) was
prepared as described. Purified proton pump was depleted of SFD by
treatment with Zwittergent 3-16, followed by glycerol gradient
centrifugation, as reported (8). Partial purification of the vacuolar
proton pump of chromaffin granules was achieved by modification of the
protocol used for preparation of the vacuolar proton pump of
clathrin-coated vesicles of bovine brain. Briefly, 500 mg of bovine
chromaffin granule membranes were incubated at 4 °C for 1 h
with 20 ml of solubilization buffer, consisting of 10 mM
Tris-HCl (pH 7.5), 1% C12E9, 0.5 mM EDTA, and 5 mM dithiothreitol. The mixture
was centrifuged at 150,000 × g for 1 h, and the
supernatant was removed and mixed with
(NH4)2SO4 to achieve 45% final
saturation. After a 30-min incubation at 4 °C, the mixture was
centrifuged at 150,000 × g for 30 min, and the
supernatant was discarded. The pellet was dissolved in 1 ml of 20 mM Tris-HCl (pH 7.5), 0.05% C12E9,
5 mM dithiothreitol, and 0.5 mM EDTA and was
layered over a 13-ml, continuous 15-30% glycerol gradient prepared in
the same buffer. The gradients were centrifuged at 38,000 rpm for
20 h at 4 °C in a Beckman SW40 rotor. Proton pump was harvested
from the bottom one-third of the gradient in 1-ml fractions.
and BL21 (DE3) pLys S and expression vector pET16b were from Novagen; radioactive reagents were from Amersham Pharmacia Biotech; nitrocellulose membranes
for plaque lift were from Millipore Corp.; and chemicals for SDS-PAGE
were from Bio-Rad. Bovine adrenal chromaffin granule membranes were the
generous gift of Dr. Joseph Albanesi (University of Texas Southwestern
Medical Center at Dallas).
and SFD
were amplified by PCR using
the cloned cDNAs SFD-RT4 and SFD21 (9), respectively, as
templates and two synthetic oligonucleotides
(5'-GGATCCGATGACCAAGATGGATATTCG-3' and
5'-GGATCCGGAGATCCAAGGGAAGCCCT-3') as primers, which were designed to contain BamHI restriction sites to enable cloning into
the bacterial expression vector pET16b. The amplification reaction was
performed in a thermal cycler using the following conditions: 1 min at
94 °C and 5 min at 65 °C, for a total of 30 cycles. The PCR
products were size-fractionated and purified by agarose gel electrophoresis; resulting fragments were identical to the parent cDNA sequences as determined by direct DNA sequencing. The PCR products and pET16b vector were separately digested with
BamHI, and the desired digestion fragments were gel-purified
and ligated by bacteriophage T4 DNA ligase. Ligation products were used
to transform E. coli DH5
. Plasmids recovered from
independent clones yielded two expression vectors, pET16b-SFD
and
pET16b-SFD
, that were screened for the correct orientation by
restriction analysis. Both of these plasmids were designed to express
fusion proteins containing 10 neighboring histidine residues and a
Factor Xa1 recognition site at the NH2 terminus. For
expression of SFD
and SFD
, E. coli strain BL21 (DE3)
pLys S was transformed with expression vectors pET16b-SFD
or
pET16b-SFD
, respectively, and grown and induced with IPTG at
37 °C, as described (23).
(9), was used to generate
a 32P-labeled RNA probe with a MAXI-Script Sp6/T7 kit from
Ambion. Designated amounts of bovine poly(A)+-enriched
mRNA were subjected to agarose (1.2%) gel electrophoresis and were
subsequently transferred to nylon filter. After baking at 80 °C for
1 h, the filters were prehybridized with 4× SSC, 5× Denhardt's
solution, 0.1 mg/ml sheared, single-stranded salmon DNA, and 0.1% SDS
at 65 °C for 3 h. Subsequently the filter was hybridized with
RNA probe (0.3 × 106 cpm/ml) for 12 h at
65 °C. Filters were sequentially washed with 2× SCC, 0.1% SDS at
65 °C for 15 min, 0.5× SSC, 0.1% SDS at 65 °C for 15 min, and
0.1× SSC, 0.1% SDS at 65 °C for 30 min. Washed filters were
exposed overnight using Amersham Pharmacia Biotech Hyperfilm.
from SFD
(9). With SFD
transcript as
template, these primers are predicted to yield a 450-bp PCR product,
and with SFD
transcript template, a 396-bp PCR product. PCR products
were separated electrophoretically on 1% agarose gels and were
visualized by ethidium bromide staining.
and SFD
--
Bacterial
cells were harvested by centrifugation at 3,840 × g
for 20 min. The pellets were resuspended in 100 ml of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl), and the
cells were broken by sonication. The lysate was centrifuged at
186,000 × g for 1 h, and the supernatants, which
contained most of the recombinant proteins, were collected and loaded
on 1 ml of Ni2+-NTA columns. After loading, the columns
were washed with 20 ml of lysis buffer and then eluted with lysis
buffer containing 20 mM imidazole (pH 7.5). The eluents
were diluted 1:10 with 5 mM Tris-HCl (pH 7.5) and loaded on
a Mono-Q (5 mm × 5 cm) column. The column was washed with 10 ml
of binding buffer (20 mM Tris-HCl (pH 7.5), and 10%
glycerol). Proteins were eluted with linear (0-400 mM)
NaCl gradients prepared in 15 ml of binding buffer. The fractions (1 ml) contained as much as 10-20 mg of the recombinant proteins,
with > 95% purity, as assessed by SDS-PAGE (24) and by the
Bradford protein assay (25).
and
SFD
--
For each assay, 0.5 pmol of SFD-depleted proton pump was
mixed with designated amounts (0-2.5 pmols) of recombinant SFD
and/or SFD
, and the final volume was brought to 10 µl with buffer
consisting of 20 mM Tris-HCl (pH 7.5), 0.05%
C12E9, 0.5 mM EDTA, and 2 mM dithiothreitol. After incubation at room temperature for
10 min, reassembled pump was assessed for ATPase and/or proton pumping activities.
-32P]ATP.
Reassembled pump (0.5 pmol/assay) was incubated with 5 µl of
phosphatidylserine (0.5 mg/ml) at room temperature for 5 min. Reactions
were initiated with addition of 200 µl of assay buffer, consisting of
50 mM Tris-MES (pH 7.0), 30 mM NaCl, 3 mM MgCl2, 0.15 mM
Na3VO4, and 3 mM
[
-32P]ATP (200-400 cpm/nmol). After incubation at
37 °C for 30 min, reactions were terminated by the addition of 1 ml
of 1.25 N perchloric acid, and liberated 32Pi
was extracted and counted as described (26).
A492-540. Reactions were initiated by addition of 1.3 mM Na-ATP and 1 µM valinomycin and were
terminated by addition of 1 µM 1799.
) and Q48 (directed against a common epitope of
SFD
and SFD
) were described previously (9). Designated amounts of
protein were subjected to SDS-PAGE and were electrophoretically
transferred to nitrocellulose filters. Protein A-purified Q50 or Q48
was incubated with filters at a 1:3000 dilution, and immunoreactivity
was assessed by ECL using donkey, anti-rabbit IgG at a 1:4000 dilution.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(lane 3) and
(lane
5) components of SFD. Panels B and
C are Western blot analysis of the two components. In
panel B, antibody Q48, directed against an epitope common to
SFD
and
, reacts with both species in lysates of E. coli transformed with expression vectors for the
(lane
2) and
(lane 4) forms of the polypeptide; lanes 3 and 5 reveal immunoreactivity
of purified rSFD
and rSFD
, respectively. Western blot analysis
(panel C) using antibody Q50, directed against the unique
18-amino acid insert of SFD
demonstrates that the antibody
recognizes rSFD
before (lane 2) and after purification (lane 3) from E. coli lysate; in contrast, the
antibody does not recognize rSFD
(lanes 4 and
5).
View larger version (23K):
[in a new window]
Fig. 1.
Expression and purification of
rSFD and rSFD
.
SDS-PAGE (panel A) followed by Coomassie Blue staining and
Western blot analysis using Q48 (panel B) or Q50
(panel C) IgG were performed as described under
"Experimental Procedures." Lanes 1, 5 µl of cell
lysate of E. coli transformed with pET16b SFD
, before
IPTG induction; lanes 2, 5 µl of cell lysate of E. coli transformed with pET16b SFD
after IPTG induction;
lanes 3, purified recombinant SFD
(150 ng); lanes
4, 5 µl of cell lysate of E. coli transformed with
pET16b SFD
after IPTG induction; and lanes 5, purified
recombinant SFD
(150 ng).
Of note was the finding that both rSFD and rSFD
were present as
soluble proteins in E. coli lysates. This differed from our
experience with production of recombinant forms of all other V1 subunits, each of which required detergents for
solubilization, and many of which required denaturation with 8 M urea to achieve dissolution of inclusion bodies that
contained the recombinant proteins.
To address whether SFD or SFD
had independent activities, or
synergistic effects when used in combination, reconstitution experiments were performed using the recombinant SFD proteins and
biochemically prepared proton pump that had been depleted of SFD. As
shown in Fig. 2, proton pump depleted of
SFD lacked significant Mg2+-activated ATPase activity.
Reassembly of recombinant forms of SFD with SFD-depleted pump resulted
in a saturable activation of MgATPase activity. No differences were
detected with the use of rSFD
alone, rSFD
alone, or equimolar
mixtures of rSFD
and rSFD
. Maximum restoration of MgATPase
activity was achieved at a molar ratio of rSFD to SFD-depleted proton
pump of 2. The final specific activity of reassembled enzyme was about
1.5 µmols of Pi·mg of
protein
1·min
1.2
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To further characterize the functional properties of rSFD and
rSFD
, we performed reconstitution experiments in which the rSFD
proteins were first reassembled with SFD-depleted pump, and the
resulting holoenzyme was then reconstituted into liposomes and assayed
for proton pumping activity. Shown in Fig.
3 are the proton pumping activities of
these preparations, as assessed by ATP-generated acridine orange
quenching. SFD-depleted pump lacked activity (trace 1),
whereas rSFD
(trace 2), rSFD
(trace 3), and
equimolar mixtures of rSFD
and rSFD
(trace 4)
activated protein pumping in SFD-depleted pump. In this particular
experiment, proton pumping, as assessed by the initial rate of
ATP-generated acridine orange quenching, was about 1.3-fold higher when
a mixture of rSFD
and rSFD
were used (trace 4), as
compared with rates attained with a single isoform of SFD (traces
2 and 3). This, however, was not a consistent
observation, and coupled with the results obtained in the experiments
of Fig. 2, we find no compelling evidence at present that there exist
functional differences between the two isoforms.
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Tissue distributions of the SFD isoforms were assessed by Northern blot
analysis, as shown in Fig. 4. The highest
copy number of transcripts was found in brain (lane 1)
followed by kidney (lane 3) and lung (lane 4);
much lower levels of SFD transcripts were detected in heart (lane
2). Because of the minor difference in molecular mass in the
transcripts for SFD and SFD
, we performed RT-PCR to determine
whether tissue differences in the transcript level of SFD
and SFD
existed. As described in "Experimental Procedures," primers were
utilized that would predictably yield products of 450 bp for SFD
and
396 bp for SFD
. Although transcripts for SFD
and SFD
were
found in all tissues, it appears that SFD
is in relative abundance
in brain (lane 2), whereas relatively more SFD
RT-PCR
product was demonstrated in all other tissues examined.
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We next sought to determine whether proton pump preparations from
sources other than brain contained both SFD and SFD
isoforms. As
shown in Fig. 5, proton pump prepared
from bovine brain (lane 1) contained roughly equimolar
amounts of SFD
and SFD
, whereas V-pump purified from bovine
chromaffin granules (lane 2) had only the SFD
isoform, as
determined by immunoblot analysis (panels B and
C). This finding allowed us to examine by another approach whether any functional differences could be attributed to the isoforms,
and we specifically sought to determine whether SFD
could substitute
for SFD
in the proton pump of chromaffin granules, which, in its
native form, contains only SFD
.
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To test this possibility, we first depleted the purified chromaffin
granule V-pump of SFD (Fig. 6) by
treatment of holoenzyme with Zwittergent 3-16, followed by glycerol
gradient centrifugations as described under "Experimental
Procedures." In panel A, SDS-PAGE reveals that the
holoenzyme was found to migrate to its usual position in the glycerol
gradient, as evidenced by the characteristic Coomassie stained bands of
70-, 58-, and 33-kDa, that can be visualized in lanes 3 and
4. In contrast, an immunoblot performed with anti-SFD antibody (panel B) demonstrate that all immunoreactive
protein was present near the top of the gradient (e.g.
lanes 8 and 9), where released SFD is typically
found (8, 9).
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Next, we utilized SFD-depleted proton pump of chromaffin granules (as
shown in Fig. 6, lanes 3 and 4) to compare the
effects of rSFD and rSFD
in restoration of enzyme activity. Shown
in Fig. 7 are the results of these
experiments. rSFD
and rSFD
were found to be equipotent in
restoration of MgATPase (panel A) and proton pumping
(panel B) activities of the SFD-depleted V-pump of
chromaffin granules.
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Finally, we have begun to investigate the structural basis of the
effects of SFD on pump function. As described previously (8, 9), and as
demonstrated in these studies, SFD activates ATPase and proton pump
activities of the holoenzyme. In addition, SFD has been shown to
activate ATPase activity of isolated, SFD-depleted V1 (8)
To determine the domain(s) through which SFD regulates pump function,
we performed binding experiments using rSFD, SFD-depleted proton pump,
SFD-depleted V1, and isolated V0. For these
studies (Fig. 8), recombinant,
histidine-tagged SFD was incubated with SFD-depleted holoenzyme,
SFD-depleted V1, or isolatedV0; the mixtures were passed over Ni2+-NTA columns; and bound proteins were
subsequently eluted with imidazole. After SDS-PAGE, the eluents were
then tested by immunoblot analysis to determine whether there had been
binding of the 116-kDa polypeptide (a V0 constituent)
and/or the 70-kDa subunit (a V1 constituent). As shown in
panel A, SFD-depleted holoenzyme binds to both rSFD
(lane 3) and rSFD
(lane 4). In addition, both
recombinant forms of SFD bind isolated V0 (panel B,
lanes 3 and 4) as well as SFD-depleted V1
(panel C, lanes 3 and 4). Important controls for
this study included passage of the biochemically prepared SFD-depleted
pump, and V1 orV0, over a Ni2+-NTA
column without preincubation with rSFD isoforms (lanes 1 of
panels A, B, and C). In addition,
histidine-tagged, recombinant (22) subunit B (a component of
V1) was substituted for rSFD and was demonstrated to not
bind either V1,V0, or holoenzyme (lanes 2 of panels A, B, and C). This study adds an
additional insight into SFD function with the demonstration that SFD
isoforms bind not only to V1, but also to V0,
thus raising the possibility that SFD may play a structural role in the
coupling of V1 toV0, and thereby, the
functional coupling of ATP hydrolysis to proton flow.
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DISCUSSION |
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Expression of functional forms of 50- and 57-kDa components of SFD
has allowed us to address several key questions regarding the role of
SFD in V-pump function. First, a lingering question from our previous
studies was whether both forms of SFD were essential to V-pump
function. A strict biochemical approach to this issue was thwarted by
our inability to separate the two isoforms (8). With the molecular
cloning of SFD and SFD
, it became apparent that their (near)
identical sequences might explain the difficulty in separating these
two species by conventional biochemical methods (9). Moreover, attempts
to determine the overall molecular mass of the putative SFD
/SFD
complex by size exclusion chromatography were equivocal, perhaps
because of the presence of detergent-protein micelles resulting from
residual Zwittergent 3-16. Two lines of evidence now demonstrate that
the
and
isoforms can function independently in activation of
ATPase and proton pumping activities.
First, we have shown that recombinant forms of either SFD or SFD
can restore the ATPase and proton pumping activities of SFD-depleted
proton pump prepared from clathrin-coated vesicles of bovine brain.
Further, titration of mixtures of rSFD
and rSFD
did not yield
synergistic effects. Second, we demonstrate that V-pump of chromaffin
granules contains only the SFD
isoform, indicating that a native
form of the enzyme functions with only one isoform. In additional
experiments, rSFD
and rSFD
had equivalent effects on
SFD
-depleted pump of chromaffin granule, further demonstrating the
independent, and interchangeable functions of the two isoforms. Additional evidence indicates that the purified recombinant SFD isoforms exist in monomeric forms prior to reassembly with the pump, as
determined by high performance liquid chromatography performed with
size exclusion columns (data not shown). We therefore believe it is
most likely that the SFD protein exist in a monomeric form if, indeed,
they are present as isolated species in the cytosol.
Our finding that optimal stimulation of pump activities occurs at a
molar ratio of SFD to holoenzyme of 2:1 suggests that there may be more
than one SFD binding site per holoenzyme. We, however, view the issue
of SFD copy number as unresolved at present, in part because
recombinant SFD isoforms may not be fully active and thus spuriously
increasing the apparent SFD/holoenzyme ratio. Also, as discussed below,
it is possible that SFD may act as a dissociable regulatory element,
and the optimal ratio required for the binding of SFD to holoenzyme may
differ from the copy number of SFD needed for activation of the enzyme,
once binding has occurred. The finding that reassembly of SFD-depleted
V-pump of CCV with mixtures of rSFD and rSFD
does not yield
additive, or synergistic, stimulation of activities indicates that the
SFD binding sites of a given V-pump molecule are promiscuous, at least under these conditions. In addition, reassembly of SFD-depleted V-pump
of chromaffin granule with either rSFD
or rSFD
provides additional evidence that SFD isoforms can act interchangeably, even in
a pump which, in its native form, has only one (SFD
) isoform. We
thus favor the view that more than one isoform can be present in a
single proton pump.
These studies add final clarification to a controversy regarding the
nature of the 50-kDa polypeptide present in the V-pump of
clathrin-coated vesicles. Previously it was reported by others (13, 14)
that this polypeptide was AP-50, a component of the AP2 complex that is
essential to the assembly of clathrin coats (15-17). Previous work
from our laboratory demonstrated that the 50-kDa polypeptide was in
fact SFD (9), and with the results of the current work, we have
functional proof that SFD isoforms are causally related to the
functional effect ascribed by others to AP-50. As we continue to note,
these observations do not exclude the possibility that AP-50 may
interact with V-pumps of clathrin-coated vesicles to induce changes in
enzyme behavior that are at present unknown (e.g.
targeting). In composite, however, our data do demonstrate that SFD components, and not AP-50, are required for V-pump function, as defined by activation of ATPase and proton pumping activities in
enzyme depleted of this key component.
Given that SFD and SFD
have interchangeable functions in
activation of ATPase and proton pumping activities, a key question remains as to the roles these isoforms might play in the overall biology of V-pumps. Although we have no direct evidence to address this
issue, it is likely that an important clue to this question resides in
our finding that SFD isoforms can bind to both the V1 and
V0 sectors of V-pumps. This observation is of some
surprise, as all evidence to date has indicated that SFD is a
V1 component, as demonstrated by its copurification with
isolated V1. Our current experiments now indicate that SFD
subunits may play a structural role in coordinating the activities of
V1 and V0 and may thus function not only as
activators of V-pumps but also as true coupling factors. In this
respect, it is notable that V-pumps (27), like F-type proton pumps
(28), appear to have two links between the ATP hydrolytic sector and
the intramembranous channel. In addition to the
subunit that plays
a key role in energy transduction, the F-type pump of E. coli has a stator arm that links F1 and F0
together in such a manner that F1 is held stationary
relative to the membrane. For the F-type pump of E. coli,
this stator arm is composed of the b subunits of Fo, and
subunit of F1, whereas in mitochondrial
F1F0 this linkage is composed of the b subunits of F0 and OSCP of F1 (28). Based upon other
investigations in our laboratory, it appears that the NH2
terminus of the 116-kDa component (subunit a) of V0 is
cytosolic in orientation and may interact with
SFD.3 We thus speculate that
subunit a with SFD may represent the stator arm of V-pumps. As such,
SFD could play a pivotal role in governing the structural and
functional assembly of V-pumps.
V-pumps, unlike F-type proton pumps, have been shown to reversibly dissociate into V1 and V0 components as a mechanism of recruitment of V-pump function in instances requiring increased proton pumping capability (29, 30). Recently, we have demonstrated that the tendency of V1 to dissociate from V0 correlates with isoform diversity of subunit a (11). Thus it is possible that the isoform diversity of the SFD subunits interplays with the isoforms of subunit a to provide a multiplicative diversity in the interactions of V1 withV0. This in turn, may represent an important means by which proton pumps undergo differential regulation in their myriad cellular locations.
We also believe that the solubility of recombinant SFD isoforms may be
highly significant. Of the 10 subunits and subunit isoforms of
V1 that we have expressed in functional, recombinant forms,
only rSFD and rSFD
exist as soluble proteins in expression cell
lysates. Although our current data speak only to the roles these
proteins play in activation of enzyme activity, and possibly coupling
functions, we believe that SFD
and SFD
may exist as soluble,
regulatory factors of V-pumps within the cytosol. Current studies are
underway to investigate this issue.
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FOOTNOTES |
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* This work was supported by Grant RO1DK-33627 from the National Institutes of Health.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.
Present address: Lilly Research Laboratories, Lilly Corporate
Center, Indianapolis, IN 46285.
§ To whom correspondence should be addressed: Division of Molecular Transport, Dept. of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9121. Tel.: 214-648-7606; Fax: 214-648-7542.
2 This is about one-tenth the specific activity of native enzyme (20) and is close to the specific activity obtained by reassembling biochemically prepared SFD with SFD-depleted proton pump. This reduced activity likely owes to treatment of the holoenzyme with Zwittergent 3-16 in preparation of SFD-depleted pump. We have found that this detergent is relatively toxic to enzyme function and is not easily removed because of its low critical micellar concentration.
3 P. Andersen, B. P. Crider, S.-B. Peng, Z. Zhou, J. Mattsson, L. Lundberg, D. J. Keeling, X.-S. Xie, and D. K. Stone, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
SFD, Sub-Fifty-eight-kDa Dimer, or
Doublet 50/57-kDa polypeptide heterodimer required for
function of the vacuolar proton pumps;
rSFD, recombinant SFD;
1799, bis(hexafluoroacetonyl) acetone;
bp, base pair;
C12E9, polyoxyethylene 9-lauryl ether;
MES, 2-(N-morpholino)ethanesulfonic acid;
NTA, nitrilotriacetic
acid;
PAGE, polyacrylamide gel electrophoresis;
PCR, polymerase chain
reaction;
V-, vacuolar;
V0, the bafilomycin-sensitive
proton channel of V-type proton pumps;
V1, the peripheral,
catalytic sector of V-type proton pumps;
CCV, clathrin-coated vesicle;
IPTG, isopropyl-1- thio--D-galactopyranoside.
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REFERENCES |
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