From the Division of Molecular Transport, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
We have identified a cDNA encoding an isoform
of the 116-kDa subunit of the bovine vacuolar proton translocating
ATPase. The predicted protein sequence of the new isoform, designated
a2, consists of 854 amino acids with a calculated
molecular mass of 98,010 Da; it has approximately 50% identity to the
original isoform (a1) we described (Peng, S.-B., Crider,
B. P., Xie, X.-S., and Stone, D.K. (1994) J. Biol.
Chem. 269, 17262-17266). Sequence comparison indicates that the
a2 isoform is the bovine homologue of a 116-kDa polypeptide
identified in mouse as an immune regulatory factor (Lee, C.-K.,
Ghoshal, K., and Beaman, K.D. (1990) Mol. Immunol. 27, 1137-1144). The bovine a1 and a2 isoforms
share strikingly similar structures with hydrophilic amino-terminal
halves that are composed of more than 30% charged residues and
hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane
regions. Northern blot analysis demonstrates that isoform
a2 is highly expressed in lung, kidney, and spleen. To
determine the possible role of the a2 isoform in vacuolar
proton pump function, we purified from bovine lung a vacuolar pump
proton channel (VO) containing isoform a2. This
VO conducts bafilomycin-sensitive proton flow after
reconstitution and acid activation, and supports proton pumping
activity after assembly with the catalytic sector (V1) of
vacuolar-type proton translocating ATPase (V-ATPase) and sub-58-kDa doublet, a 50-57-kDa polypeptide heterodimer required for V-ATPase function. These data indicate that the a2 isoform of the
116-kDa polypeptide functions as part of the proton channel of
V-ATPases.
Vacuolar-type proton translocating ATPases
(V-ATPases)1 are widely
distributed in eukaryotic cells where they are found in most
organelles. In addition, these proton pumps are localized to plasma
membranes of epithelia, macrophages, and specialized polarized cells.
V-ATPases have been shown to control osteoclast-mediated bone
reabsorption and renal acidification and are thereby involved in the
pathogenesis of osteoporosis and the systemic acidosis of uremia
(1-3). Key to understanding the regulation of these diversely
distributed proton pumps is a basic investigation of the structure of
the enzymes and delineation of the roles of the individual subunits in
pump function. The primary structures of V pump subunits, as well as
the overall subunit composition and quartenary structures of the
holoenzyme, are highly conserved through species as evolutionarily
diverse as Archaebacteria, Caenorhabditis elegans, and
Homo sapiens. In its simplest form, the V pump of Enterococcus hirae is composed of 11 subunits, some of which
are homologues of the subunits of the V-type proton pump of
clathrin-coated vesicles of bovine brain (4).
Structurally, V-ATPases resemble F1FO-type ATP
synthases in that they are complex hetero-oligomers with two functional
domains: an ATP-hydrolytic sector (V1 or VC)
that is peripheral to the membrane, and a transmembranous proton
channel (VO or VB). The V1 domain
of the V pump of clathrin-coated vesicles consists of essential, core
subunits of 70, 58, 40, 34, 33, 14, and 10 kDa, designated A, B, C, D,
E, G, and F, respectively. In addition, a key regulatory element, the
sub-58-kDa doublet (SFD), consists of polypeptides of 57 and 50 kDa,
activates V1 and functionally couples ATP hydrolysis to
proton flow through the transmembranous sector, VO (5, 6).
Separation of V1 from VO results in marked changes in the functions of these two domains. Although native holoenzyme hydrolyzes MgATP at a rate 3-fold higher than CaATP, isolated V1 hydrolyzes ATP only in the presence of
Ca2+; Mg2+, in fact, inhibits ATP hydrolysis
catalyzed by V1 (7). In addition, the proton channel,
VO, is closed after separation from V1, and
requires incubation at an acidic pH to restore proton flow, which is
inhibitable by bafilomycin A1, a V-ATPase specific inhibitor (8).
Although the subunit composition of V1 is now well defined,
there are conflicting reports regarding the components of
VO. All investigators find a 17-kDa polypeptide (subunit
c), as well as a 39-kDa subunit in VO preparations. In
addition, the VO component of the proton pump of
clathrin-coated vesicles contains a 116-kDa polypeptide, and a
polypeptide of this mass, or a smaller homologue, has been demonstrated
in most V-pump preparations. The function of the 116-kDa subunit is not
defined, but its predicted structure consists of 6-8 transmembranous
sectors, suggesting that it may function similar to subunit a of
FO.
Additional structural complexity exists in V-ATPases in the form of
subunit isoforms. Two forms of subunit A (9-11) and subunit B (12, 13)
have been identified. Subunit G, a recently identified subunit that is
required for ATP hydrolysis, has two isoforms that differ in tissue
distribution and function (14). Most recently, we have demonstrated
that the 50- and 57-kDa polypeptides of SFD are isoforms resulting
through alternative mRNA splicing (7). In addition, three forms of
the c subunit of VO have been shown to be required for
V-pump function in yeast (15).
The 116-kDa subunit of VO, the subject of this
investigation, appears to have the greatest degree of isoform diversity
of all V-pump components. This diversity arises through two mechanisms. First, alternative splicing of mRNA results in two forms of the subunit prevalent in brain (a1 isoform). This alternative
splicing results in changes within a predicted protease sensitivity
motif (PEST site, a region enriched in proline, glutamic acid, serine, or threonine residues), implying differences in the biological half
lives of the two isoforms (16, 17). Second, higher organisms have
separate genes that encode distinct isoforms of the 116-kDa subunit. In
yeast, two such genes, designated VPH1 (20) and STV1 (21), encode
proteins with amino acid sequence homology to the mammalian 116-kDa
polypeptide. Cumulative evidence suggests that three separate genes
encode forms of the 116-kDa subunit in mammalian species. In addition
to the a1 isoform of bovine brain, a related homologue has
been identified in murine T cells, and a third form in human
osteoclasts. Although interspecies comparison of primary sequence
complicates this point, it is notable that the sequence divergence
between these bovine, murine, and human forms of the 116-kDa subunit
greatly exceeds the divergence observed in all other pump subunits.
Moreover, recent experiments have demonstrated three distinct genes
encoding 116-kDa isoforms in chicken.2
Of these putative forms of the 116-kDa subunit, the isoform isolated
from murine T cells has not been identified as a V pump component. In
fact, the cDNA encoding this subunit was isolated by a strategy
designed to identify novel immune regulatory factors. To investigate
the function of this isoform and to determine its relationship to V
pump function, we have cloned and sequenced the cDNA encoding this
isoform of the 116-kDa subunit. It shares only 50% identity to the
116-kDa subunit of bovine brain that we described previously, but has
91.6% identity to the 116-kDa isoform of murine T cells. The two
polypeptides have strikingly similar structure, with hydrophilic
amino-terminal halves that are composed of >30% charged residues and
hydrophobic carboxyl-terminal halves that contain 6-8 transmembrane
regions. The new isoform, designated a2, copurifies with
vacuolar proton channel from lung. Reconstitution experiments
demonstrate that it is associated with functional VO,
indicating that it is a genuine isoform of the 116-kDa subunit of V pumps.
Materials--
Restriction enzymes, T4 DNA ligase, and a nick
translation kit for DNA probe labeling were purchased from Boehringer
Mannheim; the GeneAmp polymerase chain reaction (PCR) reagent kit with
Thermus aquaticus Taq DNA polymerase and DNA
sequencing materials and reagents were from Perkin-Elmer; a TA cloning
kit containing vector, pCR 2.1, and DNA ligase were from Invitrogen;
Escherichia coli strains XLI-Blue-MRF' and XLOLR and helper
phage R408 were from Stratagene; radioactive materials and an ECL kit
for Western blot analysis were from Amersham Pharmacia Biotech;
nitrocellulose membranes for plaque lift were from Millipore Corp., and
chemicals for SDS-PAGE were from Bio-Rad. A bovine brain cDNA
library in Synthesis of a 0.3-kb DNA Fragment by PCR--
Two
oligonucleotide primers, 5'-TCICC(G/A)AACATIACIGC(G/A)AA-3' and
5'-AAGTG(C/T)(C/T)TIATIGCIGA(A/G)GTITGGTG-3', were designed and
synthesized in accord with two regions of protein sequences of 116-kDa
subunits of vacuolar ATPases that are highly conserved in all species
(16-21). Deoxyinosine (I) was used in the third position of some
codons with a degeneracy of two or more. Screening of Bovine Brain cDNA Library--
A bovine brain
cDNA library in Subcloning and DNA Sequencing--
Inserts of all positive
clones were excised and cloned into pBluescript with the helper phage
R408, as described (10). Plasmid DNA was prepared by alkaline lysis,
and DNA sequencing reactions were performed using a Model 377 ABI PRISM
DNA sequencer and the manufacturer's reagents. All positive clones
were sequenced in both orientations using M13 reverse, M13 ( Northern Blot Analysis--
Poly (A+) RNA (2 µg/lane) from different bovine tissues was denatured and fractionated
by 1% formaldehyde-agarose gel electrophoresis, and transferred to a
Zeta-probe blotting membrane (Bio-Rad). After baking at 80 °C in a
vacuum oven for 1 h, the membrane was prehybridized for 4 h
at 50 °C in a solution consisting of 50% formamide, 1.5× saline/sodium phosphate/EDTA, 1% SDS, 0.5% nonfat dry milk, and 0.5 mg/ml of denatured salmon sperm DNA. A 0.6-kb cDNA fragment encoding the NH2-terminal portion of isoform a2
was labeled with [ Antibody Preparation and Western Blot Analysis--
Isoform
a1-specific (CVLRRQYLRRKHLGT) and a2-specific
(CGTIPSFMNTIPTKET) peptides were synthesized based upon the deduced protein sequences, coupled to keyhole limpet hemocyanin, and utilized for immunization of a New Zealand White rabbits to generate polyclonal antibodies, as described (23). The preparations of antibodies directed
against the 70-kDa (subunit A) and 39-kDa subunits have been previously
reported. For Western blot analysis, protein samples were separated by
11% SDS-PAGE and transferred electrophoretically to nitrocellulose
filters. Immunodetection was performed using immune serum at a 1:5000
dilution and an Amersham Pharmacia Biotech ECL Western blotting system.
Isolation of Vacuolar Proton Channel (VO) from Bovine
Lung--
Microsomes were prepared from bovine lung by using the
buffer solution and initial steps used to prepare clathrin-coated
vesicles from bovine brain. Briefly, bovine lung (1 kg) was homogenized in a Waring blender in 2 liters of Buffer A, consisting of 100 mM MES (pH 6.5), 3 mM azide, 1 mM
EGTA, and 0.5 mM MgCl2. The homogenate was
centrifuged at 3000 × g for 20 min, and the resulting supernatant was centrifuged at 180,000 × g for 1 h. The final crude, microsomal pellet was utilized for isolation of
vacuolar proton pump and VO, as follows. Membrane pellet (5 ml) was resuspended in 20 ml of Buffer A with 1%
C12E9 (polyoxyethylene 9-lauryl ether) and
incubated on ice for 30 min. After centrifugation at 180,000 × g for 1 h, the resulting pellet was resuspended in
Buffer A containing 1% sodium cholate. Centrifugation was repeated,
and the final pellet was resuspended in 3 ml of 1% Zwittergent 3-16, and incubated at room temperature for 1 h. After centrifugation at
180,000 × g for 1 h, the supernatant (3 ml) was
loaded on two 13-ml, linear (15-30%) glycerol gradients prepared in
Buffer G, consisting of 20 mM Tris-HCl (pH 7.5), 0.05%
C12E9, 5 mM dithiothreitol, and 0.5 mM EDTA. After centrifugation at 180,000 × g for 22 h at 4 °C, fractions of 1 ml were harvested
from the bottom of the tube, subjected to SDS-PAGE, Western blot, and
proton pumping and/or proton channel activity analysis. For further
purification, the peak fractions were combined, concentrated with a
Millipore Ultrafree-MC centrifugal filter unit, and separated by a
second 15-30% glycerol gradient centrifugation that was performed as described above.
Purification of V-pump, V1 and SFD from Bovine
Brain--
Clathrin-coated vesicles were prepared from batches of 30 bovine brains, and V pump was solubilized with 1%
C12E9 and purified to a specific activity of
14-16 µmol of Pi × mg protein Reconstitutions--
Reconstitutions of vacuolar proton pump and
proton channel (VO) were performed by the freeze-thaw,
cholate-dilution method using liposomes prepared from pure lipids.
Stock solution of liposomes composed of phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine, and cholesterol at a mass
ratio of 4.32:2.76:0.2:2.7 were prepared as described (25). Typically,
100 µl (0.5-0.8 mg of protein/ml) of purified proton pump or
VO, was mixed with 3 mg of liposomes and 26 µl of
reconstitution buffer consisting of 6% sodium cholate, 30% glycerol,
600 mM KCl, 6 µl of 600 mM dithiothreitol,
and 60 mM MgCl2. The mixture was frozen in
liquid N2 for 2 min and then incubated at room temperature
for 1 h. Proteoliposomes were diluted with 6 ml of 150 mM KCl, 20 mM Tricine (pH 7.5), 3 mM MgCl2, and 0.5 mM EDTA (dilution
buffer); concentrated by centrifugation at 110,000 × g
for 1 h at 15 °C; and then resuspended with 50 µl of dilution
buffer for proton transport assays described below. Acid activation of
the latent proton conductance of reconstituted VO was
performed as described (5), by incubation of 5 µl of proteoliposomes
containing VO with 2 µl of 0.5 M MES (pH 5.0) for 1 h on ice.
Acridine Orange Absorbance Quenching--
Proteoliposome
acidification was assessed by the measurement of acridine orange
quenching in an SLM-Amino DW2-C dual wavelength spectrophotometer as
Holoenzyme Assembly with Purified V1, VO,
and SFD--
Holoenzyme was reassembled with 3 pmol each of
V1, VO, and SFD, which were mixed with 280 µg
of lipid and 10 mM MES. The mixture was then incubated
overnight at room temperature. The reassembled holoenzyme was
reconstituted into liposomes for assessment of proton pumping as
described above.
Isolation and Identification of cDNA Encoding Isoform
a2 of the 116-kDa Subunit of Bovine Vacuolar
ATPase--
Two degenerate primers, designed to match two conserved
regions of the 116-kDa subunits, were used in polymerase chain
reactions to generate 0.3-kb DNA fragments from a bovine brain cDNA
library, as described under "Experimental Procedures." The PCR
products were cloned into pCR 2.1 vector using a commercial
TA cloning kit (Invitrogen), and 25 positive clones were selected and
sequenced. Among them, two groups of DNA sequences were obtained. The
sequences of the first group (23 clones) exactly matched the
a1 isoform sequence we have described (17). The remaining
two clones had sequences that were identical to one another, but they
encoded a protein with a predicted amino acid sequence different from
that of the a1 isoform of the 116-kDa polypeptide. The
0.3-kb insert from the second group of TA clones was used to screen a
bovine brain cDNA library in Analysis of the Deduced Amino Acid Sequence--
Translation of
the open reading frame of clone B31-1 predicts an 854-amino acid
polypeptide with calculated molecular mass of 98,010 Da, which is close
to the mass of 96,301 Da of isoform a1. Three potential
N-glycosylation sites are present at residues 43, 157, and 505. The
calculated isoelectric point is 5.89. This isoform shares 50% identity
with the a1 isoform that we described previously (16, 17).
Kyte-Doolittle (27) analysis reveals that the two isoforms have
strikingly similar structures, with two characteristic domains: a
hydrophilic amino-terminal half that is composed of more than 30%
charged residues, and a highly conserved and hydrophobic
carboxyl-terminal half that contains 6-8 transmembrane regions. Data
base searches demonstrated that isoform a2 shares 91.6%
identity at the amino acid level with mouse J6B7, a putative immune
regulatory protein from T cells (19) (Fig.
1). As shown, particularly high levels of
conservation were observed in predicted transmembranous sectors.
Expression of mRNA for Isoform a2 in Different
Tissues--
The tissue distribution of mRNA encoding the
a2 isoform was investigated by Northern blot analysis.
Although two transcripts of approximate sizes of 3.4 and 5.4 kb were
detected in all tissues (brain, heart, kidney, lung, and spleen), the
absolute and relative abundances differed between tissues. High levels
of expression were found in the kidney, lung and spleen, whereas the
brain had very low levels of a2 transcripts (Fig.
2).
Identification of Isoform a2 as a Component of Vacuolar
Proton Pump--
To determine the relationship of the cloned cDNA
to the 116-kDa component of vacuolar proton pump, we generated isoform
a1-, and a2-specific, anti-peptide antibodies
based on predicted amino acid sequence. As shown in Fig.
3, lane 3, the
a2-specific antibody reacts with a minor portion of the
116-kDa band of highly purified bovine brain vacuolar proton pump,
indicating the presence of isoform a2 in V-ATPase complex.
The same enzyme reacts heavily with isoform a1 specific,
anti-peptide antibody (Fig. 3, lane 2). This suggests that
isoform a1 is the major form and a2 is the
minor form of the vacuolar proton pump in the brain, which is in good
accord with the results of Northern blot analysis we obtained in this
study (Fig. 2) and in previous investigations (17).
Isoform a2 Is the Major Form in Lung and Co-purifies
with VO--
Whereas these findings are highly suggestive
that the a2 isoform is present in subpopulation V-type
proton pumps in bovine brain, the co-purification of the a1
and a2 isoforms precluded any direct investigation of
whether the a2 isoform was associated with a functional
proton pump. We therefore sought to find an alternative tissue source
highly enriched in the a2 isoform. As demonstrated by
Northern blot analysis in Fig. 2, mRNA for isoform a2
is present in high copy number in lung. We therefore attempted to
isolate proton pump from bovine lung using the solubilization and
purification procedure we developed for the V-type proton pump of
clathrin-coated vesicles of bovine brain. Microsomes were prepared from
freshly harvested bovine lung by homogenization and a differential
centrifugation. For comparative purposes, freshly harvested bovine
brain was processed identically. However, testing of V-pump activities
in the two microsomal preparations revealed that the vesicles from lung
had only VO Containing Isoform a2 Conducts Proton
Flow--
In order to determine whether the purified
a2-containing VO could function as a proton
channel, we performed reconstitution and acid activation experiments.
As reported previously, VO isolated form bovine brain is
closed to proton flow, but a latent proton conductance can be activated
by briefly exposing the channel to an acidic pH. As shown in Fig.
6, isolated and reconstituted
VO of bovine lung behaves similarly. Isolated
VO does not conduct protons (trace 2), but acid
pretreatment of VO activates a latent proton conductance
(trace 4), which is inhibited by bafilomycin (trace
3). This fraction cannot support proton pumping when ATP is
present in the reaction (trace 1), which further indicates that the isolated fraction does not contain functional
V1.
Reconstitution of Proton Pumping Activity with VO,
V1, and SFD--
To determine whether VO
prepared from bovine lung could function in ATP driven proton pumping,
we reassembled VO from bovine lung, with V1 and
SFD from bovine brain (5). As shown in Fig. 7, neither V1 plus SFD
(trace 1), nor VO plus SFD (trace 2),
could support ATP-dependent proton pumping as assessed by
ATP generated acridine orange quenching. However, reassembly of
V1, VO, and SFD results in a complex capable of
supporting significant ATP-dependent proton pumping
(trace 4). The reconstituted proton pumping activity was
inhibited by 3 nM bafilomycin (trace 3).
V-ATPases are distributed among most intracellular organelles of
both constitutive and regulated secretory pathways. It is thus to be
expected that these pumps are highly regulated. In this regard, a steep
intraorganelle pH gradient exists in the constitutive pathway, with
lysosomes having a pH of 4.5; endosomes, pH 5.2; and terminal stack of
Golgi membranes, a pH of almost 7.0 (29, 30). Whereas the pH of these
compartments is probably statically maintained at these set points, the
pH within organelles of the regulated secretory pathway is under a more
dynamic regulation. Thus, the pH of early mast cell granules is about
5.5, whereas the pH of these organelles rises to 7.0 after processing
of vesicle contents (31). The basis for these differences in pump
function is not well established, but may owe to molecular diversity of V-ATPases.
The role of the 116-kDa subunit in pump function remains to be
elucidated. It appears to be an essential component of pumps from
mammalian cells, and it is also present in pumps prepared from other
sources (32, 33). In yeast, disruption of the genes encoding the two
isoforms of this subunit resulted in conditional lethality (20, 21).
All isoforms of 116-kDa subunit from mammalian cells have a strikingly
similar structure, with a hydrophilic amino-terminal half and a
hydrophobic carboxyl-terminal half that contains 6-8 transmembrane
regions. Further analysis indicates that the mammalian isoforms share
about 50% overall identity. The predicted membrane-spanning regions
are even more conserved, with approximately 75% identity (Fig. 1),
perhaps reflecting that these sectors participate in an essential,
constitutive function such as transmembranous proton flow. The
hydrophilic domains of these isoforms share about 25% identity, but
all contain more than 30% charged residues.
An interesting aspect of 116-kDa isoforms is their differential
expression in tissues. Isoform a1 is highly enriched in
brain (16, 17), and a2 is more abundant in lung, kidney and
spleen (Fig. 2), although both of them are present in most tissues.
This may reflect organelle-specific distribution of isoforms within cells or cell-specific distribution within organs. An additional isoform, OC-116 kDa, (which we term a3) was
cloned by screening of a human osteoclastoma cDNA library and has
been claimed to be specific to human osteoclastomas (18). However, its
association with V-ATPases needs to be confirmed, and its distribution
needs further investigation. These differential expressions of 116-kDa isoforms may provide very important clues in investigations of the
subunit in pump function. A more detailed cytochemical analysis addressing this issue is under way in our laboratory.
Comparison of the sequences of the 116-kDa protein from bovine brain
(a1) with that of its homologue from lung (a2),
demonstrates the similarity of the two proteins and, at the same time,
indicates that these two proteins must have arisen from separate genes. In fact, analysis of the predicted primary structures of all 116-kDa homologues from vertebrates indicates that three separate genes encode
forms of these proteins. Supportive of this notion are the findings
that multiple separate genes encode 116-kDa isoforms in C. elegans and yeast (20, 21). At present, the reasons for this
extreme level of diversity of this subunit is unknown. However, we
speculate that this diversity is related to the differential targeting
and regulation of V pumps within eukaryotic cells.
It is, we believe, highly significant that the VO component
of the proton pump of lung is found in a high molar ratio relative to
the V1 component, as compared with the V1
VO constituents of bovine brain proton pump. It is possible
that the stability of the V1 VO complex
prepared from clathrin-coated vesicles of bovine brain reflects the
inherent stability of the complex in vivo. Thus, proton
pumps localized to the constitutive endocytoic pathway may not undergo
dissociation into separate V1 and VO domains, as has been described as an important regulatory mechanism for the V
pumps of Saccharomyces cerevisiae and Manduca
sexta. We are currently engaged in localizing the a2
isoform of lung to determine whether it is present in regulated
secretory compartments within the epithelia of this tissue. In such a
setting, it is possible that the V1 and VO
components undergo dissociation as a regulatory phenomenon and that
this ability of the pump to dissociate into its two components is
reflected at the biochemical level as an inherent instability in the
V1 VO complex.
In order to establish the association of cloned cDNA with
V-ATPases, we have purified a vacuolar proton channel (VO)
containing the a2 isoform from bovine lung. This
VO conducts proton flow after acid activation (Fig. 6) and
pumps protons across the membrane after assembly with catalytic sector
and SFD (Fig. 7), suggesting that isoform a2 is indeed part
of the V-ATPase.
A mouse form of the a2 isoform was previously characterized
molecularly in experiments designed to identify soluble immune regulatory factors (19). By structural predictions, neither the mouse
nor the bovine form of this protein is soluble, and biochemical
manipulations of the 116-kDa protein require detergents in our hands.
Although we do not exclude the possibility that the 116-kDa
a2 isoform (or indeed any pump component) may play a role
in eukaryotic cell functions beyond acidification, the experiments of
this study indicate that the a2 isoform is present in
VO fractions that can catalyze proton flow. In fact, it is possible that isoforms of the 116-kDa subunit may play a role in T cell
activation through its function as an essential component of the proton
pump. It is notable in this regard that inhibition of V pump function
in T cells by bafilomycin blocks antigen processing (34).
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
ZAP was the kind gift of Dr. Richard A. F. Dixon
(The University of Texas Health Science Center at Houston). All other
reagents were from Sigma.
ZAP phage DNA from
amplified bovine brain cDNA library was purified by standard
procedure (22) and used as a template for PCR, which was performed with
20 pmol of each primer and 1 µg of purified
ZAP DNA. PCR products
of 0.3 kb were cloned into pCR 2.1 vector using a TA cloning kit from
Invitrogen. The positive colonies from TA cloning were analyzed by DNA
sequencing, and a 0.3-kb insert was excised with EcoRI
digestion, purified by preparative agarose gel electrophoresis, and
used to screen a bovine brain cDNA library.
ZAP (insert size, >2.0 kb), transfected into
E. coli, XLI-Blue-MRF', was screened with the 0.3-kb PCR
product that had been labeled with [
-32P]dCTP by nick
translation. Screening of 2 × 106 individual phages
was performed using a double-lift procedure wherein plaques were
transferred to the nitrocellulose membranes for 5 min in each lift. The
membranes were prehybridized for at least 4 h at 60 °C in a
solution containing 5× SSC, 5× Denhardt's solution, 0.1 mg/ml of
sheared salmon sperm DNA, and 0.1% SDS. Hybridization was performed at
50 °C overnight with the same solution plus labeled probe, which was
added at a concentration of 5-10 × 105 cpm/ml of
hybridization solution. The membranes were then washed for 15 min at
room temperature first with 2× SSC and 0.1% SDS, then with 0.5× SSC
and 0.1% SDS, and finally for 30 min at 55 °C with 0.1× SSC and
0.1% SDS. Autoradiography was performed with an intensifying screen at
80 °C for 24-48 h. Duplicate positive clones were cored and
rescreened through one or more cycles until purified colonies were
obtained.
21), and
sequence-specific oligonucleotides as primers. DNA and protein data
base analysis was performed using PC/GENE-based programs.
-32P]dCTP by nick translation and
added to the hybridization solution at a concentration of 1 × 106 cpm/ml of solution. Hybridization was then carried out
at 50 °C overnight. The membrane was sequentially washed for 15 min at room temperature with 2× SSC and 0.1% SDS, 0.5× SSC and 0.1% SDS, and 0.1× SSC and 0.1% SDS, respectively. A final wash was carried out at 60 °C for 30 min with 0.1× SSC plus 0.1% SDS, and autoradiography was performed with an intensifying screen at
80 °C
for 3-5 days.
1 × min
1, as described (24). V1 and SFD were
prepared as reported (7, 5).
A492-540 (24, 26). Proteoliposomes containing proton
pump or VO were diluted into 1.6 ml of assay buffer,
consisting of 150 mM NaCl, 30 mM sodium-Tricine
(pH 7.5), 3 mM MgCl2, 0.5 mM EDTA,
and 6 µM acridine orange. Reactions were initiated by the
addition of ATP (final concentration, 1.3 mM) and/or
valinomycin (1 µM), and were terminated by the addition of 1.6 µg of the proton ionophore bis(hexafluoroacetonyl) acetone, as
indicated in the legends to the figures.
RESULTS
ZAP. 2 × 106
individual bacteriophages were screened, yielding six positive clones,
designated B2-2, B9-1, B17-1, B26-1, B31-1, and B34-1. DNA sequencing
demonstrated that clone B31-1 contained the full coding region, clone
B2-2 lacked coding region for 9 amino acids at the 5'-end, and the
other clones had inserts of 1.5-2.5 kb. All of the clones had
identical sequences in overlapping regions. The full sequence of clone
B31-1 includes a 2565-base pair open reading frame and untranslated
regions of 174 and 1703 base pairs at the 5'- and 3'-ends,
respectively
View larger version (105K):
[in a new window]
Fig. 1.
Alignment of isoforms of the 116-kDa subunit
and homologous sequences from mammalian cells. The predicted amino
acid sequence of bovine a2 (VBA2-BOVIN) is
compared with those of mouse J6B7 (a putative immune regulatory
factor), bovine (VBA1-BOVIN) and rat (VBA1-RAT)
isoform a1, and putative isoform from human osteoclastoma
(OC116HUMAN). Identical amino acids are designated by
asterisks, and similar amino acid residues (defined by
PC/GENE-based algorithms) are denoted by a dot. Predicted
transmembranous sectors (27) for isoform a2 are
underlined.
View larger version (64K):
[in a new window]
Fig. 2.
Northern blot analysis.
Poly(A+) RNA (2 µg) from bovine tissues was denatured and
fractionated by 1% formaldehyde-agarose gel electrophoresis and
hybridized with a 32P-labeled cDNA probe, as described
under "Experimental Procedures." Lanes 1-5 represent
poly(A+) RNA from brain, heart, kidney, lung, and spleen,
respectively.
View larger version (47K):
[in a new window]
Fig. 3.
SDS-PAGE and Western blot analysis of the
highly purified vacuolar proton ATPase from bovine brain.
Lane 1, enzyme (3 µg) was subjected to 8% SDS-PAGE
followed Coomassie Blue staining; lanes 2 and 3,
Western blot analysis utilizing a1- and
a2-specific anti-peptide antibodies, respectively.
the specific activity of the vesicles from bovine
brains, as assessed by ATP generated acridine orange quenching. To
determine whether this relatively low activity was due to an intrinsic
proteolysis of the pump from lung, we tested several different
solutions for microsomal preparation. These included variances in pH
from 6.5 to 7.5 and inclusion of proteinase inhibitors. The same
results, however, were obtained. Moreover,
C12E9, the detergent routinely utilized for the
solubilization of the V pump of clathrin-coated vesicles, was
ineffective in solubilizing bafilomycin-sensitive ATPase activity from
lung microsomes. Numerous detergents were tested and we ultimately
determined that the V-ATPase of lung was optimally solubilized with
Zwittergent 3-16. Subsequently, purification of intact V pump was
attempted by our standard protocol, which includes hydroxylapatite
chromatography, and glycerol gradient centrifugation. Repeated attempts
at purification, however, resulted in minuscule amounts of pump that
migrated to the usual position in glycerol gradient centrifugation
(data not shown). Instead, Western blot analysis using an
anti-a2 isoform antibody revealed that the a2
isoform was present at roughly the midpoint of the 15-30% glycerol
gradients, where isolated VO is typically found. As shown
in Fig. 4, Western blot analysis
demonstrated that isoform a2 and the 39-kDa-subunit (28),
an identified component of VO, were located in the same
fractions. These fractions containing the 116-kDa subunit reacted
weakly with the isoform a1-specific antibody (Fig. 4),
indicating that isoform a2 is the major form of 116-kDa
subunit in the bovine lung. In addition, none of the glycerol gradient
fractions reacted with an antibody against the 70-kDa subunit, a
component of V1 of vacuolar proton ATPases. The peak
fractions were combined, concentrated with a Millipore Ultrafree-MC
centrifugal filter unit, and further purified by a second glycerol
gradient centrifugation step. After centrifugation, relatively pure
(and active) VO was obtained, as shown in Fig. 5, lane 2. The purified
VO contained at least three polypeptides with molecular
masses of 116, 39, and 17 kDa. Also observed was a polypeptide with a
molecular mass of about 140 kDa. Whether this is a genuine
VO component or a contaminant will require additional study. To date, we have been unable to achieve a higher degree of
purification.
View larger version (43K):
[in a new window]
Fig. 4.
Western blot analysis of partial purified
proton channel (VO) from bovine lung. The fractions
from glycerol gradient centrifugation were separated by 10% SDS-PAGE
and transferred to nitrocellulose membranes for analysis. Lanes
1-12 are fractions from the bottom (lane 1)
through the top (lane 12) of the gradient;
P is purified vacuolar ATPase from bovine brain.
Panels A-D illustrate illustrate Western blots performed
with different antibodies: A, a2-specific
antibody; B, a1-specific antibody; C,
antibody against recombinant 39-kDa subunits; D, anti-70-kDa
antibody (10).
View larger version (36K):
[in a new window]
Fig. 5.
SDS-PAGE of purified, active VO
from lung and V-ATPase holoenzyme from brain. SDS-PAGE was
performed using 12.5% polyacrylamide and stained by Coomassie
Brilliant Blue; lane 1, 3 µg of V-ATPase; lane
2, 1 µg of VO.
View larger version (13K):
[in a new window]
Fig. 6.
Proton conductance catalyzed by
acid-activated, reconstituted proton channel (VO) from
bovine lung. The assays were performed as described under
"Experimental Procedures." Trace 1, no acid activation
with 1.25 mM ATP; trace 2, no acid activation
without ATP; trace 3, acid activation in the presence of
10 9 M bafilomycin; and trace 4,
acid activation only.
View larger version (11K):
[in a new window]
Fig. 7.
Assembly of proton pumping activity using
isolated catalytic sector (V1), proton channel
(VO), and SFD. The enzyme was assembled and
reconstituted as described under "Experimental Procedures." Proton
pumping was measured by acridine orange absorbance quenching. Each
reaction contained 3 pmol of V1 and/or VO and 3 pmol of SFD. Reactions were initiated by addition of 1.25 mM ATP, and 1 µM valinomycin (Val)
and were terminated by addition of 2 µl of 1 mM
bis(hexafluoroacetonyl) acetone (1799). Trace 1,
V1 plus SFD; trace 2, VO plus SFD;
trace 3, V1, VO, SFD, and 3 nM bafilomycin; and trace 4, V1,
VO, and SFD.
DISCUSSION
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant DK-33627.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF105016.
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.
The abbreviations used are: V-ATPase, vacuolar-type proton translocating ATPase; C12E9, polyoxyethylene 9-lauryl ether; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; SFD, sub-58-kDa doublet (50- and 57-kDa polypeptides required for function of V-ATPase); PCR, polymerase chain reaction; kb, kilobase.
2 J. Mattsson, X. Li, S. B. Peng, P. Andersen, B. Crider, L. Lundberg, D. K. Stone, and D. Keeling, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|