(Received for publication, September 29, 1995; and in revised form, December 5, 1995)
From the
The clathrin-coated vesicle H-ATPase is
composed of a peripheral catalytic sector (V
) and an
integral membrane proton channel (V
), both of which are
multiple subunit complexes. This study was conducted to determine if
subunit F, previously identified in vacuolar proton pumps of tobacco
hornworm and yeast, was present in mammalian pumps. Using a polymerase
chain reaction-based strategy, we have isolated and sequenced cDNA
clones from bovine and rat brain cDNA libraries. A full-length clone
from rat brain encodes a 119-amino acid polypeptide with a predicted
molecular mass of 13,370 Da and with approximately 72 and 49% identity
to subunit F of tobacco hornworm and yeast, respectively. Southern and
Northern blot analyses indicate that the protein is encoded by a single
gene. An anti-peptide antibody, directed against deduced protein
sequence, was affinity-purified and shown to react with a 14-kDa
polypeptide that is present in a highly purified pump prepared from
clathrin-coated vesicles and also isolated V
. When stripped
clathrin-coated vacuolars and purified chromaffin granule membranes
were treated with KI in the presence of ATP, the 14-kDa subunit was
released from both membranes, further indicating that it is part of the
peripheral catalytic sector. In addition, direct sequencing of this
14-kDa component of the coated vacuolar proton pump confirmed its
identity as a subunit F homologue.
Vacuolar proton pumps are responsible for the acidification of numerous cellular compartments, including clathrin-coated vesicles, endosomes, lysosomes, and Golgi membranes. In addition, these pumps acidify cellular vacuolars of the regulated secretory pathway, where they are instrumental to the packaging and processing of the contents of synaptic vesicles, mast cell granules, and insulin granules of pancreatic islet beta cells. Global loss of vacuolar pump function confers a conditionally lethal phenotype in Chinese hamster ovary and Saccharomyces cerevisiae cells, while organ-specific loss of pump activity can result in renal tubular acidosis. In contrast, relative pump over-activity is likely causal in the pathogenesis of postmenopausal osteoporosis(1, 2, 3, 4, 5, 6) .
Despite this diversity in distribution and function, there is
remarkable phylogenetic conservation of both the quaternary and primary
structures of vacuolar-type pumps. For example, the vacuolar-type pump
of clathrin-coated vesicles has a multisubunit, peripheral ATP
hydrolytic sector (7, 8) that is also identifiable in
vacuolar pumps of archaebacteria(9) , yeast(10) , and Neurospora(11) . Similar conservation exists in the
composition of the transmembranous proton channel of vacuolar-type
pumps. This sector of the clathrin-coated vacuolar proton pump is
composed of at least three subunits with molecular masses of 116, 39,
and 17 kDa and is the site of inhibition by the potent, specific
inhibitor bafilomycin A, according its designation of
V
, (
)for V
(12) .
Definition of the functional catalytic sector (V) of the
clathrin-coated vesicle pump has been an ongoing project in our
laboratory(8, 13, 14, 15, 16) .
Our strategy has been to reconstitute ATP hydrolysis from dissociated
pump components and, in so doing, both to identify genuine subunits of
the enzyme and to map their role in pump function. This has required
the cloning of candidate subunits, the large-scale production of
recombinant proteins, and the reassembly of these proteins to
biochemically prepared subcomplexes that lack the appropriate subunit
and, consequently, ATPase activity. By this approach, we have found
that ATPase activity requires four subunits of 70, 58, 40, and 33 kDa,
commonly designated subunits A, B, C, and E, respectively.
Close
examination of preparations of active V reveals several
additional proteins with molecular masses in the range of 10-15
kDa(17) . Recently, a subunit of similar mass has been
identified in vacuolar-type pumps of tobacco hornworm (18) and
yeast (19) . In these systems, evidence has been presented that
this component (subunit F) is required for pump function and that it
may serve to link the catalytic sector to the proton channel. However,
it has also been shown that antibodies directed against this 14-kDa
subunit of tobacco hornworm do not cross-react with any component of
the clathrin-coated vesicle proton pump(18) . We now report the
cloning and sequencing of a cDNA encoding a 14-kDa polypeptide of the
clathrin-coated vesicle H
-ATPase and demonstrate that
it is the mammalian homologue of subunit F of tobacco hornworm and
yeast.
Inserts from positive clones were excised and
subcloned into pBluescript with helper phage R408. Plasmid DNA was
prepared by alkaline lysis, and DNA sequencing was carried out by the
dideoxy termination method (21) using double-stranded and/or
single-stranded DNA as a template. Single-stranded DNA was recovered
from pBluescript in the presence of helper phage VCSM13. The cDNA
clones were fully sequenced in both orientations using T and T
promoter sequences and sequence-specific
oligonucleotides as primers. DNA and protein data base searches were
performed using PC/GENE-based programs.
Figure 1: Sequence of the 14-kDa rat brain cDNA clone and its deduced amino acid sequence. The nucleotide sequence of cDNA clone E-1 was determined by sequencing the full-length clone, in both directions, by the dideoxy termination method (21) as described under ``Experimental Procedures.'' The amino acid sequence obtained from direct peptide sequencing is underlined.
Figure 2: Sequence of a cDNA clone encoding the bovine brain 14-kDa component and its deduced amino acid sequence. The nucleotide sequence of cDNA clone VIII-2 was confirmed by sequencing the clone, in both directions, by the dideoxy termination method (21) as described under ``Experimental Procedures.''
Figure 4:
Northern blot analysis. Bovine poly(A) RNA (A) and rat total RNA (B) were hybridized with P-labeled 14-kDa cDNA as described under
``Experimental Procedures.'' Lanes 1-3, brain,
heart, and kidney, respectively; lane 4 (A), spleen; lane 4 (B), liver; lane 5, lung. Kb, kilobase.
In the second method, bovine brain clathrin-coated vacuolars, stripped of clathrin, and bovine chromaffin granule membranes (generous gift of Dr. David Apps, University of Edinburgh) were suspended in 50 mM NaCl, 30 mM KCl, 20 mM HEPES (pH 7.0), 0.2 mM EDTA, and 5 mM ATP and incubated for 30 min on ice. Subsequently, 300 mM KI (final concentration) was added to the mixtures, and after a 1-h incubation on ice, the mixtures were centrifuged for 1 h at 45,000 rpm in a Beckman Ti-60 rotor. Supernatants containing inactive peripheral components were concentrated by trichloroacetic acid precipitation and analyzed by SDS-PAGE (23) and Western blotting.
Four partial
cDNA clones were obtained from the bovine cDNA library. The longest,
VIII-1, consists of 603 base pairs, which contain a
poly(A) end and an incomplete 5`-end (Fig. 2).
Bovine and rat DNA sequences are identical at 92% of the bases within
available coding regions.
Figure 3: Alignment of 14-kDa subunit sequences. The predicted amino acid sequences of the rat (VATF RAT) and bovine (VATF BOVIN) 14-kDa subunits are compared with those of tobacco hornworm (VATF MANSE), D. melanogaster (VATF DROME), C. elegans (VATF CAEEL), and yeast (VATF YEAST). Identical amino acids are designated by asterisks, and ``similar'' amino acid residues (defined by PC/GENE-based algorithms) are denoted by periods.
Figure 5:
Southern blot analysis of bovine genomic
DNA. Genomic DNA, isolated from bovine brain, was digested with the
restriction enzymes indicated below and separated on a 1% agarose gel.
The digests were transferred to a Zeta-Probe membrane and hybridized
with P-labeled 14-kDa cDNA as described under
``Experimental Procedures.'' Restriction enzymes used for lanes 1-7 were BamHI, XbaI, BglII, XboI, ApaI, SmaI, and EcoRI, respectively. Kb,
kilobase.
Figure 6:
Western blot analysis of the
clathrin-coated vesicle proton pump. SDS-PAGE (A) and Western
blot (B) analyses of the purified proton pump (lane
1), purified V (lane 2), and purified V
(lane 3) were performed as described under
``Experimental Procedures'' using purified IgG directed
against the 14-kDa polypeptide.
Figure 7: Western blot analysis of the clathrin-coated vesicles (A) and chromaffin granule membranes (B) before and after dissociation of peripheral pump components by treatment with KI and ATP. Antibodies directed against the 70-, 39-, and 14-kDa subunits were used for immunoblotting as indicated. Lane 1, purified proton pump; lane 2, supernatants of vesicles incubated with KI and ATP; lane 3, control supernatants of vesicles incubated without KI and ATP as described under ``Experimental Procedures.''
In the decade since a vacuolar-type proton pump was first
isolated and reconstituted(7) , considerable efforts have been
directed toward defining the composition of these pumps as well as
understanding the role of defined subunits in pump function.
Investigations of these issues by the approach of resolution and
reconstitution have led to the identification of two general sectors: a
proton channel, V(12) , and a catalytic domain,
V
(8) . Both of these sectors, when separated from
one another, have activities that are probably latent under physiologic
conditions, and this is likely of considerable importance.
V
, when purified and reconstituted, cannot conduct protons
until it is activated by acidity (12) . In a cellular context,
this property may be essential to preservation of organellar pH
gradients during the biogenesis (or regulation) of vacuolar-type pumps;
specifically, a closed proton channel would prevent rapid proton leaks
from acidic compartments. Likewise, the subunits responsible for ATP
hydrolysis undergo a marked transition when released from V
by select procedures. Namely, the isolated, functional,
catalytic sector, termed V
, can no longer hydrolyze MgATP,
and it hydrolyzes CaATP only in the presence of millimolar
concentrations of calcium. This property potentially prevents idle
hydrolysis of ATP when the catalytic sector is not
membrane-associated(8, 17) .
We have utilized the
partial reactions catalyzed by isolated V and V
to define the components of each of these sectors and thereby the
structure and function of the holoenzyme. In a series of studies,
biochemically prepared V
was selectively depleted of
individual polypeptides, and these subunit-depleted V
preparations were assessed for ATPase activity before and after
readdition of the missing component. To assure purity of the latter,
each of four subunits was cloned, expressed, purified, and renatured.
Collectively, these experiments demonstrated that all four polypeptides
of 70, 58, 40, and 33 kDa are subunits of V
, and each is
required for Ca-ATPase activity (13, 14, 15, 16) . Attempts to
reassemble Ca-ATPase activity solely from these four recombinant
subunits, however, have not been successful. (
)As all of
these subunits were shown to be active by reconstitution to
subunit-depleted complexes, it appears that another component(s) is
required for catalytic activity.
Potential candidates for such
function(s) are several small polypeptides with molecular masses in the
range of 10-15 kDa that are present in biochemical preparations
of both the holoenzyme and V (Fig. 6A, lane 2). Close inspection of these components reveals the
presence of three distinctive polypeptides within this mass range.
Collectively, the experiments of this study identify one of these
polypeptides as subunit F, thus demonstrating for the first time the
presence of this component in vacuolar-type proton pumps of mammalian
organelles. It is likely that the previous failure to identify this
component by immunoblot analysis (18) owed to differences in
the primary structures of subunit F of bovine and hornworm
vacuolar-type pumps.
It remains to be determined what role this
polypeptide plays in overall pump function. Studies conducted with the
vacuolar pumps of tobacco hornworm and yeast indicate that subunit F
may structurally couple the ATP hydrolytic sector to the proton
channel(18, 19) . Whether this entails an involvement
in ATP hydrolysis per se remains to be determined, although
inhibitory antibodies and gene knockout experiments indicate an
essential role for subunit F in the net reaction of ATP-driven proton
flow. Current experiments are directed toward the identification and
characterization of the remaining two small polypeptides in V and toward ultimately defining the roles of these components and
subunit F in overall pump function.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U43175 [GenBank]and U43176[GenBank].