From the Zentrum der Biologischen Chemie,
Universitätsklinikum Frankfurt, D-60590 Frankfurt, Germany and
the § Department of Biochemistry, University of Edinburgh,
Edinburgh EH8/9XD, United Kingdom
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vacuolar proton-translocating ATPase (holoATPase and free membrane sector) was isolated from bovine chromaffin granules by blue native polyacrylamide gel electrophoresis. A 5-fold excess of membrane sector over holoenzyme was determined in isolated chromaffin granule membranes. M9.2, a novel extremely hydrophobic 9.2-kDa protein comprising 80 amino acids, was detected in the membrane sector. It shows sequence and structural similarity to Vma21p, a yeast protein required for assembly of vacuolar ATPase. A second membrane sector-associated protein (M8-9) was identified and characterized by amino-terminal protein sequencing.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proton-translocating adenosine triphosphatases have fundamental roles in energy conservation, secondary active transport, the acidification of intracellular compartments, and cellular pH homeostasis. They fall into three broad classes, called F, P, and V (1), of which the vacuolar type (V-ATPases)1 is both the most recently recognized and the least well characterized. ATPases of this class occur in endomembranes bounding the acidic compartments of animal, plant, and fungal cells (2) and also in the plasma membranes of some specialized cell types. They have been purified from several mammalian sources, including adrenal secretory vesicles (3, 4), brain clathrin-coated vesicles, (5, 6), and kidney medulla microsomes (7), as well as from the vacuoles of fungi and higher plants. Most V-ATPases contain some 6-10 different subunits (2), but subunit composition depends on the source of the enzyme, and tissue-specific isoforms exist (8). The V-type ATPases are structurally similar to those of the F-type, having a transmembrane proton-conducting sector and an extramembrane catalytic sector. By analogy with the two sectors of F-ATPases (9-12), these are termed V0 and V1, respectively. For a recent review, see Ref. 13.
In this work, the recently developed technique of blue native polyacrylamide gel electrophoresis (BN-PAGE; Refs. 14-17) was employed to purify vacuolar ATPase holoenzyme (V1V0) and free membrane sector (V0) simultaneously from adrenal secretory vesicle membranes. Combined with high resolution Tricine-SDS-PAGE in the second dimension, the subunit composition, particularly with respect to small polypeptides, was determined. Two novel proteins, 8-9 and 9.2 kDa in size, were found in the membrane sector. Here we report the detailed analysis of the larger of these two polypeptides.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
Restriction enzymes and T4-DNA ligase were
obtained from New England Biolabs. Taq DNA polymerase was
from Stratagene, and TA Cloning Kit® was from Invitrogen.
Sequenase version 2.0 sequencing kit, [-35S]dATP and
Hybond N+ membranes were obtained from Amersham Pharmacia
Biotech. ABI PrismTM dye terminator cycle sequencing kit was purchased
from Perkin Elmer. The cDNA library from bovine adrenal medulla was a kind gift from Leonora Ciufo (University of Edinburgh, Edinburgh, Scotland, United Kingdom). Human EST clone ID 143553 (GenBankTMaccession number R75754) was obtained from the IMAGE
Consortium (18). Bovine tissues were frozen in liquid nitrogen several
minutes after the death of the animal. The probe against human
glyceraldehyde-3-phosphate dehydrogenase was a kind gift from J. Altschmied (Physiologische Chemie I, Universität,
Würzburg). Anti-subunit G1 antibody was kindly
provided by Bill P. Crider (University of Texas Southwestern Medical
Center, Dallas).
Isolation of V0 and V1V0 ATPase from Chromaffin Granule Membranes by BN-PAGE-- Chromaffin granule membranes were prepared according to Apps et al. (19). The membranes (11 mg of protein in 1.5 ml of 10 mM Hepes/NaOH, pH 7.4) were solubilized at 4 °C by addition of 1 ml of 1.75 M 6-aminohexanoic acid, 50 mM BisTris-Cl, pH 7.0, and 500 µl of 10% dodecyl maltoside. After 30 min centrifugation at 100,000 × g, 200 µl of 5% Serva Blue G in 500 mM 6-aminohexanoic acid was added to the supernatant. One ml of supernatant was loaded onto each of three 3-mm-thick preparative 5-13% acrylamide gradient gels for BN-PAGE (14). After BN-PAGE, the blue bands were excised and the native complexes electroeluted. About 300 µg of V1V0 complex and 900 µg of V0 membrane sector were recovered from 11 mg of membrane protein.
Isolation of V0 and V1V0 ATPase from a Triton X-114 Extract-- Triton X-114-extraction was used for enrichment of V0 and V1V0-ATPase (20). Chromaffin granule membranes (1 mg protein in 0.17 ml of 10 mM Hepes/NaOH, pH 7.4) were centrifuged for 30 min at 100,000 × g. The pellet was resuspended in 0.2 ml 150 mM KCl, 10 mM Tris, pH 7.5, and solubilized by addition of 50 µl 10% (w/v) Triton X-114. V0 and V1V0 were precipitated by a 15-min incubation on ice. After a 10-min centrifugation at 100,000 × g, the pellet was washed with 0.5 ml of 10 mM Na+/Mops, pH 7.2, and centrifuged as before. The pelleted proteins were solubilized by addition of 90 µl 1 M 6-aminohexanoic acid, 50 mM BisTris/HCl, pH 7.0, and 21 µl of 10% dodecyl maltoside, and centrifuged again for 10 min at 100,000 × g. After addition of 10 µl of 5% Serva Blue G in 500 mM 6-aminohexanoic acid, 50 µl were applied to 10-mm gel wells for analytical BN-PAGE. After blue native electrophoresis, individual lanes were cut from the gel and processed in a second dimension by Tricine-SDS-PAGE. Electrophoretic techniques, staining techniques, and densitometric quantification followed the protocols described previously (17, 21).
Partial Protein Sequencing-- V0 and V1V0 complexes were electroeluted from blue native gels, resolved by Tricine-SDS-PAGE, and electroblotted onto polyvinylidene difluoride (PVDF) membranes. Individual bands were sequenced directly using a 473A protein sequencer (Applied Biosystems), or after various chemical treatments, e.g. cyanogen bromide cleavage, partial acidolysis by 80% formic acid (24 h at 37 °C), incubation in a 1:1 (v/v) mixture of trifluoroacetic acid and methanol (16 h at 37 °C) for partial deacylation (22), cleavage between asparagine and glycine (23) by 3 M hydroxylamine, pH 9.6 (7 h at 37 °C), or cleavage at tryptophan (24) by 0.7% iodosobenzoic acid dissolved in 80% acetic acid (24 h at room temperature). For searching genomic data bases with amino acid query sequences, the TFASTA computer program of the Husar package of the German Cancer Research Center (Heidelberg, Germany) was used. Protein secondary structures were calculated using the ANTHEPROT program (25-28).
Screening of a Bovine cDNA Library by PCR-- The NH2-terminal amino acid sequences of bovine M9.2 and the sequences of two corresponding human cDNA clones, IMAGE consortium clone 143553 (GenBankTM accession number R75754; Ref. 18) and murine MM85D12 (GenBankTM accession number D21772; Ref. 29) were used to deduce a pair of degenerate primers for PCR with the plasmid DNA of the whole bovine adrenal medulla cDNA library: VATPB9.2c, 5'-AT(C/T) GTG ATG AGC GTG TTC TGG GG-3'; and VATPB9.2n, 5'-GCC AAA AIA GAT AGC AGC AIA C-3'. PCR was performed in 50 mM KCl, 1.5 mM MgCl2, 0.1% gelatin, 200 µM of each dNTP, 0.5 µM of each primer, 10 mM Tris/HCl, pH 8.8. The temperature cycle was as follows: 94 °C for 1 min, 42 °C for 1 min, 70 °C for 30 s for 30 cycles and a single step of 72 °C for 10 min. The PCR product was cloned into the pCR II vector (Invitrogen) and sequenced. A third perfect match primer was deduced from this sequence: VATPB9.2a, 5'-GGG GCA TCG TCG GCT TCC TGG TGC-3'. The bovine cDNA library was then screened by PCR using the combination of primers VATPB9.2a and VATPB9.2n and the same temperature profile. Six pools, comprising a total of 1500 colonies, were taken as the template for PCR, and examined for the occurrence of a 106-bp PCR product. The positive pool was divided into subpools, and the procedure was repeated until a single clone (BVATPM9.2) was obtained.
Sequencing of Human and Bovine Clones--
The insert from clone
BVATPM9.2 was cut out with BamHI and cloned into
pBluescriptTM II SK(). The new clone pBBM9.2 was subcloned by using the BstEII site at nt 69 and the XbaI
site at nt 246. The insert of human cDNA clone 143553 was cut out
with EcoRI and HindIII and cloned into
pBluescriptTM II SK(
). Clones pBBM9.2 and pBHM9.2 were
sequenced in both directions.
RNA Isolation and Northern Blotting--
Total RNA was prepared
according to the method of Chomczynski and Sacchi (30). RNA was
separated by formaldehyde agarose gel electrophoresis using 5 mM sodium acetate and 0.1 mM EDTA in running
and loading buffers, capillary-blotted on Hybond N+
membranes (31), and fixed by UV irradiation. DNA probes were labeled
with [-32P]dCTP by random priming, and
QuikHyb® solution from Stratagene was used for
hybridization. A 900-bp cDNA was excised from pBBM9.2 with
BamHI and used as a probe for M9.2. The probe against bovine
V1V0-ATPase subunit c (proteolipid c),
GenBankTM accession number J03835 (32), was made by PCR using primers
BVch 5'-TCA GCC GCC ATG GTC TTC AG -3' and BVcr 5'-CGG CGA AGA TGA GGA
TGA GG-3'. Using the bovine adrenal medulla cDNA library as a
template, a 358-bp fragment corresponding to positions 190-547 of
V-ATPase proteolipid c was amplified by PCR, tested by restriction
analysis, and cloned into pCR 2.1 (Invitrogen).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Characterization of V-ATPase by BN-PAGE and Two-dimensional Electrophoresis-- Analytical BN-PAGE (Fig. 1) was used for separation of V1V0 holocomplex and free V0-membrane sector from solubilized chromaffin granule membranes (lane M), and from a fraction prepurified by Triton X-114 extraction/precipitation (lane P). The oxidative phosphorylation complexes from solubilized bovine heart mitochondria served as molecular mass standards (17, 33, 34). The molecular masses assigned to complex I and complex V are minimal values, inasmuch as the copy number of some subunits is not exactly known (35, 36). The prominent band with an apparent mass around 440 kDa was identified as V0-membrane sector by the characteristic polypeptide patterns in two-dimensional electrophoresis, and by amino-terminal protein sequencing (see below). A faint protein band with an apparent mass of about 1000 kDa was identified as holo V1V0-ATPase. The position of free V1 sector is also indicated in Fig. 1, although the amounts were too low for detection in BN-PAGE (see below).
|
|
Identification of Protein Subunits of V-ATPase and Membrane Sector-- Analysis of polypeptide composition by SDS-PAGE was performed directly from lanes of BN-PAGE (Fig. 2) and after electroelution of the complexes from blue native gels (Fig. 3).
|
|
Amino-terminal Protein Sequences-- The complexes resolved by preparative BN-PAGE were electroeluted, and the protein subunits resolved by Tricine-SDS-PAGE and electroblotted onto PVDF membranes for direct amino-terminal protein sequencing (Fig. 4). Only a few of the proteins had free amino termini accessible to Edman degradation. Among these proteins were the major bovine brain subunit B, identified by the sequence MRGIVNGAAPELPV (39, 40); M45, also called glycoprotein IV or Ac45 protein (41, 42); and proteolipid c (32). However, more than 90% of proteolipid c appeared to be amino-terminally blocked, because the signal intensities of phenylthiohydantoin amino acids from cyanogen bromide fragments were up to 10-fold higher than after direct sequencing. The novel M9.2 and M8-9 proteins were also directly accessible to Edman degradation (cf. Table II). The amino-terminal sequences obtained from the four bands of the M8-9 protein (Fig. 3) suggested that M8-9 might be present in a "full length" form (largest band 1) and three amino-terminally shortened forms (smaller bands 2-4).
|
|
Primary Structure and Properties of the M9.2 Protein-- TFASTA computer searching using the amino-terminal protein sequence of the bovine M9.2 protein revealed a high degree of homology to several human as well as one murine cDNA clone. The function of these proteins was not known. The M9.2 cDNA from one of the human cDNA clones, IMAGE Consortium Clone 143553 (GenBank accession no. R75754), was sequenced (Fig. 5). The sequence around the initiator codon matches exactly the optimal sequence for initiation by eukaryotic ribosomes ACCATGG as described by Kozak (52). The sequenced M9.2 cDNA clone from a bovine adrenal medulla cDNA library was incomplete (Fig. 5). However, the full bovine M9.2 protein sequence, except the amino acids at positions 4, 14, and 17, was obtained by Edman degradation (Fig. 4A), which also showed that the amino-terminal methionine residue was processed in the mature protein. The almost perfect conservation between human and murine proteins, which differed only at position 22, strongly suggests that the three unidentified residues may be conserved in the bovine protein as well. In this case, the bovine protein would be completely identical to the human protein.
|
|
Tissue Distribution of M9.2 mRNA--
A Northern blot using
RNA from various bovine tissues was hybridized with a
32P-labeled 900-bp cDNA probe against bovine M9.2,
which was excised from pBBM9.2 with BamHI. A 900-bp
transcript was present in all tissues, but in low concentrations in
skeletal muscle, heart muscle, and cortex (Fig.
7A). The same blot was
rehybridized with a probe against human glyceraldehyde-3-phosphate
dehydrogenase, which is present in every tissue (Fig. 7B),
and with a probe against the bovine V1V0-ATPase
proteolipid c (Fig. 7C). Comparable M9.2/proteolipid c
signal ratios were observed in most tissues, including skeletal and
heart muscle with weak hybridization signals, but not in brain. The
proteolipid c signal in brain was strong, whereas the M9.2 signal was
hardly detectable. Quantification indicated an approximately 100-fold
lower M9.2/proteolipid c signal ratio in brain.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two-dimensional electrophoresis (BN-PAGE/Tricine-SDS-PAGE) was
used to identify the proteins associated with
V1V0 holocomplex and V0 membrane
sector. The novel M9.2 and M8-9 proteins were identified as proteins
associated with the V0 membrane sector for the following
reasons. (i) Because BN-PAGE separates membrane proteins according to
their molecular masses (17), contaminants of
V1V0 and V0 complexes should also
be multiprotein complexes or oligomeric forms of smaller complexes.
However, in the 29-115-kDa range, the only proteins detectable have
already been identified in different V-ATPase preparations (3-6),
which makes the presence of significant amounts of contaminating
protein complexes unlikely. (ii) The M9.2 and M8-9 proteins were found
both in the membrane sector and in the holoenzyme. Their staining
intensities relative to the M39 subunit were almost identical in the
membrane sector and in the holoenzyme (Table I). (iii) Proteins that
precipitate during BN-PAGE could contaminate the V-ATPase complexes.
However, in two-dimensional gels, these contaminants would appear as
smearing bands crossing the polypeptide columns of the complexes as was found with dopamine--monooxygenase. This is not the case for the
M9.2 and M8-9 proteins, as they are found only as discrete spots at the
positions of the V0 and V1V0
complexes.
The assignment of subunit G to V0 or V1 is still a matter of debate. A protein homologous to subunit G was first discovered as a component of the yeast V-ATPase, encoded by the VMA10 gene (57). It was named M16, and was suggested to belong to V0 on the basis of its sequence homology with subunit b of F-ATPase, from the characteristics of VMA10 knockouts, and from cold inactivation of the V-ATPase, which failed to release it from the membrane. Similar results were obtained on cold inactivation of the chromaffin granule V-ATPase (49); however, Tomashek et al. (58) have shown recently that Vma10p interacts with subunit E and classified it as a stalk subunit, belonging to V1. Subunit G in the midgut V-ATPase of Manduca sexta could be released from the membrane by cold inactivation or by treatment with chaotropic anions (59). Cold inactivation studies suggested also that subunits G and H from bovine brain clathrin-coated vesicles (60), which were later shown to be isoforms and renamed G1 and G2, belong to V1 rather than to V0 (48). In the present work, we could identify subunit G in the holo-V-ATPase, but not in the V0 membrane sector, by using an anti-G1 antibody (Western blot not shown). This direct approach again suggests that subunit G is a V1 component.
The electrophoretic separation of the holoenzyme (V1V0) from its subcomplexes (V1 and V0) allowed the determination of the molar ratio of the various species. We found a V0/V1V0 ratio of 5 after solubilization of chromaffin-granule membranes and resolution by BN-PAGE. It is hard to exclude the loss of V1 subcomplexes during membrane isolation, particularly as dissociation of V1V0 is promoted by MgATP at low temperatures. Nevertheless, we consider this unlikely for the following reasons: 1) 2 mM EDTA was included in all buffers during membrane isolation; 2) release of subunit B, a component of V1, was not detectable by immune blotting of soluble fractions obtained during membrane isolation.
A large excess of V0 over V1V0 has been reported before in chromaffin granule membranes (61), although in this case the ratio was determined after a prepurification step, which may have selected for the membrane sector. There have, however, been several other reports of the occurrence of free V0 and V1: After solubilization of stripped bovine brain clathrin-coated vesicles with the nonionic detergent C12E9, a V0/V1V0 ratio of about 2 was found by glycerol-gradient velocity centrifugation (62), and free V1 was detected in cytosol from bovine brain and from Madin-Darby bovine kidney cells (63). Convincing evidence for the regulation of V-ATPase activity by the reversible dissociation of V1V0 has been presented. This occurs in the vacuoles of S. cerevisiae in response to glucose deprivation (64), and in goblet cell apical membranes of M. sexta during moulting or starving of the larvae (65, 66). Whether reversible dissociation of V1V0 might also have a regulatory role in chromaffin cells, or whether V0 itself might have an independent function, for example in exocytosis, is still a matter for speculation (67, 68). It is noteworthy that in synaptic vesicles V0 appears to exist in a complex with the vesicle membrane proteins synaptobrevin and synaptophysin (69).
Coomassie staining intensities of V0-subunits (Table I) did not indicate a high copy number for any V0 protein except for proteolipid c. Assuming 1:1 stoichiometries for all V0 proteins except six copies of proteolipid c as determined by Arai et al. (70), and neglecting the extent of glycosylation, a total mass of 288 kDa was calculated from the masses of the proteins listed in Table I. It was impossible to assign a monomeric or dimeric state to the major band of the V0 membrane sector, because it had an apparent mass of 440 kDa in BN-PAGE, which was between the calculated masses of a monomeric (288 kDa) and a dimeric state (576 kDa). There are no data at present on the effects of protein glycosylation on the apparent masses in BN-PAGE. However, we speculate that the major V0 form was the monomeric form, because glycosylation of M115 and M45 subunits should increase the Stokes radius and the apparent mass.
The holoenzyme seemed to be present in monomeric form, inasmuch as the calculated mass of 815 kDa was close to the apparent mass of around 1000 kDa in BN-PAGE, assuming 3 copies each of subunits A and B (11, 70). Furthermore, 3 copies of subunit G were assumed for calculation, because the normalized staining intensity of subunit G was about 3-4 times higher than that of M39 (cf. Table I).
In the mammalian M9.2 protein a CSVCC sequence resembles potential metal-binding motifs (54), but only a cysteine doublet is retained at corresponding positions in the C. elegans and D. melanogaster sequences. If the C. elegans and D. melanogaster sequences were equivalent to the mammalian sequences, this would argue against the presence of a functional metal binding site.
The sequence similarity of human M9.2 and the yeast Vma21 proteins is not very high (45% similarity, 19% identity), but corresponding proteins of yeast and mammalian origin can have low sequence similarity, as shown by comparison of the 6.4-kDa protein of bovine bc1 complex and the homologous yeast 8.5-kDa protein (71). The sequence and structural similarities of M9.2 and Vma21p indicate that the two proteins are potential homologues, and that assembly of mammalian V-ATPase might follow a pathway similar to that of the yeast V-ATPase. However, yeast Vma21p, which is required for assembly of V-ATPase, is not a subunit of V-ATPase, but instead localizes to the endoplasmatic reticulum membrane (56), whereas M9.2 protein was found to be associated with V0 and V1V0 complexes in adrenal glands. Because antibodies against M9.2 are not yet available, we cannot exclude that M9.2 additionally or mainly localizes to the endoplasmic reticulum membrane. It seems conceivable that the mammalian protein is integrated into the complex after exerting its function in assembly, whereas yeast Vma21p is not.
M9.2 mRNA was detected in all tissues, but the M9.2/proteolipid c transcript level was about 100-fold lower in brain than in other tissues. This tissue-specific variation is not yet understood, but could indicate altered translational control, or decreased M9.2 protein degradation in brain. Alternatively, one could speculate that an undetected brain-specific analogue of M9.2 exists.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank J. Altschmied for the human glyceraldehyde-3-phosphate dehydrogenase clone, Leonora Ciufo for the cDNA-library of bovine adrenal medulla, Bill P. Crider for the kind gift of anti-subunit G1 serum, and Thomas A. Link for helpful advice with structural analyses.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant SFB 472 from the Deutsche Forschungsgemeinschaft (to H. S.).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) Y15285 (bovine M9.2 protein) and Y15286 (human M9.2 protein).
The protein sequence data have been submitted to the SWISS-PROT data base with accession numbers P81103 (bovine M9.2 protein) and P81134 (bovine M8-9 protein).
¶ To whom correspondence and reprint requests should be addressed: Zentrum der Biologischen Chemie, Universitätsklinikum Frankfurt, Theodor-Stern-Kai 7, Haus 25B, D-60590 Frankfurt am Main, Germany. Tel.: 49-69-6301-6927; Fax: 49-69-6301-6970; E-mail.: schaegger{at}zbc.klinik.uni-frankfurt.de.
1 The abbreviations used are: V-ATPase, vacuolar type ATPase; BN-PAGE, blue native polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; EST, expressed sequence tag; V1V0, V-ATPase, vacuolar proton pumping ATPase (holoenzyme); F1F0, complex V, mitochondrial proton pumping ATPase (holoenzyme); V1 and V0, hydrophilic (catalytic) and hydrophobic (transmembrane) sectors of V-ATPase, respectively; proteolipid c, subunit c of V-ATPase; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; bp, base pair(s); BisTris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; Mops, 3-(N-mor pholino)propanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|