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(Received for publication, November 9, 1995; and in revised form, January 12,
1996) From the
A prominent 16-kDa protein copurifies with the V-ATPase isolated
from both posterior midgut and Malpighian tubules of Manduca sexta larvae and thus was believed to represent a V-ATPase subunit.
[
H V-ATPases are heteromultimeric enzymes composed of peripheral
V Taken together, up to six genuine V-ATPase
subunits have now been identified at the molecular level. However,
although recent years have seen considerable progress in elucidating
the molecular structure of V-ATPases, we are still far from knowing the
actual number or the proper function of V-ATPase subunits, irrespective
of their origin. Several subunits, which may not be universal
constituents of V-ATPases, have been described (see Nelson(1992));
moreover, several unidentified polypeptides copurify with the
holoenzyme in many V-ATPase preparations (Adachi et al., 1990;
Gluck and Caldwell, 1987; Perez-Castineira and Apps, 1990; Ward and
Sze, 1992). In particular several polypeptides have been detected in
the range of 10-20 kDa. For instance, a 16-kDa polypeptide was
released, together with known V
Figure 1:
V-ATPase purified from Malpighian
tubules and midgut of M. sexta. V-ATPase from Malpighian
tubule brush border membranes was prepared as indicated under
``Experimental Procedures.'' Midgut V-ATPase was prepared
from highly purified goblet cell apical membranes using previously
published procedures (Wieczorek et al., 1990). Proteins were
stained with Coomassie Blue. First lane, standard proteins
with molecular masses of 94, 67, 43, 30, 20, and 14 kDa; second
lane, V-ATPase prepared from Malpighian tubule brush border
membranes; third lane, V-ATPase prepared from highly purified
goblet cell apical membranes. The molecular masses of V-ATPase subunits
are indicated in kilodaltons. Names of known subunits are given in brackets.
Initially, Schweikl et al.(1989) had assumed that the
16-kDa band represented the proteolipid that forms the proton channel
and is a genuine and universal V-ATPase subunit. However, several
findings contradicted this assumption. First, the 16-kDa protein was
stripped from the membrane by treatment with chaotropic iodide as well
as by the gentler method of cold inactivation (Fig. 2; see also Fig. 5in Gräf et al. (1994b)), and
therefore it could not be a membrane protein; although the release of
peripheral subunits by cold inactivation was less efficient than by
iodide stripping, the 16-kDa protein was stripped to the same extent as
the established V
Figure 2:
Stripping of Malpighian tubule brush
border membranes with chaotropic iodide or by cold inactivation.
Treatment with 0.8 M KI as well as separation and recovery of
soluble and membrane-bound proteins were performed as described
previously (Gräf et al., 1994b). Cold
inactivation was performed as described by Moriyama and Nelson(1989). A
portion of membranes was treated prior to cold inactivation with 1.2%
sodium cholate to produce inside-out vesicles (Noel et al.,
1993; Lepier et al., 1994) and thus to improve the efficiency
of the cold inactivation procedure. Proteins were stained with
Coomassie Blue. Lane 1, standard proteins with molecular
masses of 94, 67, 43, 30, 20, and 14 kDa; lanes 2 and 3, KI stripping experiment, membrane pellet (lane 2)
and supernatant containing soluble proteins (lane 3); lanes 4 and 5, cold inactivation of untreated
membranes, pellet, and supernatant, respectively; lanes 6 and 7, cold inactivation of membranes treated before with sodium
cholate as described above, pellet and supernatant, respectively. Known
V-ATPase subunits are indicated by capital letters and the
16-kDa band by an arrow.
Figure 5:
Deduced primary and predicted secondary
structures of the 13-kDa V-ATPase subunits from M. sexta and
yeast and of F-ATPase subunit b from bacteria. a, alignment of
the amino acid sequence of the M. sexta 13-kDa subunit (M) to the yeast 13-kDa subunit (Y) and to amino
acids 55-156 of the F-ATPase subunit b from V. alginolyticus (F). The alignment was composed manually, based on pair
comparisons by the Gap program. Identities are indicated by vertical bars and similarities by dots. b, predicted
secondary structure of the complete sequences of all three proteins.
Prediction of turns,
Figure 3:
[
Figure 4:
cDNA
sequence and predicted amino acid sequence for the M. sexta 13-kDa subunit. The epitope shared with the E subunit of the
V-ATPase is boxed. The stop codon is marked by an asterisk.
The deduced M.
sexta amino acid sequence was 37% identical and 63% similar to the
13-kDa protein Vma10p, which had been identified very recently as a
V-ATPase subunit in yeast (Fig. 5a;
Supekováet al., 1995); most of the
conserved amino acids occur in the N-terminal half of the protein (48%
identity, residues 1-59). Some similarity appeared to exist to
subunit b of bacterial F-ATPases (Fig. 5a; 26% identity
in a 100-amino acid overlap to the F-ATPase subunit b of Vibrio
alginolyticus; Krumholz et al., 1989), but also to the
N-terminal part of tropomyosins (27% identity in a 73-amino acid
overlap to non-muscle tropomyosin from African clawed frog; Hardy et al., 1991). The secondary structure of the deduced M. sexta 13-kDa protein may be helpful in deducing its
function. The sequence of the N-terminal half predicts that it forms a
continuous, highly hydrophilic
Figure 6:
Western blot of E. coli periplasmic proteins. Blots from SDS-polyacrylamide gel
electrophoresis of the cold osmotic shock supernatant from induced
p13MBPp2 cells. First lane, standard proteins with molecular
masses of 94, 67, 43, 30, 20, and 14 kDa, Ponceau S staining; second lane, periplasmic proteins, Ponceau S staining; third lane, immunoblot of periplasmic proteins stained with an
antiserum to the M. sexta V-ATPase
holoenzyme.
Figure 7:
Western blots of V-ATPases from various
sources. Immunostainings of proteins after SDS-polyacrylamide gel
electrophoresis and blotting. Lane 1, M. sexta V-ATPase prepared from goblet cell apical membranes, stained with
an antiserum to the holoenzyme; lanes 2-5, samples
stained with affinity-purified monospecific antibodies to the
recombinant 13-kDa protein; lane 2, 5 µg of M. sexta V-ATPase prepared from midgut goblet cell apical membranes; lane 3, 5 µg of M. sexta V-ATPase prepared from
Malpighian tubule brush border membranes; lane 4, 20 µg of
a membrane preparation from crab (Eriocheir sinensis) gills
(kindly provided by M. Putzenlechner); lane 5, 80 µg of a
crude membrane pellet from mouse kidney; lane 6, 5 µg of M. sexta V-ATPase prepared from goblet cell apical membranes,
stained with the monoclonal antibody 47-5. Known V-ATPase
subunits are indicated by capital letters and the 16-kDa band
by an arrow.
However, it was still not
clear whether the remarkable difference between the calculated and the
apparent molecular masses of 13 and 16 kDa, respectively, was due to
intrinsic properties of the protein or to an incomplete open reading
frame in the cloned cDNA. Therefore the open reading frame, starting at
base position 45, together with the 3`-untranslated region, was cloned
into the translation vector pSPUTK which contained the 5`-untranslated
sequence of
Figure 8:
In vitro transcription/translation of the recombinant 13-kDa protein. First lane, V-ATPase prepared from M. sexta goblet
cell apical membranes, Coomassie Blue staining; second lane,
protein translated from the p13SPUTK plasmid, after
immunoprecipitation; autoradiography. Known V-ATPase subunits are
indicated by capital letters and the 16-kDa band by an arrow.
Figure 9:
Reassociation of the V
The cDNA encoding a novel subunit of the M. sexta V-ATPase was cloned by immunoscreening. The hydrophilic protein
consisted of 117 amino acids with a calculated molecular mass of 13,692
Da. It showed 37% sequence identity to the recently published 13-kDa
subunit Vma10p of the yeast V-ATPase (Supeková et al., 1995) as well as some similarity to subunit b of
bacterial F-ATPases. In Western blots, monospecific antibodies to the
13-kDa protein cloned from M. sexta midgut specifically
recognized the 16-kDa band of the purified V-ATPase from M. sexta midgut and Malpighian tubules. In vitro translation
revealed that the recombinant 13-kDa protein exhibits the mobility of a
16-kDa protein in SDS-electrophoresis gels (Fig. 8). Thus, the
recombinant 13-kDa protein and the 16-kDa protein of the purified
V-ATPase are identical.
We presume
by analogy to our case, that some of the strongly stained bands in the
16-kDa range found in published SDS-electrophoresis gels of purified
V-ATPases from other sources (see Introduction) may not represent the
proteolipid as alleged. Thus, strong staining of the proteolipid with
Coomassie Blue would not be expected because of its very low content of
positively charged amino acids. Instead, these bands may represent
mainly the novel 13-kDa subunit. However, the novel subunit may also be
represented by other low molecular mass proteins copurifying with the
respective V-ATPases.
Our conclusion that the
13-kDa subunit G is a member of the V The
function of the 13-kDa subunit is enigmatic so far, but the predicted
unusual secondary structure with one continuous, highly charged
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
X92805[GenBank].
Volume 271,
Number 14,
Issue of April 5, 1996 pp. 8502-8508
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
C]N,N`-dicyclohexylcarbodiimide
labeling and its position on SDS-electrophoresis gels revealed that
this protein was different from the 17-kDa proteolipid. A cDNA clone
encoding a highly hydrophilic protein with a calculated molecular mass
of 13,692 Da was obtained by immunoscreening. Monospecific antibodies,
affinity-purified to the 13-kDa recombinant protein expressed in Escherichia coli, specifically recognized the 16-kDa protein
of the purified V-ATPase, confirming that a cDNA encoding this protein
had been cloned. In vitro translation of the cRNA showed that
the cloned 13-kDa subunit behaved like a 16-kDa protein on
SDS-electrophoresis gels. The cloned protein showed 37% amino acid
sequence identity to the 13-kDa V-ATPase subunit Vma10p recently cloned
from yeast and some similarity to subunit b of bacterial F-ATPases. In
contrast to the Vma10p protein, which behaved like a V
subunit, the M. sexta 13-kDa protein behaved like a
V
subunit, since it could be stripped from the membrane by
treatment with the chaotropic salt KI and by cold inactivation. When KI
dissociated V-ATPase subunits were reassociated by dialysis that
removed the KI, a soluble, 450-kDa complex of the M. sexta V-ATPase could be purified by gel chromatography. This V complex consisted of subunits A, B, E, and th
e 13-kDa subunit,
confirming that the cloned protein is a new V-ATPase subunit and a
member of the peripheral V complex of the V-ATPase. We
designate this new V component subunit G.
-translocating vacuolar-type ATPases
(V-ATPases) occur in endomembranes as well as in various plasma
membranes of eukaryotic cells (see Harvey(1992)). The tobacco hornworm (Manduca sexta) midgut V-ATPase is highly concentrated in the
apical plasma membrane of the goblet cells (Wieczorek et al.,
1986; Schweikl et al., 1989; Klein et al., 1991). In
contrast to most other V-ATPases, it does not drive acid or fluid
transport, but energizes electrophoretic
K/2H-antiport by generating a
transmembrane voltage of more than 200 mV (Wieczorek et al.,
1991; Wieczorek, 1992; Azuma et al., 1995). The resulting
K electrochemical potential drives the absorption of
amino acids by K coupled symport (Giordana and
Parenti, 1994; Martin and Harvey, 1994). Plasma membrane VATPases have
also been found in other insect organs, such as Malpighian tubules (Drosophila hydei: Bertram et al., 1991; M.
sexta: Klein et al., 1991; Formica polyctena:
Van Kerkhove, 1994), where they are involved in the energization of
salt and fluid secretion (for review, see Nicolson (1993)). and membrane integral V complexes, which
together, in analogy to F-ATPases, form ball and stalk structures known
as portasomes (see Harvey(1992)). The V part consists of at
least two subunits, a 43-kDa subunit and the highly conserved 17-kDa
proteolipid, subunit c, which binds DCCD ()(Bowman et
al., 1986) and which, probably as a hexamer, forms the
proton-conducting pore (Arai et al., 1988). A 14-kDa protein,
first shown to be a constitutive V-ATPase subunit in M. sexta (Gräf et al., 1994b) and subsequently
found in yeast and D. melanogaster (Graham et al.,
1994; Nelson et al., 1994; Guo et al., 1996),
exhibits some affinity to the V part
(Gräf et al., 1994b), but also appears to
be involved in the assembly and stability of the V complex
(Graham et al., 1994). Chaotropic salts (Rea et al.,
1987) or cold treatment in the presence of ATP (Moriyama and Nelson,
1989) lead to the dissociation of various V-ATPase subunits from the
membrane; hence these polypeptides were defined as constituents of the
peripheral V complex. Among them, three subunits, A, B, and
E, are major components of the V complex and occur in every
V-ATPase, including that of M. sexta (67-kDa subunit A:
Gräf et al., 1992; 56-kDa subunit B: Novak et al., 1992; 28-kDa subunit E: Gräf et al., 1994a). subunits, by chaotropic
treatment of Neurospora crassa vacuolar membranes (Bowman et al., 1989). Again, protein bands in the range of
10-15 kDa, obtained by SDS-polyacrylamide gel electrophoresis,
have been discussed as putative peripheral subunits of the V-ATPase
from bovine clathrin coated vesicles (Puopolo et al., 1992).
Very recently, a 13-kDa subunit Vma10p was identified as a putative
constituent of the V complex in the yeast V-ATPase
(Supekováet al., 1995). Here we report
the identification of a V-ATPase subunit, which has a calculated
molecular mass of 13 kDa and an apparent molecular mass of 16 kDa. This
novel subunit is a major V component of the M. sexta V-ATPase, and we designate it subunit G.
Insects
Larvae of M. sexta (Lepidoptera, Sphingidae) were reared under long day conditions
(16 h of light) at 27 °C using a synthetic diet (modified according
to Bell and Joachim (1974)).[
Labeling
reactions were performed in a volume of 500 µl containing
5-10 µg of purified V-ATPase in 50 mM Tris-MOPS (pH
8.0), 1 mM MgClC]DCCD Labeling
, 1 mM 
-mercaptoethanol, 0.1 mM EDTA, 0.003%
C
E
, and 10 µM [C]DCCD (50-60 Ci/mol). Sample
s were
incubated for 30 min at 30 °C. Labeled protein was collected by
trichloroacetic acid precipitation; the pellet was washed twice with
ice-cold acetone, air-dried, resuspended in 15 µl of SDS buffer
(125 mM Tris-HCl (pH 6.8), 5% sucrose, 2% SDS, 2%
-mercaptoethanol, 0.005% bromphenol blue) and incubated for 1 h at
37 °C. After SDS-polyacrylamide gel electrophoresis and Coomassie
Blue staining, the gel was incubated in enhancer solution
EN
HANCE, DuPont) for 1 h, washed in cold water for 1 h, and
dried on Whatman filter paper. The dried gel was exposed to Kodak x-ray
film at -70 °C for a minimum of 3 days.Purification of Monospecific Antibodies to the 16-kDa
Protein
Purified V-ATPase (0.5 mg) was electrophoresed on a
SDS-agarose gel (ProSieve, FMC) with only one broad slot. The gel was
briefly fixed in 50% methanol, 10% acetic acid, until opaque bands were
visible in the clear gel against a black background. The 16-kDa band
was excised, and the protein was extracted from the agarose matrix by
repeated freeze-thaw/centrifugation steps, according to the
manufacturer, using an extraction buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 0.2% sodium lauryl
sarcosine. Collected supernatants were concentrated on Centricon 10
ultrafilters, and the protein was coupled to CNBr-activated Sepharose
4B, following the manufacturer's instructions. The slurry was
filled into a MicroSpin column (Pharmacia Biotech Inc.) resulting in a
final bed volume of approximately 0.3 ml, and equilibrated in TBS (0.5 M NaCl and 20 mM Tris-HCl, pH 7.5). 0.5 ml of
filtered rabbit polyclonal antiserum to the purified V-ATPase
holoenzyme (Wieczorek et al., 1991) was diluted 5-fold with
TBS, added to the column, and incubated for 1 h at room temperature
under gentle agitation to keep the slurry inside the column in
suspension. The column was then washed with TBS until A
of the eluate was <0.1, and incubated twice
with 0.5 ml of TBS containing 0.2% Triton X-100 for 1 h each, under
gentle agitation as described above. After removal of Triton X-100 by
TBS, antibodies were eluted from the column using 100 mM glycine-HCl (pH 2.7). Three fractions of 0.5 ml each were pooled
and neutralized. Gelatin was added to a final concentration of 1% to
improve the antibody stability. Monospecificity was confirmed on
Western blots of purified goblet cell apical membrane vesicles using a
1:3 dilution of the final antibody solution (data not shown).Immunoscreening of a cDNA Library and
Sequencing
400,000 plaque-forming units from a cDNA library
prepared in 
-ZAP II from M. sexta posterior larval midgut
were screened as described previously (Gräf et
al., 1992), using a 1:10 dilution of the monospecific antibodies.
After three screening steps, one positive clone was obtained and
subcloned into pBluescript SK(-) by in vivo excision
(Short et al., 1988). Single-stranded plasmid DNA was prepared
(Katayama, 1990) and sequenced by the dideoxynucleotide chain
termination method (Sanger et al., 1977) using custom
synthesized primers, Sequenase 2.0 and 
-
S-dATP.Expression and Purification of the 13-kDa Fusion Protein
in E. coli
The 13-kDa fusion protein was expressed in E.
coli as described previously (Gräf et
al., 1994b). In brief, the complete coding sequence was amplified
by polymerase chain reaction employing an 20-mer upstream primer
starting with ATG (base positions 45-64) and a 32-mer downstream
primer (base positions 351-370), including an added BamHI recognition site and a 6 base nonsense sequence. After
blunting with T4 polymerase and digestion with BamHI, the
resulting fragment was ligated into the expression vector pMAL c2 (New
England Biolabs) which had been predigested with XmnI and BamHI to yield p13MBPc2. For subcloning in pMAL p2, which
contains the malE signal sequence for transport into the periplasmic
space, the insert of p13MBPc2 was excised by digestion with SacI and PstI. After ligation into pMAL p2, the
p13MBPp2 obtained by this procedure was transformed into competent
UT5600 E. coli cells. Expression of the fusion protein was
induced by supplementing 800-ml cultures (OD
0.5)
grown at 37 °C with 0.3 mM isopropyl-1-thio--D-galactopyranoside. After 2 h,
cells were harvested by centrifugation at 4,000 
g for
10 min and resuspended in 30 mM Tris-HCl (pH 8) containing 20%
sucrose. NaEDTA (pH 8) was added to a final concentration of 1
mM, cells were stirred for 10 min at room temperature and
centrifuged at 8,000 g for 20 min at 4 °C. The
pellet was resuspended in ice-cold 5 mM MgSO
and
stirred for 10 min on ice. The cold osmotic shock supernatant that
contained the fusion protein was collected by centrifugation at 8,000
g for 20 min at 4 °C, and the protein was
concentrated by ammonium sulfate precipitation (80% saturation).Purification of Monospecific Antibodies to the 13-kDa
Fusion Protein
After dialysis, the whole protein of the cold
osmotic shock supernatant (approximately 3 mg of protein in 0.6 ml) was
coupled to CNBr-activated Sepharose 4B. The antiserum to the purified
V-ATPase holoenzyme (Wieczorek et al., 1991) was used for
purification of monospecific antibodies to the cloned 13-kDa protein.
Affinity chromatography was performed as described above for the
purification of monospecific antibodies to the 16-kDa protein purified
by gel electrophoresis from the original V-ATPase preparation.Coupled in Vitro Transcription/Translation of
cDNA
The putative coding sequence and the complete
3`-untranslated region of the 13-kDa protein was amplified by
polymerase chain reaction (Lion and Haas, 1990) using a 27-mer upstream
primer that started with a six-base nonsense sequence followed by two
cytosine residues and the first 19 bases of the coding sequence
(5`-TATCAGCCATGGCGAGTCAGACACATG-3`; the generated NcoI site is
underlined) and a 20-mer downstream primer directed to the T7 site of
pBluescript SK(-). The resulting DNA fragment was digested with NcoI at the 5` end and with ApaI at the 3` end and
was electrophoresed on a 1% low melting point agarose gel. The fragment
was cloned into the translation vector pSPUTK (Stratagene), which had
been predigested with NcoI and ApaI, and transformed
into competent E. coli AG-1 cells to yield p13SPUTK. This
plasmid was purified by a conventional miniprep procedure (Sambrook et al., 1989). To destroy residual RNase the plasmid DNA
preparation was treated with proteinase K. After phenol/chloroform
extraction and ethanol precipitation the plasmid DNA was used for
coupled in vitro transcription/translation (TNT-rabbit
reticulocyte system from Promega). Reactions were performed according
to the manufacturer by adding SP6 polymerase,
[S]methionine (20 µCi) and p13SPUTK-DNA (1
µg) to the reticulocyte mixture and incubating for 2 h at 30 °C
in a volume of 25 µl. For immunoprecipitation of the translated
protein, 0.5 ml of blocking buffer (0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 0.05% Tween 20, 3% gelatin) and 2 µl of
anti-V-ATPase holoenzyme serum (approximately 30 µg of IgG) were
added to the mixture, followed, after 1-h incubation, by 5 µl of
protein G-Sepharose. Binding to protein G was achieved during 4 h on
ice. Sepharose beads were collected by centrifugation and washed three
times with washing buffer (0.5 M NaCl, 20 mM Tris-HCl
(pH 7.5), 0.05% Tween 20). For SDS-gel electrophoresis, the translated
protein that was bound to the beads was eluted by boiling in 20 µl
of SDS buffer for 3 min. The whole sample was used for one lane of an
SDS-polyacrylamide electrophoresis gel. The dried gel was exposed to
Kodak XAR 5 film for 5 h at -80 °C.Preparation of V-ATPase from Malpighian
Tubules
For one preparation, Malpighian tubules from 20 fifth
instar larvae were pooled in 6 ml of ice-cold M buffer (0.3 M mannitol, 17 mM Tris-HCl (pH 7.5), 5 mM EGTA).
Brush border membranes from Malpighian tubules were isolated by the
magnesium precipitation procedure (Booth and Kenny, 1974; Wolfersberger et al., 1987). In brief, after homogenization of the tissue
for 45 s at 20,000 rpm using an Ultraturrax homogenizer, 6 ml of
ice-cold 24 mM MgSO was added. After 15 min on
ice, the sample was centrifuged for 15 min at 2,500 g and 4 °C, and the supernatant containing the brush border
membranes was transferred into a new tube and centrifuged at 30,000
g for 30 min at 4 °C. For further purification the
entire procedure was repeated after resuspending the 30,000 g pellet in 2 ml of M buffer and adding an equal amount of 24
mM MgSO. The resulting brush border membrane
pellet was frozen in liquid nitrogen and stored at -20 °C.
The V-ATPase was solubilized and purified as published previously
(Schweikl et al., 1989; Wieczorek et al., 1990).Isolation of the V
V Complex of the
V-ATPase subunits were stripped from V by incubating 0.6 ml of Malpighian tub
ule brush border membranes,
suspended in M buffer (approximately 1 mg of protein), in the presence
of 0.8 M KI for 1 h on ice. After centrifugation at 100,000
g for 1 h at 4 °C, the dissociated V subunits in the supernatant were reassoc
iated by dialysis against
500 ml of a buffer (pH 7.0) consisting of 50 mM KCl, 10 mM Tris-MOPS, 3 mM -mercaptoethanol, 0.5 mM EGTA. The sample was concentrated to approximately 0.1 ml on a
Centricon 10 concentrator and fractionated by fast protein liquid
chromatography on a Superdex 200 HR 10/30 gel chromatography column
using the dialysis buffer. The V complex was found in the
fraction containing proteins of approximately 450 kDa (using ferritin
as standard).Other Methods
Isolation and purification of the
V-ATPase from Manduca midgut goblet cell apical membranes,
protein determination with Amido Black, standard SDS-polyacrylamide gel
electrophoresis, Western blotting on nitrocellulose membranes (BA85),
and immunostaining were performed as described previously (Schweikl et al., 1989; Wieczorek et al., 1990, 1991;
Gräf et al., 1994b). For preparation of
the crude membrane pellet from mouse kidney, one kidney was homogenized
for 45 s at 20,000 rpm using an Ultraturrax homogenizer. The homogenate
was cleared by centrifugation at 1000 g for 5 min at 4
°C, and the supernatant was centrifuged again at 100,000 g for 30 min at 4 °C. The membrane pellet was resuspended
in M buffer.
The Purified M. sexta V-ATPase Contains Two Proteins in
the 16-kDa Range
A prominent protein band with an apparent
molecular mass of 16 kDa, appearing in SDS-polyacrylamide gels of the
purified M. sexta midgut V-ATPase with a highly reproducible
staining intensity relative to the other protein bands (Fig. 1),
had already been noticed by Schweikl et al.(1989). Since a
plasma membrane V-ATPase had been demonstrated previously
immunocytochemically in Malpighian tubules of M. sexta (Klein et al., 1991; Russell et al., 1992), we purified the
enzyme from brush border membranes and found a strongly stained 16-kDa
band in SDS gels of this preparation, too (Fig. 1). The
enrichment of the 16-kDa protein in the same fraction as all other
established V-ATPase subunits, together with the fact that it is one of
the major proteins in the preparation of the purified V-ATPase, argue
strongly that it is a constitutive subunit of the insect V-ATPase.
subunits A, B, and E. Second,
[C]DCCD labeling showed that, in
SDS-polyacrylamide gel electrophoresis, the proteolipid exhibited an
apparent molecular mass of 17 kDa (Fig. 3). The 17-kDa band was
only weakly stained by Coomassie Blue in ordinary SDS gels and was not
stripped from the membrane by treatment with chaotropic iodide (Fig. 2, lanes 2 and 3; see also Fig. 5in Gräf et al. (1994b)).
Third and finally, the strong staining of the 16-kDa band observed with
Coomassie Blue would not be expected for a highly hydrophobic protein
such as the proteolipid, since it possesses only a very low content of
positively charged amino acids (Dow et al., 1992).
-helices and -sheets by the method of
Chou and Fasman(1978), modified according to Nishikawa(1983), were
performed using the Peptidestructure and Plotstructure programs.
Elevations from the base line indicate a high probability for the
respective type of secondary structure. Prediction of hydrophobicity
according to the method of Kyte and Doolittle(1982) was performed using
the Pepplot program. Thickness of the black boxes indicates
the degree of hydrophobicity. Sequences were aligned corresponding to
their similarity overlaps. The bar indicates the length of 10
amino acids.
C]DCCD labeling of
the V-ATPase proteolipid. To improve resolution in the low molecular
mass range, a 22% acrylamide gel (22% T, 0.5% C) was used. First
lane, V-ATPase purified from goblet cell apical membranes after
Coomassie Blue staining and enhancer treatment, sample incubated at 37
°C; second lane, fluorography of a DCCD-labeled V-ATPase
sample incubated at 37 °C; third lane, fluorography of a
DCCD-labeled V-ATPase sample heated to 95 °C. When samples were
heated prior to electrophoresis, the proteolipid formed large
aggregates accumulating at the top of the gel (see also Finbow and
Pitts(1993)). Known V-ATPase subunits are indicated by capital
letters and the 16- and 17-kDa bands by their molecular
masses.
Isolation and Sequencing of cDNA Encoding a 13-kDa
Protein
The antiserum against the purified V-ATPase was very
rich in 16-kDa-specific antibodies, enabling us to purify monospecific
antibodies by affinity chromatography on a Sepharose column that had
been conjugated with the 16-kDa protein eluted from SDS-agarose gels
(not shown). The monospecific antibodies were used for immunoscreening
of a M. sexta larval posterior midgut cDNA library
(Gräf et al., 1992). One positive clone
was isolated after three screening steps. The 808 base pairs of cDNA
sequence obtained included an open reading frame (base position
45-395) encoding a very hydrophilic protein with 117 amino acids,
a calculated molecular mass of only 13,692 Da (Fig. 4), and an
isoelectric point at pH 9.88 (DNAsis). The initiator ATG was chosen due
to its sequence environment which is similar to other cloned cDNAs
encoding M. sexta V-ATPase subunits (Gräf et al., 1992, 1994b; Novak et al., 1992; Dow et
al., 1992) and matches closely the consensus sequence for the
translation start site in eukaryotes (Kozak, 1989). Using the putative
coding region of the cDNA for hybridization screening of a cDNA library
from the silk gland of Bombyx mori, we detected a clone
exhibiting an almost identical DNA sequence; the identity of the
deduced amino acid sequence was 94%. ()
-helix, followed by two further
-helices covering approximately 20% of the whole sequence (Fig. 5b). The predominantly hydrophilic 
-helical
secondary structure predicted for the N-terminal half of the insect
13-kDa protein is also predicted for the yeast 13-kDa subunit Vma10p
and the F-ATPase subunit b (Fig. 5b).Expression of the 13-kDa Protein
To confirm that
the cDNA encoding the protein corresponding to the 16-kDa band of
SDS-electrophoresis gels had been cloned and that it contained the
complete coding sequence, the recombinant protein was expressed using
two different strategies. First, the postulated coding sequence was
cloned into the E. coli expression vector pMAL p2. Periplasmic
expression in a protease-deficient strain (UT5600) rather than
cytoplasmic expression was chosen because we had observed strong
degradation when we used pMAL c2 and standard E. coli strains
in preliminary experiments. Although the fusion protein was the main
protein constituent of the periplasmic fraction (Fig. 6), it was
partially degraded. Therefore it was not appropriate to cleave the
fusion protein at the fusion site with factor Xa protease in order to
determine the apparent molecular mass of the recombinant 13-kDa protein
by SDS-polyacrylamide gel electrophoresis. However, the periplasmic
fraction seemed to be pure enough to be used for the purification of
antibodies to the recombinant protein from the anti-holoenzyme serum by
affinity chromatography. Western blots with purified V-ATPase showed
that the resulting 13-kDa specific antibody preparation was highly
specific for the 16-kDa protein (Fig. 7, lanes 2 and 3). Only after long incubation times for the alkaline
phosphatase reaction or at high antibody concentrations could a weak
cross-reaction with the 28-kDa subunit E be observed (Fig. 7, lane 3). These results clearly indicate that the 13-kDa
recombinant protein used for antibody purification corresponds to the
16-kDa protein of the purified V-ATPase.
-globin to achieve efficient in vitro transcription/translation by using SP6 polymerase and rabbit
reticulocyte lysates. Since the crude lysate contained an enormous
amount of low molecular mass proteins that prevented any exact size
determination of the [S]Met labeled, translated
protein on SDS-electrophoresis gels, we immunoprecipitated the
translated protein using the anti-holoenzyme antiserum. Subsequent
SDS-polyacrylamide gel electrophoresis, followed by autoradiography,
clearly indicated an apparent molecular mass of 16 kDa. This result
confirmed that the cDNA sequence, which predicted a 13-kDa protein,
included the complete open reading frame for the 16-kDa protein (Fig. 8). The unexpectedly high apparent molecular mass of this
protein in SDS-polyacrylamide gel electrophoresis may be a consequence
of its high, almost 41%, content of charged amino acids. By contrast,
subunits A and B, whose apparent molecular masses match closely their
calculated molecular masses, have a content of charged amino acids of
less than 25%.
Identification of the 13-kDa Protein as a Subunit of the
V
Although the 13-kDa
protein copurified with established V-ATPase subunits and appeared as a
prominent band in SDS-polyacrylamide gels of the purified V-ATPase
preparation, it was desirable to obtain further evidence that it is a
genuine subunit of the V-ATPase holoenzyme. First we tried to inhibit
ATP-dependent proton transport by the monospecific antibodies, in
experiments analogous to those reported for the 14-kDa subunit
(Gräf et al., 1994b). However, the
antibodies did not influence the reaction. Although this result was
disappointing, it was not unexpected, since subunit-specific antibodies
do not necessarily inhibit the function of a complex holoenzyme (see
also Nelson et al., 1994). If the 13-kDa protein is a genuine
V-ATPase subunit and, moreover, a constituent of the V Complex of the V-ATPase complex, it should be present in the isolated V
complex in quantities similar to those in the purified V-ATPase.
Therefore we stripped peripheral membrane proteins, including the
V subunits, from V-ATPase-containing brush border membranes
of Malpighian tubules by treatment with a high concentration of
chaotropic iodide. After centrifugation, the supernatant was dialyzed
against an iodide free buffer and, thereafter, subjected to gel
chromatography. Analysis by SDS-polyacrylamide gel electrophoresis
revealed V subunits in low molecular mass fractions, but
also in a fraction corresponding to an apparent molecular mass of 450
kDa, the approximate calculated molecular mass of the V complex (Fig. 9). Fractions corresponding to higher
molecular masses contained no protein. Since no protein could be
detected in the 450-kDa fraction without dialysis (Fig. 9), we
concluded that the V subunits had been dissociated by the
iodide treatment to subcomplexes and monomers and that a small portion
(approximately 2% of total protein in the stripping supernatant) of
V proteins had reassociated during dialysis, forming large
complexes of approximately 450 kDa. This dissociation/reconstitution
was similar to that which Puopolo et al.(1992) obtained with
V-ATPase from bovine clathrin-coated vesicles. However, the composition
of the reconstituted M. sexta 450-kDa V complex
was unique. The main proteins constituting the complex were the 67-kDa
subunit A, the 56-kDa subunit B, the 28-kDa subunit E, and the 13-kDa
(apparent 16-kDa) protein (Fig. 9). Furthermore, the staining
intensity of the 16-kDa band was highly reproducible relative to the
three other V subunits and was similar to that observed for
the purified V-ATPase. Other proteins seemed to be present only in
substoichiometric amounts and therefore were probably impurities.
complex. First lane, Malpighian tubule brush border
membrane proteins; second lane, 450-kDa fraction of the KI
stripping supernatant after dialysis and gel chromatography; third
lane, 450-kDa fraction of the stripping supernatant without
dialysis. Proteins were stained with Coomassie Blue. Known V-ATPase
subunits are indicated by capitals, the 16-kDa band by an arrow.
Cross-reaction of the 13-kDa Subunit-specific Antibodies
with V-ATPase-containing Membrane Preparations of Xenic
Origin
The 13-kDa specific antibodies, originally directed to
the plasma membrane V-ATPase of M. sexta midgut, cross-reacted
not only with the 16-kDa band of the Malpighian tubule V-ATPase, but
also with xenic proteins in the 16-kDa size range. We detected
respective staining (Fig. 7) in a crab gill membrane preparation
enriched with V-ATPase (Onken and Putzenlechner, 1995) as well as in a
crude membrane pellet from murine kidney consisting, among others, of
membranes expected to contain high amounts of V-ATPase (Gluck et
al., 1992). To obtain staining similar to that of M. sexta samples, the Western blots of the two xenic membrane preparations
had to contain more membrane protein, and higher concentrations of
antibodies had to be used.
The 13-kDa Protein Is a V-ATPase Subunit
Several lines
of evidence indicate that the 13-kDa protein is a genuine V-ATPase
subunit. First, it copurifies in strictly reproducible amounts with the
V-ATPase purified from both the midgut goblet cell apical membrane and
the Malpighian tubule brush border membrane. Second, it is a major
component not only of the holoenzyme, but also of the reassociated
V complex. Third, in Western blots monospecific antibodies
to the recombinant 13-kDa protein cross-reacted with a 16-kDa protein
band in V-ATPase containing membrane preparations of both crab gills
and mouse kidney. Finally, a fairly homologous 13-kDa protein was
recently identified as a subunit of the yeast V-ATPase
(Supekováet al., 1995).The 13-kDa Subunit Shares an Epitope with Subunit
E
Western blots of both midgut and Malpighian tubule V-ATPase
from M. sexta which were probed with the 13-kDa specific
antibodies showed a cross-reaction with a band in the range of 30 kDa (Fig. 7). This result was not surprising, since our monoclonal
antibody 47-5 (Klein et al., 1991) binds to the 13-kDa
subunit and to the 28-kDa subunit E of the purified midgut V-ATPase (Fig. 7, lane 6), suggesting that both proteins share a
common epitope. Indeed, a five-amino acid sequence, EARKR (boxed in Fig. 4), is found in both the 13-kDa subunit and subunit
E (Gräf et al., 1994a). Thus, the epitope
reacting with the monoclonal antibody appears to be clearly defined.
The 13-kDa specific antibodies also cross-reacted with a protein in the
30-kDa range of a membrane preparation from crab gills but not from
mouse kidney (Fig. 7). This epitope may be common to
invertebrates, especially since the EARKR sequence is found neither in
mammalian and yeast subunits E nor in the yeast 13-kDa protein Vma10p.The 13-kDa Subunit Is a Member of the V
In our recent report on the 14-kDa subunit we had
already shown that the 16-kDa protein of the membrane bound V-ATPase
was released from midgut goblet cell apical membranes upon treatment
with chaotropic iodide ( Fig. 5in Gräf et al. (1994b)). In this paper, we obtained the same result
using Malpighian tubule brush border membranes. Moreover, cold
inactivation experiments also indicated that the 13-kDa protein is a
peripheral V-ATPase subunit. Furthermore, upon dialysis of the
stripping supernatant from the chaotropic treatment, the 13-kDa subunit
as well as the A, B, and E subunits of the V-ATPase, all present in the
complex mixture of dissociated proteins, specifically reassembled to
form a high molecular mass protein complex of approximately 450 kDa,
which we call the V Complex complex. This V complex,
like the catalytic complex of the V-ATPase purified from
clathrin-coated vesicles (Xie and Stone, 1988), exhibits
Ca
-dependent ATPase activity
(Gräf et al., 1995). Moreover, the 16-kDa
protein is also a major component of a highly purified cytoplasmic
V complex isolated from molting M. sexta larvae. (
)Taken together, our results provide compelling evidence
that the novel 13-kDa subunit is a member of the peripheral V complex. Since the 14-kDa subunit first cloned from M. sexta (Gräf et al., 1994b) has a
lready been
designated as subunit F (Nelson et al., 1994), we suggest the
term subunit G for the 13-kDa subunit. complex seems not to
be in line with results obtained in yeast, where cold inactivation
experiments and the properties of the yeast null mutant suggested that
the homologous 13-kDa subunit Vma10p is a member of the membrane bound
V sector of the V-ATPase (Supeková et al., 1995). However, the alignment of the two derived
amino acid sequences may explain this apparent contradiction (Fig. 5a): sequence identities are clustered in the
N-terminal parts (48% identity from residues 1 to 59), whereas the
C-terminal parts share only 24% identical amino acids. The sequence
similarity to subunit b of bacterial F-ATPases argues neither for nor
against the V or V membership of the 13-kDa
subunit from both M. sexta and yeast, since subunit b appears
to be anchored to the membrane by its apolar N-terminal region
(Deckers-Hebestreit and Altendorf, 1992), for which no equivalent
exists in the 13-kDa subunits (Fig. 5b).-helix covering the N-terminal half of the protein, and its
similarity with the F-ATPase subunit b regarding both sequence and
predicted secondary structure of the N-terminal part may provide a clue
to the understanding of its role in the V-ATPase holoenzyme. For
example, the location of the 13-kDa insect subunit G in the V sector and the homologous 13-kDa yeast subunit Vma10p in the
V sector suggests that the 13-kDa subunits may connect
V to V in both cases.
)))
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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