From the Department of Biology, University of
Osnabrück, D-49069 Osnabrück, Germany and the
§ Whitney Laboratory, University of Florida,
St. Augustine, Florida 32086
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ABSTRACT |
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Plasma membrane V-ATPase isolated from midgut and
Malpighian tubules of the tobacco hornworm, Manduca sexta,
contains a novel prominent 20-kDa polypeptide. Based on N-terminal
protein sequencing, we cloned a corresponding cDNA. The deduced
hydrophobic protein consisted of 88 amino acids with a molecular mass
of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide,
using a monoclonal anti- body to the 20-kDa polypeptide, confirmed
that the correct cDNA had been cloned. The 20-kDa polypeptide is
glycosylated, as deduced from lectin staining. Treatment with
N-glycosidase A resulted in the appearance of two
additional protein bands of 16 and 10 kDa which both were
immunoreactive to the 20-kDa polypeptide-specific monoclonal antibody.
Thus, extensive N-glycosylation of the novel Vo
subunit M9.7 accounts for half of its molecular mass observed in
SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities
to the yeast protein Vma21p which resides in the endoplasmic reticulum
and is required for the assembly of the Vo complex.
However, as deduced from immunoblots as well as from activities of the
V-ATPase and endoplasmic reticulum marker enzymes in different membrane
preparations, M9.7 is, in contrast to the yeast polypeptide, a
constitutive subunit of the mature plasma membrane V-ATPase of M. sexta.
H+ V-ATPases are a class of ion transport proteins
that couple ATP hydrolysis to the movement of protons across membranes. In endomembranes they function, in concert with chloride channels, as
acidifiers of intracellular compartments, whereas in plasma membranes
their roles are dependent on the cell type. V-ATPases consist of a
peripheral V1 complex, which is responsible for the hydrolysis of ATP, and a membrane-bound Vo complex which is
responsible for the translocation of protons. Although the subunit
composition may depend upon the source of the enzyme, at least seven
subunits of the V1 complex, subunits A to G, appear to be
universal V-ATPase components (1). By contrast, the subunit composition
of the Vo complex is less clear. There is no doubt that a
16-17-kDa proteolipid, the proton "channel," is a major
constituent of the Vo complex. A membrane-associated
subunit in the 40-kDa range and an ~100-kDa transmembrane subunit may
be two additional essential Vo components (1). Recently, a
novel 9.2-kDa membrane sector-associated polypeptide was reported from
bovine chromaffin granules (2). Its sequence and structure show some
similarity to Vma21p, a yeast protein involved in the assembly of the
V-ATPase; whether or not it is a constitutive V-ATPase subunit remains
an open question.
In the larval midgut epithelium of the model insect, Manduca
sexta (Lepidoptera, Sphingidae), a plasma membrane V-ATPase is present in the apical membranes of goblet cells where it energizes the
alkalinization of the gut lumen to a pH of more than 11 (3). For the
V1 complex, amino acid sequences of five insect subunits A,
B, E, F, and G have been deduced from cloned cDNAs (4), and
evidence for the existence of subunit D has been derived from partial
amino acid sequencing.1 For
the Vo complex, only sequences of the 17-kDa proteolipid and of the subunit M40 have been derived from cDNAs to date (5, 6),
although evidence for a 100-kDa subunit is appearing on the horizon.
Based on a partial amino acid sequence obtained from a 20-kDa
polypeptide band that is present in gels after
SDS-PAGE2 of the insect
holoenzyme, we have cloned and sequenced the cDNA encoding a
9.7-kDa protein that is remarkably similar to the bovine 9.2-kDa
Vo-associated protein. The insect protein is glycosylated extensively, with sugar residues contributing to half of its apparent molecular mass. We provide evidence here that the 9.7-kDa protein is a
constitutive subunit of the Vo complex of the mature
V-ATPase holoenzyme.
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 et
al. (7).
N-terminal Protein Sequencing--
V-ATPase was isolated from
the goblet cell apical membranes of larval M. sexta midgut
as described previously (8, 9). Three hundred µg of purified V-ATPase
were subjected to preparative SDS-PAGE and stained with Coomassie Blue.
The 20-kDa band was excised from the gel and concentrated as described
by Rider et al. (10) but using 5% polyacrylamide as spacer
and a 15% polyacrylamide gel underneath. The resulting protein spot
was blotted onto a polyvinylidene difluoride membrane (Immobilon P)
using a buffer system consisting of 10 mM
NaHCO3 and 3 mM Na2CO3.
After staining with Amido Black, the spot was excised and installed
into the blot cartridge of a model 473A protein sequencer (Applied
Biosystems), and its amino acid sequence was determined as described
previously (11).
Screening of an M. sexta cDNA Library and Sequencing of
Clones--
The N-terminal amino acid sequence of the M. sexta 20-kDa protein was used to design the degenerate primer
pM20-deg (5'-ATGGC(T/C)TTCTTCGTICC(TCA)AT(TC)AC(TC)GTITTC-3') which was
optimized according to the codon usage of M. sexta
proteins (12). Direct PCR (13) was performed in the presence of the primers pM20-deg (100 pmol) and pT7 (20 pmol,
5'-AATACGACTCACTATAGGGC-3'), the latter corresponding to the T7
promoter of the RNA Isolation and Northern Blotting--
Poly(A) RNA was
prepared from either the midgut or Malpighian tubules of fifth instar
larvae using the Quickprep Micro mRNA purification Kit from
Amersham Pharmacia Biotech. Aliquots of 3.5 µg were loaded onto a 1%
agarose, 2% formaldehyde gel. Sample preparation, gel electrophoresis,
and Northern transfer on Hybond N membranes (Amersham Pharmacia
Biotech) were performed as described previously (6). After UV
irradiation, the poly(A) RNA was hybridized with a
digoxigenin-11-dUTP-labeled single-stranded RNA probe. This probe had
been generated by in vitro transcription using the DIG RNA
labeling kit (Roche Molecular Biochemicals), T7 polymerase, and 1 µg
of the plasmid pcM20BSK-A, linearized by SacI restriction. Hybridization was performed for 14 h at 68 °C in 50%
formamide, 5× SSC buffer, 0.02% SDS, 0.1%
N-laurylsarcosine, and 2% (w/v) blocking reagent (Roche
Molecular Biochemicals) at a probe concentration of approximately 100 ng/ml. Stringency washing was carried out at 68 °C in low salt
buffer (0.1× SSC buffer, 0.1% SDS). Labeled poly(A) RNA was detected
by use of the chemiluminescent substrate CSPD® (Roche Molecular
Biochemicals) according to the manufacturer's protocol. The membranes
were exposed to a Kodak XAR 5 film for 30 min.
Detection of Glycoproteins--
Deglycosylation of the V-ATPase
isolated from partially purified goblet cell apical membranes was
performed in a buffer containing 50 mM Tris-HCl (pH 8.0)
and 0.1% SDS. One hundred µg of V-ATPase were treated with 0.3 units
of endoglycosidase F/N-glycosidase F (Roche Molecular
Biochemicals) in 200 µl of incubation buffer for 12 h at
37 °C. The proteins were precipitated by trichloroacetic acid,
subjected to SDS-PAGE, then blotted and stained with 10 µg
ml Isolation and Deglycosylation of the 20-kDa
Protein--
V-ATPase was purified according to Schweikl et
al. (8) and Wieczorek et al. (9), modified as follows.
First, the whole midgut tissue was used for solubilization instead of
partially purified goblet cell apical membranes. Second, the first
steps of preparation were performed in the presence of 5 mM
Pefabloc® Sc (Biomol). Finally, centrifugation on the discontinuous
sucrose density gradient was carried out in the presence of 0.2 M KCl. 0.8 mg of purified V-ATPase was subjected to
preparative SDS-PAGE and negative-stained by the precipitation of a
white imidazole-zinc complex (21). The 20-kDa protein band was excised
from the gel and shredded into small pieces, leading to a total volume
of approximately 0.4 ml. Four hundred µl of a buffer consisting of
0.2 M sodium acetate (pH 5.2), 0.2% Triton X-100, and 5 mM Pefabloc® Sc were added to one-half of the shredded
gel, and 400 µl of the same buffer, in addition containing 3 milliunits of N-glycosidase A (Roche Molecular
Biochemicals), to the second half. Both samples were incubated under
gentle agitation for 36 h at 37 °C. The eluates obtained after
separation from the gel pieces were precipitated in trichloroacetic
acid and washed in acetone. Dried pellets were resuspended in Laemmli
buffer and subjected to SDS-PAGE. After Western blotting on a
nitrocellulose membrane (BA85), the protein bands were either
immunostained with the monoclonal antibody 224-3 (22) or silver-stained
using the kit from Amersham Pharmacia Biotech.
Coupled in Vitro Transcription/Translation of cDNA--
The
putative coding region together with the complete 3'-untranslated
region of the 20-kDa protein was amplified by PCR. The 27-mer upstream
primer started with a 6-base nonsense sequence followed by two cytosine
residues and the first 19 bases of the coding sequence
(5'-TATCAGCCATGGGTGCTTCCTTTGTCC-3'; the generated NcoI site is underlined) and the 22-mer downstream primer
(5'-GTAATACGACTCACTATAGGGC-3') corresponding to the T7 site of
pBluescript SK( Membrane Fractionation--
Midguts from fifth instar larvae
were homogenized with a glass Teflon homogenizer in a buffer containing
0.32 M sucrose, 0.02 M Tris-HCl (pH 7.6), 1 mM EGTA, 3 mM MgCl2, and 5 mM Pefabloc® Sc. After filtration through cotton gauze the
homogenate was centrifuged at 700 × g for 10 min at
4 °C. The resulting supernatant was again centrifuged at 7000 × g for 10 min at 4 °C. The microsomal pellet was
obtained by spinning the 7000 × g supernatant at
100,000 × g for 100 min at 4 °C. It was resuspended
in a buffer containing 0.02 M Tris-HCl (pH 7.6), 1 mM EGTA, 3 mM MgCl2, and 5 mM Pefabloc® Sc, layered onto a discontinuous 20-60%
(w/w) sucrose density gradient (10% steps), and centrifuged overnight
in a swing-out rotor (SW41Ti, Beckman) at 25,000 rpm and at 4 °C.
Fractions obtained were spun down and frozen in liquid nitrogen.
Other Methods--
Purification of goblet cell apical membranes
was performed according to Wieczorek et al. (9). Isolation
of V-ATPase from partially and from highly purified goblet cell apical
membranes of the M. sexta larval midgut as well as from
Malpighian tubules, protein determination by Amido Black, SDS-PAGE,
[14C]DCCD labeling, Western blotting, and immunostaining
were performed as described previously (8, 9, 23, 24). V-ATPase was assayed as enzyme activity sensitive to 1 µM bafilomycin
B1 (Fluka) according to Wieczorek et al. (9). The activities
of NADPH-cytochrome c reductase and glucose-6-phosphatase
were determined following modified protocols (25, 26). To inhibit
unspecific hydrolysis of glucose 6-phosphate by alkaline phosphatases,
1 mM levamisol was added to the assay mixtures. Cytoplasmic
expression of the 9.7-kDa polypeptide as an MBP fusion protein in
E. coli was performed according to Gräf et
al. (27) using the pMal-c2 expression system from New England
Biolabs. The vector that was obtained by inserting the coding sequence
and the 3'-untranslated region of pcM20BSK-A into the multiple cloning
site of pMal-c2 was named pM9.7Mal-c2.
The M. sexta V-ATPase Contains a 20-kDa Polypeptide as Part of Its
Vo Complex--
In immunoblots of V-ATPase isolated from
highly purified goblet cell apical membranes, a polyclonal antiserum to
the M. sexta V-ATPase revealed a prominent protein band with
an apparent molecular mass of ~20 kDa (lane 1 in Fig.
1, see also Ref. 28). The polypeptide could also be visualized by silver staining (lane 2 in Fig.
1; see also Ref. 22), whereas staining with Amido Black (lane
3 in Fig. 1) as well as with Coomassie Blue (not shown)
usually did not reveal detectable amounts of polypeptide. In
immunoblots of V-ATPase purified from either midgut goblet cell apical
membranes or Malpighian tubule brush border membranes, the 20-kDa
polypeptide was recognized by the monoclonal antibody 224-3 (lanes 4 and 5 in Fig. 1, respectively), which is
specific for this polypeptide but also shows slight cross-reactivity to
subunit B (22). Since the 20-kDa polypeptide was isolated, together
with known V-ATPase subunits, from two different types of highly
purified plasma membranes originating from two different tissues,
midgut and Malpighian tubules, it appears to be a constitutive subunit
of the insect V-ATPase.
Two previous experiments already had indicated that the 20-kDa
polypeptide may be a member of the Vo complex. First, it
remained in the membrane fraction after peripheral subunits were
stripped off by chaotropic iodide (24). Second, its relative amount in goblet cell apical membranes was enriched during moult when the V1 complex was released from the membrane (22, 29).
If the 20-kDa polypeptide is a Vo subunit, it should be
present in the free Vo complex together with the
established M. sexta Vo subunits c (5) and M40
(6). Partially purified goblet cell apical membranes from starving
larvae turned out to be a good source for the isolation of the
Vo complex because, as in membranes from moulting larvae,
they contain enriched free Vo complexes from which the
V1 complexes have been
detached.3 After
solubilization of goblet cell apical membranes and zonal centrifugation
in a discontinuous sucrose density gradient (8, 9), the two established
Vo subunits c and M40 as well as the putative novel 20-kDa
subunit were found not only in the upper 30% fraction as part of the
remaining V1Vo holoenzyme but also in the upper
20% fraction as part of the integral Vo complex (Fig. 2). The strictly similar distribution of
the 20-kDa polypeptide and the Vo subunits c and M40
indicates that the novel polypeptide is a member of the Vo
complex.
Protein Sequencing of the 20-kDa Polypeptide Leads to the Isolation
of a cDNA Encoding a 9.7-kDa Protein--
The 20-kDa polypeptide
was isolated by SDS-PAGE, and its N-terminal protein sequence was
determined to be
(M/G)AXFVPITVF(L/T)ILXGXVGI. The first 10 amino acids (underlined) were chosen to design a codon-optimized, degenerate primer pM20-deg, assuming that the initial
amino acid was methionine and that the third one was possibly phenylalanine (see "Experimental Procedures"). PCR using the
M. sexta
To obtain a full-length cDNA clone, the M. sexta
Although the protein deduced from cDNA cloning was only
approximately half the size of the 20-kDa polypeptide identified by SDS-PAGE, four findings suggest that the 20-kDa protein was encoded by
the open reading frame. First, the deduced N-terminal 19 amino acids
were in entire agreement with the data obtained from sequencing of the
20-kDa protein (Fig. 4), except for the missing N-terminal methionine
in the mature protein. Moreover, the initiator ATG (nucleotide
positions 64-66) of the open reading frame was embedded in a sequence
environment that is similar to that of other cloned cDNAs encoding
V-ATPase subunits from M. sexta (5, 14, 24, 27, 36) and
matched closely the Kozak consensus sequence for translational
initiation by eucaryotic ribosomes (37). Second, cDNA sequencing
was performed three times with cDNAs cloned independently, each in
both directions, rendering unlikely sequencing mistakes or cloning
artifacts leading to a frameshift. Even if nucleotides were deleted or
inserted, the resulting reading frames would not increase substantially
the molecular masses of the corresponding polypeptides. Third, the
initial PCR performed on the
To supply direct and definitive evidence that the cloned cDNA
encoded the 20-kDa polypeptide, we expressed the recombinant 9.7-kDa
protein as a fusion protein in E. coli. After SDS-PAGE the
fusion protein was blotted and immunostained with the monoclonal antibody 224-3. In contrast to the unfused maltose-binding protein (MBP) with a molecular mass of 42.7 kDa, the 9.7-kDa/MBP-fusion protein
had an electrophoretic mobility corresponding to 53 kDa and was
immunoreactive to the 20-kDa-specific, monoclonal antibody 224-3 (see
Fig. 7, lanes 5 and 6).
A BLASTP search revealed that the deduced M. sexta amino
acid sequence was 46% identical and 69% similar to the 9.2-kDa
membrane sector-associated protein of V-ATPase subunit recently cloned from human, bovine, and murine sources (Fig. 4), which differ from each
other in only one amino acid at position 22 (2). Similarities were also
detected to the unidentified open reading frames of
Caenorhabditis elegans chromosome IV (44% identity, 70%
similarity) and of Drosophila melanogaster chromosome III (29% identity, 55% similarity), both reported recently (2), and to
the yeast Vma21p protein (38) (24% identity, 51% similarity) and the
E. coli UncI gene product (39) (21% identity, 56%
similarity). In general, all similarities observed were accompanied by
a similarity of the predicted hydropathic properties (not shown).
The 20-kDa Polypeptide Is Glycosylated--
The deduced amino acid
sequence of the M9.7 protein contains two potential
N-glycosylation sites near the C terminus. Thus it appeared
plausible that the discrepancy between the molecular mass of 9.7 kDa
calculated from the open reading frame and that of 20-kDa determined
from SDS-PAGE of the V-ATPase was due to the posttranslational
processing of the 9.7-kDa protein by N-glycosylation.
Glycosylation of the 20-kDa protein had already been suggested
previously (28), because the band detected by immunostaining after
SDS-PAGE consistently appeared rather broad and diffuse (Fig. 1). To
check for glycosylation of the 20-kDa polypeptide, we deglycosylated
purified M. sexta V-ATPase by treatment with a mixture of
endoglycosidase F and N-glycosidase F (40). After SDS-PAGE
and Western blotting, putative glycoproteins were visualized by
staining with concanavalin A. Whereas lectin staining of the untreated
control resulted in the appearance of two major bands at 40 and 20 kDa,
respectively, deglycosylation of the V-ATPase resulted in a complete
loss of reactivity to concanavalin A (Fig. 6). Thus both lectin staining and the
susceptibility to endoglycosidase F/N-glycosidase F
treatment suggested that the 20-kDa polypeptide of the M. sexta V-ATPase is an N-linked glycoprotein.
Deglycosylation of the 20-kDa Polypeptide Leads to 16- and 10-kDa
Products--
The 20-kDa polypeptide band from SDS-PAGE was excised
from the gel and treated with glycosidase. To cleave
N-linked glycans carrying a fucose The 20-kDa Polypeptide Is a Subunit of the Mature
V-ATPase--
Copurification of the 20-kDa polypeptide with
established V-ATPase subunits in preparations of the
V1Vo complex and the partially purified
Vo complex already had indicated that it is a member of the
plasma membrane V-ATPase of M. sexta (see above). By
contrast, the putative yeast homologue Vma21p evidently is not a
constituent part of the V-ATPase. Instead, it resides in the membranes
of the endoplasmic reticulum, where it is required for the assembly of
the integral membrane sector of the V-ATPase (38). To exclude the
possibility that the presence of the Manduca polypeptide in goblet cell apical membranes results from contaminating ER membranes, we partially purified midgut microsomal membranes and assayed V-ATPase
activity and the activities of the ER marker enzymes, NADPH-cytochrome
c reductase and glucose-6-phosphatase in the various
fractions from sucrose density gradient centrifugation as well as in
purified goblet cell apical membranes. The highest activities of both
NADPH-cytochrome c reductase and glucose-6-phosphatase were
found in the 20/30% sucrose fraction, whereas V-ATPase activity in
this fraction was less than 10% that in purified goblet cell apical
membranes (Fig. 8A). By
contrast, the 30/40% sucrose fraction contained less ER marker enzyme
activity and considerably higher V-ATPase activity than the 20/30%
sucrose fraction. The highest V-ATPase activity was found in purified
goblet cell apical membranes, which exhibited only ~20% of the
activity of the ER marker enzymes. Immunoblots after SDS-PAGE using
equal protein amounts of the different membrane preparations indicated
that the 20-kDa polypeptide, which was detected by the monoclonal
antibody 224-3, was heavily enriched in the goblet cell apical
membranes, whereas the 20/30% sucrose fraction contained only minor
amounts (Fig. 8B). Since the same result was observed for
the constitutive V-ATPase subunit E, which was detected by monoclonal
antibody 90-7 (43), we conclude that, in contrast to its yeast
homologue Vma21p, the 20-kDa polypeptide is not retained in the
endoplasmic reticulum. We designate the 20-kDa polypeptide as subunit
M9.7, in accordance with the common nomenclature for Vo
subunits, because these results demonstrate that it is a constituent
part of the mature holoenzyme that resides predominantly in the plasma
membrane.
M9.7 Is a Constitutive Vo Subunit of the Mature
V-ATPase--
The cDNA encoding a prominent 20-kDa polypeptide in
V-ATPase preparations from two different tissues of M. sexta
larvae was cloned after N-terminal sequencing. Although the deduced
hydrophobic protein exhibits a molecular mass of only 9.7 kDa,
expression of the recombinant M9.7 protein in E. coli
revealed immunoreactivity to a monoclonal antibody directed to the
20-kDa polypeptide and thus confirmed that the cloned cDNA encoded
the 20-kDa polypeptide. The remarkable difference between the
theoretical molecular mass of 9.7 kDa and the electrophoretic mobility
at 20 kDa was shown to be caused by extensive
N-glycosylation.
The poor reactivity of the 20-kDa band to Coomassie Blue or Amido Black
may be a consequence of the less than 5% of positively charged amino
acids in the 9.7-kDa protein. In this respect the 9.7-kDa protein
resembles the proteolipid, subunit c, which is also poorly detectable
by both dyes, due to a low (4.5%) content of lysine and arginine. In
any case, the faint staining behavior of the M9.7 (20-kDa) protein does
not argue against our conclusion that it is a constitutive V-ATPase
subunit. By contrast, it appears to be a genuine Vo subunit
of the mature V-ATPase, since it copurifies in strictly reproducible
amounts with highly purified V-ATPase from midgut and Malpighian
tubules and since it is a significant component not only of the
holoenzyme but also of the partially purified Vo complex.
M9.7 and Its Mammalian Counterpart May Differ in
Glycosylation--
Deglycosylation of the 20-kDa polypeptide with
N-glycosidase A resulted in the weak immunostaining with the
monoclonal antibody 224-3 of a 10-kDa polypeptide and a strong staining
of a 16-kDa polypeptide, whereas the staining intensity of the 20-kDa
band decreased. This finding suggests that deglycosylation by
N-glycosidase A may occur at two different reaction rates as
follows: a fast step resulting in the decrease of the molecular mass
from 20 to 16 kDa, and a slow step reducing the molecular mass by an
additional 6 kDa. This conclusion is consistent with the existence of
two potential N-glycosylation sites on the deduced amino
acid sequence of the 9.7-kDa protein, one at positions 68-70 and the
other at positions 84-86, close to the C terminus. By contrast, the
amino acid sequences of the mammalian 9.2-kDa protein contain only one of the two potential N-glycosylation sites present in the
M. sexta protein (see Fig. 3). The bovine 9.2-kDa protein
was reported not to be affected by glycosidase F treatment (2).
However, several observations suggest that the V-ATPase isolated from
chromaffin granules may also contain a glycosylated 9.2-kDa protein.
Thus SDS-PAGE of bovine V-ATPase obtained by electrophoresis in blue native gels revealed a membrane-bound 16-kDa polypeptide that was not
susceptible to internal protein sequencing (2). It was tentatively
assigned to subunit M20, a putative Vo polypeptide first
reported for the coated vesicle V-ATPase (44, 45). The results obtained
from deglycosylation of the M. sexta 20-kDa polypeptide suggest that the 16-kDa protein represents an intermediate product resulting from preferential deglycosylation at only one glycosylation site. Thus, the bovine 16-kDa polypeptide may represent the
N-glycosylated version of the 9.2-kDa polypeptide, being
processed at the glycosylation site that is also conserved in the
9.7-kDa protein of M. sexta at amino acid positions 68-70.
Consequently, the conserved glycosylation sites could contribute to
approximately 6 kDa of the total molecular mass of both the bovine and
the M. sexta polypeptides, whereas the unique C-terminal
site of the M. sexta protein may contribute to an additional
4 kDa, resulting in a total molecular mass of 20 kDa in SDS-PAGE.
M. sexta Subunits M9.7 and B May Share an Epitope--
The
monoclonal antibody 224-3 not only cross-reacts with the Vo
subunit M9.7 but also with the V1 subunit B of the M. sexta V-ATPase (22). However, no immunocytochemical labeling was
detected in goblet cell apical membranes of the moulting midgut, which lacks V1 subunits, including subunit B, but contains all
subunits of the Vo complex, including M9.7. The
cross-reactivity of antibody 224-3, observed in immunoblots after
SDS-PAGE, with subunits B and M9.7 may be the result of the similar
epitope L(A/M)LTAA, with five of six identical amino acids that are
present in subunit B (positions 261-266) as well as in subunit M9.7
(positions 39-44). The epitope of subunit M9.7 is localized in a
region that is predicted to be in the transmembrane The Function of Subunit M9.7 Remains Enigmatic--
Sequence and
structural similarities of bovine M9.2 with yeast Vma21p already had
suggested that these proteins may be potential homologues (2). The
yeast protein is not a V-ATPase subunit but has been assigned to the
endoplasmic reticulum membrane where it may be involved in the assembly
of Vo subunits (38). By contrast, the extensive
glycosylation of the M. sexta polypeptide argues against
such a localization. Moreover, we have clearly demonstrated its
presence in high amounts in two preparations of purified target membranes of the insect V-ATPase, the goblet cell apical membrane from
midgut, and the brush border membrane from Malpighian tubules, and its
presence in low amounts in preparations enriched with endoplasmic
reticulum membranes.
Although the localization of the bovine polypeptide remains
questionable (2), like the M. sexta 9.7 polypeptide, it
lacks a di-lysine motif (46), the signal for retention in the
endoplasmic reticulum; on the other hand, this motif occurs in the
yeast protein (38). In addition, the topology of the yeast protein
seems to be inverse to that of the mammalian and insect proteins:
glycosylation at the C terminus of the M. sexta protein
indicates that the C terminus is exposed to the extracellular surface,
whereas the C terminus of Vma21p appears to be localized on the
cytosolic side of the membrane (38).
Significant similarities were also observed to the uncI gene
product of E. coli. uncI precedes the eight ATP synthase
genes and encodes a hydrophobic protein of 14.5 kDa which may be part of the F0 complex (39, 47). However, no function for
uncI has been described to date with the exception that it
may specifically affect the expression of the F-ATPase subunit a
encoded by uncB (48).
The M. sexta 9.7-kDa protein is more similar to the
mammalian 9.2-kDa V-ATPase subunit than to the putatively homologous
protein from D. melanogaster that was deduced from an
unidentified open reading frame. Since Manduca and
Drosophila V-ATPase subunits usually exhibit identities and
similarities of 77-98 and 86-99%, respectively, the putative
Drosophila protein may not be the exact correlate of the
M. sexta 9.7-kDa protein.
Prediction of the secondary structure of the mammalian V-ATPase subunit
M9.2 and its putative homologous counterparts in C. elegans,
D. melanogaster, and yeast as well as that of the UncI protein of E. coli revealed two membrane-spanning helices.
However, prediction of secondary structure of the M9.7 subunit of the
M. sexta V-ATPase revealed a high probability for only one
hydrophobic
The proton-conducting Vo complex consists of six copies of
possibly three varying proteolipids, subunit M40, and subunit M100 (1).
By the identification of the novel Vo subunits M9.7 and M9.2 from M. sexta and mammals, respectively, the total
number of genuine Vo subunits has increased. Whether the
list of Vo subunits is complete and in which way the
components form a functional Vo complex are exciting
questions that await further investigation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Zap II DNA and 2 × 105
plaque-forming units from an M. sexta larval midgut
-Zap
II cDNA library (14). The reaction was carried out with AmpliTaq DNA polymerase (Perkin-Elmer) in a buffer consisting of 50 mM KCl, 1.5 mM MgCl2, 0.01%
gelatin, 200 µM of each dNTP, and 10 mM
Tris-HCl (pH 8.3). After initial denaturation at 94 °C for 2 min,
temperature cycles were set as follows: 94 °C for 30 s, 53 °C for 60 s, 73 °C for 40 s, 40 repeats. The
resulting PCR product was cloned into the pUC18 vector using the
SureClone Ligation Kit from Amersham Pharmacia Biotech and sequenced to
verify the origin of the PCR product. To obtain full-length clones,
4 × 105 plaque-forming units of the
-Zap II
cDNA library (14) were screened using a digoxigenin-11-dUTP labeled
probe that had been generated by PCR (15) in the presence of the
primers pM20-1FWD (5'-GCATTGTGTGCCCTATCTTT-3') and pM20-3REV
(5'-CAGTAGTGGAATGACATCGG-3'). This approach led to the isolation of
five independent phage clones. The phagemid sections of two clones were
rescued by in vivo excision and subsequent infection of
Escherichia coli XL1-Blue (16). The derived pBluescript
SK(
) plasmid clones pcM20BSK-A and pcM20BSK-B were purified using the
QIAprep Spin Miniprep Kit from Qiagen and sequenced in both directions.
Sequencing of all plasmids was performed by the dideoxynucleotide chain
termination method (17) using Sequenase 2.0 (18) from Amersham
Pharmacia Biotech and appropriate sets of custom-synthesized 20-mer
oligonucleotides prepared by MWG Biotec, Germany.
1 concanavalin A and 20 µg ml
1
horseradish peroxidase modified according to Clegg (19) and Hawkes
(20).
). The PCR product was digested with NcoI
at the 5' end and with KpnI at the 3' end, purified by gel
electrophoresis in 0.75% agarose, and extracted from the gel using the
QIAquick gel extraction kit from Qiagen. The fragment was cloned into
the NcoI/KpnI-predigested translation vector
pSPUTK (Stratagene) and transformed into competent E. coli XL1-Blue. The resulting plasmid pM9.7SPK was prepared using the QIAprep
Spin Miniprep Kit from Qiagen; residual RNase was destroyed by a
treatment with proteinase K followed by phenol/chloroform extraction
and ethanol precipitation. Coupled in vitro
transcription/translation with SP6 polymerase and
[35S]methionine (20 µCi; Amersham Pharmacia
Biotech) was performed using the TNT-rabbit reticulocyte
system from Promega. After SDS-PAGE and Coomassie Blue
staining, the gel was incubated in enhancer solution
(EN3HANCE, DuPont) for 1 h, washed in cold water
for 1 h, and dried on Whatman filter paper.
[35S]Methionine-labeled protein bands were visualized by phosphorimaging.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
V-ATPase purified from midgut and Malpighian
tubules of M. sexta. SDS-PAGE and
immunoblots of different V-ATPase preparations from fifth instar
larvae. Lane 1, V-ATPase isolated according to Schweikl
et al. (8), except for the use of highly purified goblet
cell apical membranes (9) for solubilization of the holoenzyme. Blot
was stained with an immune serum to the purified V-ATPase (23);
lanes 2-4, V-ATPase isolated from a membrane fraction
enriched with goblet cell apical membranes as described by Schweikl
et al. (8). Lane 2, silver-stained gel;
lane 3, blot stained with Amido Black; lane 4, blot stained with the monoclonal antibody 224-3; lane 5,
V-ATPase isolated from purified Malpighian tubule brush border
membranes (24). Blot was stained with the monoclonal antibody 224-3. Lanes 1-5 were loaded with 5 µg of V-ATPase. Molecular
masses as derived from SDS-PAGE with standard proteins are shown in
kDa.
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Fig. 2.
Immunoblots and DCCD labeling of V-ATPase
subunits from the midgut of starving M. sexta
larvae. V-ATPase was prepared according to Schweikl et
al. (8). During the last purification step, carried out on a
sucrose density gradient, the V1Vo holoenzyme
and the Vo complex are enriched in the upper 30% and the
upper 20% fractions, respectively. Lanes 1 and
2, blots of V-ATPase stained with an immune serum directed
to the purified V-ATPase but obtained from another rabbit than that
used in Fig. 1. It showed almost no immune reactivity to the 20-kDa
polypeptide. The proteins derived from either the upper 30%
(lane 1) or the upper 20% (lane 2) sucrose
fraction. Capital letters at the left indicate
identified V1-subunits or the putative
V1-subunits C and D (marked by asterisks);
lanes 3 and 4, specific labeling of
Vo subunits in the upper 30% fraction (lane 3)
and in the upper 20% fraction (lane 4) as follows:
a) monospecific antibodies to the Vo-subunit M40
(6); b) the monoclonal antibody 224-3 to the 20-kDa
polypeptide; and c) [14C]DCCD to detect the
proteolipid, subunit c. The corresponding protein bands are indicated
by arrows. SDS-PAGE was performed with 3 µg of protein in
case of immunoblotting, and with 6 µg of protein in case of
fluorographic [14C]DCCD detection.
[14C]DCCD-labeled protein bands were visualized by
phosphorimaging.
-Zap II cDNA library as a template resulted
in the specific amplification of a 750-bp fragment that was obtained
only in the presence of both primers, pM20-deg and pT7 (Fig.
3). Sequencing of the cloned PCR fragment
revealed that the correct cDNA encoding the 20-kDa protein had been
amplified; the sequence and position of the deduced N-terminal amino
acids perfectly matched with those that were determined from protein
sequencing at positions 11-19, a section that could not be attributed
to the upstream primer.
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Fig. 3.
Amplification of a specific DNA fragment by
direct PCR. A 750-bp fragment of a -Zap II cDNA library
from the midgut of M. sexta was amplified in the presence of
a T7 primer and a degenerate primer (pM20-deg) corresponding to the
amino acid sequence obtained by N-terminal sequencing of the putative
20-kDa V-ATPase subunit. PCR products were separated by agarose gel
electrophoresis and stained with ethidium bromide. Lane 1,
0.5 µg of standard DNA fragments (sizes indicated in kilobase pairs);
lane 2, PCR product obtained in the presence of both
primers; lanes 3 and 4, control reactions
exclusively performed with either primer pM20-deg (lane 3)
or primer pT7 (lane 4); lane 5, control reaction
performed in the presence of both primers but without template
DNA.
-Zap
II cDNA library was screened by hybridization with a
digoxigenin-11-dUTP-labeled probe corresponding to the PCR fragment
that was related to the 20-kDa polypeptide. After three screening
steps, two independent cDNA clones were isolated and turned out to
be identical in their nucleotide sequences. The cDNA sequence
comprised 765 base pairs and was terminated by a poly(A) tail of 31 bp
(Fig. 4). Unexpectedly, the open reading
frame which corresponded to the data obtained from protein sequencing
was only 264 bp in length, encoding a hydrophobic protein of 88 amino
acids with a calculated molecular mass of 9.67 kDa and an isoelectric
point at pH 9.04, determined according to Skoog and Wichman (30).
Prediction of hydropathic properties and secondary structure based on
different algorithms (31-35) showed a high probability for one
membrane-spanning
-helix in the range of amino acid positions
40-60. A second predicted hydrophobic
-helix within the first 20 amino acids may possibly be too short to span the membrane. The C
terminus was more hydrophilic than the N terminus and is likely to be
located at the extracellular surface since, according to the PROSITE
data base, it contains two potential glycosylation sites at positions
68-70 and 84-86 (Fig. 4).
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Fig. 4.
CDNA sequence and predicted amino acid
sequence of the M. sexta M9.7 polypeptide. The
M. sexta protein (GenBankTM accession number
AJ006029) is compared with the recently identified human V-ATPase
subunit M9.2 (GenBankTM accession number Y15286) and with
an unidentified open reading frame of D. melanogaster
(GenBankTM accession number L07835). Identical amino acids
are indicated by vertical bars and similar ones by
dots. Identities between all three sequences are highlighted
by gray shading. Putative polyadenylation signals in the
3'-untranslated region of the cDNA sequence are
underlined. Possible N-glycosylation sites of the
M. sexta protein are double underlined. In
addition, the N-terminal amino acid sequence obtained from protein
sequencing is shown in the gray box.
-Zap II DNA resulted in only one PCR
fragment of the indicated length even at relatively low stringency.
Thus, a possible splice variant encoding an expanded open reading frame
seems not to exist in the cDNA library. This conclusion was
supported by Northern blots of poly(A) RNA isolated from midgut and
Malpighian tubules; in both cases only one mRNA species of
approximately 800 bp in length was observed (Fig.
5). Moreover, this experiment placed the
identified mRNA precisely in those tissues in which the putative
20-kDa V-ATPase subunit was expressed (Fig. 1). Fourth and finally,
both the deduced 9.7-kDa protein and the 20-kDa polypeptide appear to
be hydrophobic membrane proteins.
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Fig. 5.
Northern blots of poly(A) RNA isolated from
midgut and Malpighian tubules of M. sexta. A
Northern blot was hybridized with a digoxigenin-labeled single-stranded
RNA probe against the cDNA encoding the 9.7-kDa polypeptide.
Lane 1, 5 µg of standard RNA fragments (sizes indicated in
kilobase pairs); lane 2, 3.5 µg of poly(A) RNA isolated
from Malpighian tubules; lane 3, 3.5 µg of poly(A) RNA
isolated from midgut.
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Fig. 6.
Detection of glycosylated proteins in a
V-ATPase preparation from M. sexta midgut.
Western blots after SDS-PAGE of V-ATPase isolated from goblet cell
apical membranes according to Schweikl et al. (8).
Lane 1, staining with an immune serum to the purified
V-ATPase; lanes 2 and 3, staining with
concanavalin A. Protein bands containing sugar residues were visualized
by the reaction of horseradish peroxidase with aminoethylcarbazole. The
gels were loaded with 5 µg each of untreated V-ATPase (lane
2) or of V-ATPase incubated with a mixture of endoglycosidase F
and N-glycosidase F (lane 3).
1,3-linked to
asparagine N-acetylglucosamine, a motif present in
lepidopteran glycoproteins (41), we used N-glycosidase A
which is capable of hydrolyzing most types of asparagine-bound
N-glycans including
1,3-bound core fucose residues (42).
After a further SDS-PAGE separation and Western blotting, deglycosylation products were detected by the monoclonal antibody 224-3. Treatment of the isolated 20-kDa polypeptide with
N-glycosidase A resulted in the appearance of two additional
immune reactive bands at approximately 16 and 10 kDa, whereas the
untreated, fully glycosylated polypeptide still migrated at 20 kDa
(Fig. 7). Since the control polypeptide
showed no signs of degradation, and since N-glycosidase A
did not exhibit protease activity (not shown), these results provided
clear evidence that the 20-kDa polypeptide was glycosylated to a high
degree. In addition, the lower deglycosylated protein band migrated in
SDS-PAGE at exactly the same molecular mass as the recombinant 9.7-kDa
protein obtained from coupled in vitro
transcription/translation of the cloned open reading frame (Fig. 7).
Finally, the recombinant 9.7-kDa protein obtained as a fusion protein
with maltose-binding protein by expression in E. coli
exhibited the same immunoreactivity as the 20-kDa polypeptide (see
above). Thus the remarkable difference between the calculated and the
apparent molecular masses of 10 and 20 kDa appears to be due to
extensive posttranslational processing of the 9.7-kDa protein by
N-glycosylation, contributing to half of the molecular mass
of the 20-kDa polypeptide observed in SDS-PAGE.
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Fig. 7.
Identification of the 9.7-kDa polypeptide as
the deglycosylated version of the 20-kDa polypeptide. Lane
1, 10 µg of standard proteins with molecular masses indicated in
kDa. After SDS-PAGE the proteins were transferred to a nitrocellulose
membrane and stained with Ponceau S; lanes 2 and
3, immunoblots after SDS-PAGE of the gel-excised 20-kDa
polypeptide, stained with the monoclonal antibody 224-3. Both
preparations in lanes 2 and 3 were treated in exactly the
same way except for the presence of N-glycosidase A which
was added only in the preparation shown in lane 3;
lane 4, autoradiography of the recombinant 9.7-kDa
polypeptide translated from the plasmid pM9.7SPK. Alignment with the
Western blots of lanes 1-3 was done on the basis of protein
standards; lanes 5 and 6, immunoblots from
SDS-PAGE of 2 µg of a crude E. coli homogenate after
induction of protein expression. The proteins were stained with the
monoclonal antibody 224-3; lane 5, control expression in
E. coli cells transformed with pMal-c2 encoding the unfused
maltose-binding protein (MBP); lane 6, expression in
E. coli cells transformed with pM9.7Mal-c2 encoding the
9.7-kDa MBP fusion protein.
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Fig. 8.
Immunoblots and specific activities of
V-ATPase and ER marker enzymes in different membrane preparations.
A, specific activities of NADPH-cytochrome c
reductase (I), glucose-6-phosphatase (II), and
V-ATPase (III) were determined for microsomal membrane
vesicles obtained from the 20/30% fraction (a) and the
30/40% fraction (b) of a sucrose density gradient as well
as for highly purified goblet cell apical membranes (c). The
specific maximal enzyme activities were 9.1 × 10 2 ± 1.1 × 10
2 units per mg of protein for
NADPH-cytochrome c reductase (n = 4),
8.4 × 10
3 ± 2.2 × 10
3 units
per mg of protein for glucose-6-phosphatase (n = 4),
and 0.72 ± 0.07 units per mg of protein for V-ATPase activity
(n = 3); values are means ± S.E. with numbers of
independent preparations (n) shown in parentheses.
B, each 1 µg of microsomal membranes obtained from the
20/30% (a) and the 30/40% sucrose fraction (b)
as well as highly purified goblet cell apical membranes (c)
were separated by SDS-PAGE, blotted and stained with the monoclonal
antibodies 90-7 to subunit E (43) and 224-3 to subunit M9.7 (22).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix.
Consequently, the lack of immunoreactivity of subunit M9.7 in
cryosections of M. sexta midgut may result from insufficient
accessibility of the epitope.
-helix at a central position similar to that known for
the homologous proteins. A second transmembrane helix at the N
terminus, such as that predicted for its homologues, remains uncertain
for the M. sexta protein because the predicted hydrophobic
-helix consists of 13-17 amino acids and thus may be too short to
span the membrane. Possibly this finding reflects a real structural
difference between the M. sexta and mammalian proteins, and
therefore subunit M9.7 could have another function than its mammalian
homologues. However, the N-terminal region is highly hydrophobic, and
therefore it could be embedded in the membrane by a different type of
secondary structure.
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FOOTNOTES |
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* This work was supported by Grant Wi 698 from the Deutsche Forschungsgemeinschaft and by National Institutes of Health Grant AI 22444.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) AJ006029.
¶ To whom correspondence and reprint requests should be addressed: University of Osnabrück, Dept. of Biology, Section of Animal Physiology, D-49069 Osnabrück, Germany. Tel.: 49-541-9693501; Fax: 49-541-9693503; E-mail: wieczorek{at}biologie.uni-osnabrueck.de.
1 M. Huss, R. Schmid, W. R. Harvey, and H. Wieczorek, unpublished data.
3 M. Huss, H. Merzendorfer, W. R. Harvey, and H. Wieczorek, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; MBP, maltose-binding protein; bp, base pair; DCCD, dicyclohexylcarbodiimide.
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