(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.
[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.
H-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)).
V-ATPases are heteromultimeric enzymes composed of peripheral
V 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).
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 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.
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 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).
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, -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.
Figure 3:
[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.
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 -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).
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 -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%.
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 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.
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 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).
The
function of the 13-kDa subunit is enigmatic so far, but the predicted
unusual secondary structure with one continuous, highly charged
-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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X92805[GenBank].