Plant Physiol. (1998) 118: 137-147
Initial Steps in the Assembly of the Vacuole-Type
H+-ATPase1
Richard K. Frey and
Stephen K. Randall*
Department of Biology, Indiana University-Purdue University at
Indianapolis, 723 West Michigan Street, Indianapolis, Indiana
46202-5132
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ABSTRACT |
The plant vacuole is acidified by a
complex multimeric enzyme, the vacuole-type H+-ATPase
(V-ATPase). The initial association of ATPase subunits on membranes was
studied using an in vitro assembly assay. The V-ATPase assembled onto
microsomes when V-ATPase subunits were supplied. However, when the A or
B subunit or the proteolipid were supplied individually, only the
proteolipid associated with membranes. By using poly(A+)
RNA depleted in the B subunit and proteolipid subunit mRNA, we
demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest
that poly(A+) RNA-encoded proteins are required to catalyze
the A subunit membrane assembly. Initial events were further studied by
in vivo protein labeling. Consistent with a temporal ordering of
V-ATPase assembly, membranes contained only the A subunit at early
times; at later times both the A and B subunits were found on the
membranes. A large-mass ATPase complex was not efficiently formed in
the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B
subunit.
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INTRODUCTION |
The accumulation of solutes in the vacuole, largely driven by two
vacuole H+ pumps, the pyrophosphatase and the
V-ATPase, allows the development of an osmotic gradient, which
ultimately leads to turgor pressure, a requisite for cell elongation
and the development of the mature plant cell. The V-ATPase, although
predominantly localized at the vacuole, has also been detected in a
variety of organelles that constitute the endomembrane system,
including the ER (Herman et al., 1994
), Golgi apparatus (Chanson and
Taiz, 1985; Ali and Akajuuza, 1986), and coated vesicles (Depta et al.,
1991). The V-ATPase is a large multimeric enzyme consisting of one or
more of 7 to 10 distinct subunits (Parry et al., 1989; Sze et al., 1992). The molecular mass of the holoenzyme has been estimated at 600 to 700 kD (Randall and Sze, 1986; Bremberger and Luttge, 1988;
Parry et al., 1989). The 69- to 72-kD (A subunit) and the 57- to
60-kD (B subunit) proteins are major subunits of the V-ATPase, with an
apparent stoichiometry of three each per holoenzyme (Sze et al., 1992).
There also appear to be six copies of a 16-kD proteolipid (Kaestner,
1988) and all other subunits are thought to exist in single copies
(Mandala and Taiz, 1986; Randall and Sze, 1986; Sze et al., 1992).
The V-ATPase is considered to have two distinct sectors (Rea et al.,
1987a). The soluble peripheral sector (V1)
contains the regulatory (B subunit; Manolson et al., 1985) and
catalytic subunits (A subunit; Mandala and Taiz, 1986; Randall and Sze,
1986). The integral membrane sector (V0) contains
the proteolipid and is thought to form the pore through which protons
are passed. Electron micrographs show a "ball-and-stalk" structure
with a diameter of 9 to 10 nm resting on a narrower stalk that connects
the ball and membrane (Klink and Luttge, 1991; Moore et al.,
1991; Taiz and Taiz, 1991; Sze et al., 1992). The
V1 sector (containing the A and B subunits and
several other polypeptides) is dissociated from the holoenzyme by
chaotropic anions (Lai et al., 1988; Ward et al., 1992) and can be
reassociated into a functional complex (Ward et al., 1992). This
indicates that the reassembly of the peripheral subunits with the
membrane sector does not require de novo protein synthesis. Reversible
assembly of the ATPase appears to be a regulatory mechanism in yeast
(Kane, 1995).
The V0 sector is dominated by the 16-kD
proteolipid, so called because of its solubility in chloroform-methanol
(Rea et al., 1987b). The deduced amino acid sequence of the oat 16-kD
subunit predicts four membrane-spanning domains (Lai et al., 1991).
Along with the 16-kD proteolipid, 32-, 13-, and 12-kD proteins have been identified as V0 sector proteins in oat
(Ward and Sze, 1992). A larger integral membrane protein that often
associates with the purified ATPase has been observed in a variety
of plant species and yeast (Arai et al., 1988; Parry et al., 1989;
DuPont and Morrissey, 1992; Manolson et al., 1992, 1994) and varies in
apparent molecular mass from 95 to 115 kD. This subunit appears
unnecessary for V-ATPase H+ pumping and ATP
hydrolysis activity in mung bean, oat, Kalanchoe, and corn
vacuoles (Ward et al., 1992). Disruption of this gene in yeast,
however, leads to a blockage in ATPase assembly on the tonoplast
(Manolson et al., 1992).
Yeast genomic deletion mutants have been useful tools in determining
requirements for assembly and targeting of the V-ATPase. Mutants
lacking the 69-, 60-, or 27-kD V1 subunits failed
to assemble into soluble complexes (Doherty and Kane, 1993), and
mutants lacking the 69-, 60-, 42-, 32-, or 27-kD
V1 subunits also failed to assemble the
V1 subunits onto the vacuole membrane (Kane et
al., 1992; Ho et al., 1993b; Graham et al., 1994). Mutants
lacking the 54-kD subunit could assemble and target the peripheral
complex to the tonoplast but could not translocate protons (Ho et al.,
1993a). Although complete assembly of the holoenzyme requires
all but the 54-kD subunit, a number of "intermediate" complexes
have been identified using a combination of native and denaturing gel
electrophoresis (Tomashek et al., 1996). The
V0 sector appears to be assembled as a separate
entity and is not affected by the presence or absence of the peripheral
sector subunits (Kane et al., 1992; Manolson et al., 1992; Bauerle et
al., 1993; Doherty and Kane 1993; Ho et al., 1993a, 1993b);
Graham et al., 1994). The 95-, 36-, and 16-kD subunits are all required
for the assembly of a stable integral membrane sector (Kane et al.,
1992; Doherty and Kane, 1993) and for tonoplast targeting of the
peripheral complex (Kane et al., 1992; Manolson et al., 1992; Bauerle
et al., 1993; Doherty and Kane, 1993). In addition to the ATPase
subunits, a 25.5-kD protein (Hirata et al., 1993) and a 21-kD
(ER-associated) protein (Hill and Stevens, 1995) are necessary for
V-ATPase assembly. These studies have thus demonstrated the
requirements for various subunits for final localization to the vacuole
and for the functionality of the proton pump. It is important to note
that all of these analyses address the steady-state situation in cells,
and thus may not address the mechanisms or requirements for the
initial steps of assembly.
Because little is known about the early events in biosynthesis and
assembly of the ATPase, we have developed an in vitro assay with which
we can observe the initial events of V-ATPase assembly in plants. This
assay exploited a heterologous translation system and ER membranes
(nonplant) so that the role of plant-specific factors could be
determined. Using this assay we show that although the
V1 sector of the ATPase holoenzyme may form in
the absence of membranes, the A subunit efficiently associates with
microsomal membranes even in the absence of stoichiometric amounts of
the 16-kD proteolipid and the B subunit. These experiments point to the
presence of a possible receptor or assembly factor that recognizes the
A subunit. Furthermore, this evidence suggests that assembly of the
holoenzyme in planta may first require binding of the A subunit to the
membrane. These data are consistent with a model of assembly in which
the A subunit is first assembled on the membrane (perhaps in
conjunction with V0- sector proteins) followed by the B subunit. This suggests a potential regulatory mechanism by which
the proton pump would be inactive until it reaches an appropriate
intracellular destination (possibly a subdomain of the ER or perhaps
the medial or trans Golgi), at which time it would be activated by
assembly of the appropriate remaining subunits.
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MATERIALS AND METHODS |
Plant Material
Oat (Avena sativa L.) seeds were germinated in the dark
at 25°C on cheesecloth suspended over an aerated solution of 0.5 mM CaSO4. Plants were harvested after
3 to 5 d of growth.
RNA Extraction
Oat roots were frozen in liquid nitrogen and pulverized to a fine
powder. The powder was then extracted with 4 M guanidinium thiocyanate and RNA was sedimented through a 5.66 M cesium
chloride cushion (Chirgwin et al., 1979).
Poly(A+) RNA was purified with
oligo(dT)-cellulose in a batchwise fashion (Maniatis et al.,
1982).
In Vitro Transcription of Cloned V-ATPase Subunit Genes
In vitro transcription of cDNAs encoding V-ATPase subunits was
performed using a commercially obtained transcription kit (Promega) according to the manufacturer's instructions. A cDNA (accession no.
J03769; Zimniak et al., 1988) encoding the A subunit from carrot
(Daucus carota) was graciously provided by Dr. Lincoln Taiz;
a cDNA (accession no. J04185; Manolson et al., 1988) encoding the B
subunit from Arabidopsis was graciously provided by Dr. Morris
Manolson; and a cDNA (accession no. M73232; Lai et al., 1991) encoding
the 16-kD subunit from oat was graciously provided by Dr. Heven Sze.
Fractionation of Poly(A+) RNA on Suc Gradients
Poly(A+) RNA purified as described above was
fractionated on linear 30% to 60% (w/w) Suc gradients. Suc solutions
were prepared in 10 mM Hepes, pH 7.5, 1 mM EDTA
(pH 7.5), 0.25% Sarcosyl, and treated overnight with 0.1% DEPC. After
20 min of autoclaving, the volumes were adjusted to the original volume
with DEPC-treated water. To reduce possible RNase contamination, the
gradient maker was cleaned with 5 M hydrochloric acid,
rinsed extensively with DEPC-treated water, and then rinsed with
the 30% and 60% Suc stocks. Ultracentrifuge tubes were treated with
3% (v/v) hydrogen peroxide for 1 h and rinsed three times with
DEPC-treated water. Poly(A+) RNA (100 µL,
approximately 40 µg) was loaded on top of a 5.3-mL Suc gradient.
Gradients were centrifuged for 6 h at 70,000 rpm in an NVT90 rotor
(Beckman). Fractions (200 µL) were collected dropwise from the bottom
of the tube. After dilution to 400 µL with DEPC-treated water, 2.5 µg of yeast tRNA was added as a carrier, and the RNA was precipitated
with 0.3 M ammonium acetate and 2.5 volumes of ethanol. The
final precipitate was resuspended in 10 µL of DEPC-treated water.
Generally, 0.5 µL was used in a 12-µL translation assay.
Quantitation of the 16-kD Proteolipid RNA
RNA fractions separated on Suc gradients (1 µL each) were
blotted onto Nytran Plus membranes (Schleicher & Schuell) using a
Minifold II slot-blot apparatus (Schleicher & Schuell). Blots were
probed with a 32P-labeled antisense riboprobe
generated from the cloned 16-kD proteolipid sequence (Lai et al., 1991)
under conditions that revealed a single-sized message on northern blots
(not shown). The autoradiograms were quantitated by densitometry using
the National Institutes of Health Image 1.44 image-processing and -analysis program in conjunction with a scanner (Apple, Cupertino, CA).
In Vivo Labeling of Oat Roots
Labeling and extraction of
[35S]Met-labeled oat roots was performed as
described previously (Randall and Sze, 1989).
In Vitro Translation and Immunoprecipitation of ATPase
Subunits
In vitro translations of poly(A+) RNA and of
in vitro-synthesized mRNAs were performed using a commercially obtained
rabbit reticulocyte lysate (Promega) and
[35S]Met (Amersham) according to the
manufacturers' instructions. Where cDNA-encoded mRNAs were used as the
template, 9 µL of reticulocyte lysate, 2 µL of
[35S]Met (10 µCi/µL), 0.5 µL of cold
amino acids (1 mM each), and 0.5 µL of RNA were used in
the translations. In general, the amount of RNA in a translation
mixture was not quantified, but the amount of RNA added to translations
was optimized for the most efficient translation in the presence of
microsomes for each batch. When mixtures were treated with proteinase
K, 11.5 µL of translation mixture was diluted to 100 µL with KMH
(10 mM Hepes, 80 mM KCl, and 2 mM
magnesium acetate, pH 7.5) and treated with 0.5 mg/mL proteinase K for
30 min on ice. The reaction was stopped by the addition of 5 mg/mL PMSF
(in DMSO, final concentration 0.1% [v/v]) for 10 min on ice. Samples
were then processed as in the assembly assay described below.
For immunoprecipitation, proteins labeled with
[35S]Met were solubilized in 4% SDS, 50 mM Tris/HCl (pH 7.4), heated at 95°C for 4 min, and then
cooled to 4°C. The extracts were diluted 2-fold with water and then
further diluted 4-fold with 2.5% Triton X-100, 190 mM
NaCl, 6 mM EDTA, and 50 mM Tris (adjusted with
hydrochloric acid to pH 7.4). Proteinase inhibitors were added (1 mM PMSF, 0.3 mg/mL leupeptin, and 10 units/mL aprotinin) to
the extract. After a pretreatment with 20 µL of a suspension of 10%
(w/v) protein A-Sepharose (Sigma) for 30 min at 4°C, the extract was
centrifuged in a tabletop microcentrifuge at maximum speed for 2 min to
remove nonspecifically binding proteins. Immune or preimmune sera
(anti-ATPase; Randall and Sze, 1989) were generally used at a 1:1000
dilution for immunoprecipitation. The mixtures were rotated
end-over-end overnight at 4°C. The antigen-antibody complex was
incubated with 20 µL of 10% protein A-Sepharose for 30 min at 4°C.
Samples were centrifuged in a tabletop microcentrifuge at maximum speed
for 2 min and the resultant pellets were washed five times with 0.75 mL
of 50 mM Tris, 5 mM EDTA, and 150 mM NaCl (adjusted to pH 7.4 with hydrochloric acid)
containing 0.1% Triton X-100, and two times with the same wash buffer
minus the detergent. The immunoprecipitated ATPase subunits were
resuspended in 2× electrophoresis sample buffer minus DTT (Laemmli,
1970). SDS-PAGE (generally 10% acrylamide) was performed according
to the method of Laemmli (1970).
In Vitro Assembly Assay
Translations were performed as described previously in the
presence or absence of commercially obtained canine microsomes (Promega). One microliter (20 equivalents) of microsomes per 12 µL of
translation mixture was added either at the time of translation (cotranslational) or after the translation process was terminated by
the addition to a final concentration of 5 µg/mL cyclohexamide (posttranslational). After a 1-h incubation at 37°C, translation mixtures were diluted to 100 µL with 0.25 M Suc in KMH.
The diluted translations were loaded onto a cushion consisting of 500 µL of 0.5 M Suc in KMH. After centrifugation in a TLA
100.3 rotor (Beckman) for 35 min at 75,000 rpm (245,000g at
maximal radius), the membranes were recovered in the pellet. The top
200 µL of the overlay was retained and considered nonmembrane
associated (supernatant). The pellet was resuspended in 200 µL of
0.25 M Suc in KMH. If necessary, immunoprecipitations were
conducted as described above.
Suc-Gradient Fractionation of in Vitro-Synthesized
ATPase Subunits
Translation mixtures were loaded onto a 20% to 60% (w/w)
continuous gradient of Suc in KMH. The gradients were centrifuged in an
NVT90 rotor for 2.25 h at 70,000 rpm (390,000g at
maximal radius). Fractions of approximately 300 µL were collected by
piercing the bottom of the tubes with a syringe needle and collecting
drops manually. Proteins ([35S]Met labeled)
derived from translations using poly(A+) mRNAs
were immunoprecipitated as described previously. Proteins derived from
in vitro-synthesized mRNAs were analyzed directly. BSA (66 kD),
amylase (200 kD), catalase (250 kD), and apoferritin (443 kD), run in
parallel gradients, were used as standards to estimate the size of the
protein complexes present at various Suc densities.
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RESULTS |
Assembly of V-ATPase Subunits onto Microsomes
To investigate the initial interactions of the V-ATPase with
membranes, we developed an in vitro assembly assay. Using total cellular poly(A+) mRNA from oat roots as a
template, radiolabeled proteins were synthesized in vitro in the
presence or absence of microsomes. The translation mixtures were
centrifuged through a Suc cushion, yielding a particulate fraction
containing membranes and membrane-associated proteins in the pellet and
soluble proteins (nonmembrane associated) on top of the cushion. To
determine the location of the V-ATPase subunits, the overlay and the
particulate fraction were subjected to immunoprecipitation using
anti-V-ATPase antisera followed by SDS-PAGE and fluorography. In
immunoprecipitation reactions this antibody is reactive primarily to
the A and B subunits (Randall and Sze, 1989). When the mRNA was
translated in the absence of microsomes, the A (69 kD) and the B (60 kD) V-ATPase subunits were found only in the overlay (Fig.
1A), indicating that they were soluble
(i.e. not aggregated). When translations were performed in the presence
of microsomes, the A and B ATPase subunits were localized primarily in
the membrane pellet, consistent with a membrane association of these
V-ATPase subunits (Fig. 1A). Thus, poly(A+) RNA
from oat root homogenates contained sufficient information to direct in
vitro membrane association of the A and B V-ATPase subunits. It should
be noted that although we cannot visualize all of the ATPase subunits
under these conditions, it is likely that all are represented in the
poly(A+) RNA.
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| Figure 1.
The V-ATPase associates with membranes in vitro.
A, Poly(A+) programmed translations were conducted in the
presence (+) or absence ( ) of microsomal membranes as described in
``Materials and Methods''. Membranes that passed through a Suc
cushion (lanes P [pellet]) and components remaining on top of
the cushion (lanes S [supernatant]) were immunoprecipitated
with anti-ATPase and separated by SDS-PAGE. The gels were soaked in
Amplify (NEN-Dupont), dried, and then exposed to Kodak XAR-5 film. B,
Assays were conducted as in A except for the posttranslational
assembly. In that case, translation was conducted in the absence of
microsomal membranes, terminated by the additional of 5 µg/mL
cyclohexamide, and then the mixture was incubated with
microsomal membranes for an additional hour. Only the membrane pellets
are shown. C, Assays were conducted as in A. After translations in the
presence of microsomal membranes, the entire mixture was further
incubated in the presence (+) or absence () of 0.5 mg/mL proteinase K
(as described in ``Materials and Methods''). i., Poly(A+)
programmed the translation and the membrane pellets obtained after
Suc-gradient centrifugation are shown. ATPase subunits were then
immunoprecipitated. ii., The RNA encoding the translocated positive
control,  -lactamase (supplied by Promega), programmed the
translation. To demonstrate that the membrane conferred protection from
degradation on -lactamase, the entire mixtures were analyzed
directly (no Suc-gradient fractionation) after protease treatment as in
i.
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Previously, it was observed that mRNA encoding the A subunit was
associated with membrane-bound ribosomes, whereas the mRNA encoding the
B subunit was not membrane associated (Randall and Sze, 1989). This
suggested that the A subunit might become membrane associated at the
time of synthesis (cotranslational association). To determine if
membrane association of the subunits could occur cotranslationally or
posttranslationally (after protein synthesis), translations of
poly(A+) mRNAs were performed in the presence and
absence of microsomes. In the posttranslational association
experiments, protein synthesis was terminated by the addition of 5 µg
mL
1 cyclohexamide before the addition of
membranes. Both the A and B subunits became membrane associated whether
membranes were present during or after translation (Fig. 1B). This
raises the possibility that in vivo, assembly of these ATPase
subunits onto the membrane could occur posttranslationally.
The A subunit is not an integral membrane protein, yet it is apparently
synthesized by membrane-associated ribosomes (Randall and Sze, 1989).
Proteins synthesized in this manner typically are translocated into the
lumen of the ER or are inserted into the membrane (Singer et al.,
1987). To determine whether the A and B subunits, when
synthesized in vitro, were located on the extralumenal or the lumenal
side of the microsome, their susceptibility to proteinase K was tested.
When pre--lactamase (a protein known to be translocated to the lumen
of the ER) was translated in the presence of microsomes, it became
proteinase K resistant (Fig. 1C). On the other hand, both ATPase
subunits associated with the microsomes were degraded by proteinase K
(Fig. 1C), consistent with an extralumenal location. These results
indicate a topology consistent with a cytosolic exposure of the
V1 sector, as is found on the vacuole membrane.
When the 16-kD V-ATPase proteolipid subunit (the product of an mRNA
transcript derived from a cDNA; see ``Materials and Methods'') was
translated in the presence of microsomes, it associated with the
membrane pellet (Fig. 2). The proteolipid
was soluble in chloroform:methanol (2:1, v/v), which is consistent with
the hydrophobic nature of this protein (Kaestner et al.
[1988]; Fig. 2). The majority of the higher-molecular-mass
protein products observed in some translations (Fig. 2, Total
Translation) do not appear in chloroform-methanol extracts. This
suggests that they likely represent aggregations of the proteolipid
that are mostly disrupted by chloroform-methanol treatment (Kaestner et
al., 1988). Because the translation of the proteolipid was programmed
by a unique mRNA (derived from its cDNA), this membrane association
does not require the presence of any additional plant proteins.
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| Figure 2.
The 16-kD proteolipid alone is sufficient for
membrane association. Assembly assays were conducted as described in
Figure 1, except that only the RNA (transcribed from the cDNA; Lai et
al. [1991]) encoding the 16-kD proteolipid was used to program the
translation. Total Translation shows the entire translation mixture
before extraction in chloroform-methanol. Additional bands greater than
16 kD are likely aggregates of the extremely hydrophobic 16-kD
proteolipid. The remaining lanes were all extracted in
chloroform:methanol (2:1, v/v) before loading on the gel. S, Soluble;
P, pellet.
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The proteolipid also associated with the microsomes whether membranes
were present during or after translation (Fig.
3). Membrane association of the
proteolipid appeared to be significantly more efficient, however, when
synthesis occurred in the presence of microsomes. This suggests that
this integral membrane protein is cotranslationally assembled in vivo.
It also appears that the nonmembrane-associated protein was sensitive
to proteolysis, since the total amount of proteolipid recovered was
consistently much less when the translation was conducted in the
absence of membranes. An alternative explanation may be a decreased
rate of synthesis caused by translational arrest occurring in the
absence of membranes.
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| Figure 3.
The 16-kD subunit associates with microsomal
membranes cotranslationally but inefficiently posttranslationally.
Assembly assays were conducted as described in Figure 1. RNA for the
16-kD proteolipid (as in Fig. 2) programmed translations. All samples
were extracted in chloroform-methanol before separation on the gel. S,
Soluble; P, pellet.
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To test the competency of the individual A and B subunits to associate
with membranes, unique mRNAs transcribed in vitro from cDNAs encoding
these subunits were translated in the presence of microsomes. These
subunits did not associate with microsomes, whether translated alone or
together (Fig. 4). Note that the largest molecular mass, B subunit protein likely represents the full-length B
subunit, whereas the smaller protein may be the result of internal initiation of translation (Manolson et al., 1988). Simultaneous translation of the 16-kD proteolipid with the A and B subunits did not
catalyze assembly of the latter two peripheral subunits to the membrane
(Fig. 5). Because the A and B subunits
associate with the membrane when they are supplied as total cellular
poly(A+) RNA (Fig. 1), but not when they are
supplied to the assembly assay individually (Fig. 5), it is likely that
components other than the A and B subunits and the 16-kD proteolipid
are required to achieve assembly (see ``Discussion'').
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| Figure 4.
The A and B subunits, either singly or together,
do not associate with the membrane. Assays were conducted as described
in Figure 1 except that translations were programmed with RNAs (derived
from cDNAs) for the A or B subunit. S, Soluble; P, pellet.
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| Figure 5.
The A and B subunits translated in concert will
not assemble on membranes even in the presence of the
membrane-associated 16-kD subunit. Assays were conducted as described
in Figures 1 and 2. Note that radioactivity at the gel front in lane S
is caused by unincorporated [35S]Met in that fraction. S,
Soluble; P, pellet.
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The A Subunit Can Become Membrane Associated in the
Presence of Substoichiometric Amounts of Several of the ATPase Subunits
It was clear that the in vitro assembly assay could sensitively
measure membrane association of ATPase subunits. When
poly(A+) mRNA served as the template, very
efficient assembly was detected (Fig. 1); however, when only the A, B,
or 16-kD proteolipid subunit was supplied, no assembly of the A or B
subunit occurred. Because previous results suggested that the A subunit
might be assembled on the membranes in the absence of the B subunit
(Randall and Sze, 1989), and because the A subunit itself did not
appear to be sufficient for self-association, we were interested in
whether a distinct entity was required for catalysis of membrane
association of the A subunit. To address this question, total mRNAs
were first fractionated. The mRNA encoding the A, B, and proteolipid
subunits were largely separated from each other by Suc-gradient
centrifugation (Fig. 6). The fractionated
mRNA was translated in vitro (Fig. 6A). Fraction 5 was substantially
depleted in both translatable mRNA for the B subunit (approximately
3.1-fold) and mRNA for the 16-kD proteolipid (approximately 5-fold).
Fraction 6 was slightly enriched in the B subunit over the A subunit,
but was depleted in 16-kD proteolipid mRNA. We have assumed that a
depletion in proteolipid mRNA resulted in a commensurate decrease in
protein synthesis in vitro, which is a reasonable assumption in that
translations were linearly dependent on mRNA concentrations. The
partially purified mRNAs were then translated in vitro and assayed for
in vitro assembly (Fig. 7). In the
fraction most highly enriched in the A-subunit RNA (fraction 5 in Fig.
6A), the A subunit associated efficiently with membranes (Fig. 7). In
fraction 6, both the A and B subunits were efficiently associated.
Because the A-subunit protein derived from a unique RNA alone did not
associate with the membrane in the presence of the B subunit or the
16-kD proteolipid (Fig. 4), yet the poly(A+) RNA
enriched in the A subunit did (Fig. 7), we must conclude that an
additional, as-yet-unknown plant mRNA-encoded component(s) catalyzes
the association of the A subunit with the membrane. The establishment
of the identity of this component (or components) will be essential to
obtain an understanding of the assembly of the V1
sector.
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| Figure 6.
Suc-gradient centrifugation of total
poly(A+) RNA results in a fraction enriched in A-subunit
RNA and depleted in B-subunit RNA and 16-kD proteolipid RNA. A, An
aliquot from each fraction was translated in the presence of
[35S]Met and the incorporation (protein synthesis) into a
portion was determined (Randall and Sze, 1989). The remaining
translation mixture was immunoprecipitated with anti-ATPase, separated
on a 12% gel, and fluorographed (inset). B, The individual
immunoprecipitated subunits were quantitated as described in
``Materials and Methods'' and plotted as the percentage of maximal
peak. The 16-kD message was determined by hybridization analysis of
each fraction on a slot-blot apparatus using a 32P-labeled
antisense transcript from the oat 16-kD proteolipid clone (Lai et al.,
1991) as the probe.
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| Figure 7.
The A subunit in the presence of substoichiometric
amounts of the B subunit and 16-kD proteolipid associates with
membranes. Fractionated poly(A+) RNA fractions 5 (F5) and 6 (F6) were obtained as shown in Figure 6. Although the RNAs were
obtained from a single experiment, this assembly experiment using those
fractionated RNAs is representative of two experiments.
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V1 Subunits Inefficiently Form a
High-Molecular-Mass Complex
The peripheral subunits of the plant V-ATPase have been
solubilized from vacuole membranes by chaotropic anions, and after removal of the chaotrope in the presence of membranes, they reassembled into a functional enzyme (Ward et al., 1992). Thus, it is possible to
reassemble the V1-sector proteins with the
membrane components in vitro. Furthermore, studies of yeast V-ATPases
have shown that the A, B, and 27-kD peripheral subunits can be
found in a soluble complex, the V1 sector (Kane
et al., 1992). The yeast V1 complex has an
apparent molecular mass of 400 to 500 kD, as determined by glycerol
gradients (Doherty and Kane, 1993). To test whether the plant enzyme
could be assembled into a soluble V1 complex, we
translated the individual subunits, a combination of the A and B
subunits, or total poly(A+) mRNA, all in the
absence of microsomes (but otherwise under conditions similar to those
in the translation and assembly assays). The sizes of the complexes
were estimated by ultracentrifugation in Suc gradients. When the A and
B subunits were translated together (Fig.
8) and fractionated on a Suc gradient,
the estimated molecular masses for the A and B subunits in both cases
were less than 100 kD. This indicated that these subunits did not
aggregate and could not form partial complexes. The failure of the A
and B subunits alone to form a stable complex is perhaps not
surprising, given the requirement for the additional 27-kD subunit in
yeast (Kane et al., 1992). This is also consistent with the observation
(by two-hybrid analysis) that the A and B subunits can only weakly interact (Tomashek et al., 1996).
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| Figure 8.
The A and B subunits alone do not assemble into a
high-molecular-mass complex. After translation, the synthesized
proteins were fractionated on 30% to 60% (w/w) Suc gradients.
Aliquots were directly loaded onto SDS-PAGE gels and analyzed by
fluorography. Note that the B subunit typically runs as a doublet after
translation of cDNA-derived message. The migration positions of
molecular-mass markers, run in a parallel gradient, are indicated at
the top of the panels. Data are representative of two experiments.
|
|
The in vitro-synthesized subunits (poly[A+]
programmed) have been shown in this study to assemble efficiently on
membranes (Fig. 1), but do not appear to efficiently form a
high-molecular-mass complex in the absence of membranes (Fig.
9). The majority of the A and B subunits
migrated in the gradient at a molecular mass of less than 100 kD.
However, a small proportion of the A and B subunits migrated at a
molecular mass of greater than 443 kD, suggesting the inefficient
formation of the V1 sector (not discernible in
Fig. 9). It is possible that this complex could also contain chaperone
proteins. Note that these same subunits are very efficiently associated
when membranes are present under virtually identical conditions (i.e.
the same buffers and centrifugation through a Suc cushion; Fig. 1).
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| Figure 9.
The poly(A+)-encoded A and B subunits
do not assemble into a high-molecular-mass complex. After translation
of total poly(A+) RNA, the synthesized proteins were
fractionated on 30% to 60% (w/w) Suc gradients and immunoprecipitated
with anti-ATPase antibody. Aliquots were loaded onto SDS-PAGE gels and
analyzed by fluorography. A and B designate the positions of the A and
B subunits. Band 1 represents a 91-kD polypeptide that reacts with the
polyclonal antibody, and band 2 represents a 64-kD polypeptide that is
likely to be calnexin. The migration positions of molecular-mass
markers, run in a parallel gradient, are indicated at the top of the
panels. Data are representative of two experiments.
|
|
Discontinuity of A- and B-Subunit Membrane Assembly in
Vivo
The data described above suggest that the A subunit is capable of
associating with the membrane in the absence of stoichiometric amounts
of the B subunit and the 16-kD proteolipid. However, the Suc-gradient
analysis indicated that in the presence of all subunits (poly[A+] programmed translation), a portion of
the A subunit was found in a higher-molecular-mass complex containing
the B subunit. The former observation suggested that an initial step in
V-ATPase assembly could be A-subunit association with the membrane,
whereas the latter observation suggested that the formation of a
soluble V1 sector containing the A subunit might
be an initial step.
To determine which mechanism would be the most likely in vivo, we
tested whether the A and B subunits were simultaneously associated with
the membrane immediately after synthesis. Oat roots were briefly (1 h)
labeled with [35S]Met. After this labeling
period roots were immediately homogenized using conditions previously
shown to maintain ATPase activity (Randall and Sze, 1986), and membrane
and soluble fractions were obtained. Under these isolation conditions,
no A or B subunits in the soluble fraction have ever been observed
(data not shown), indicating the stability of the assembled complex.
ATPase subunits were immunoprecipitated from both membrane and soluble
fractions and analyzed by SDS-PAGE. After this short labeling period a
significant portion of the A subunit, but not the B subunit, was found
to be associated with the membrane fraction (Fig.
10). In the soluble fraction all of the
B subunit and a portion of the A subunit were present. Based on
longer-time-course labeling experiments, by 2 h approximately
similar amounts of the A and B subunits were present on the membranes
(data not shown).
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| Figure 10.
The A subunit assembles on membranes before the B
subunit in vivo. Oat roots were labeled with [35S]Met for
1 h as described in ``Materials and Methods''. Roots were
homogenized in a buffer designed to maintain a functional ATPase
complex (Randall and Sze, 1986), and membranes were fractionated on a
Suc cushion, immunoprecipitated with anti-ATPase, resolved by SDS-PAGE,
and analyzed by fluorography. Shown is one of two representative
experiments.
|
|
| |
DISCUSSION |
This study reports the results of the development of an in vitro
membrane-assembly system, using either poly(A+)
RNAs or unique RNAs derived from cDNAs as the source of ATPase subunits. The role of canine microsomes and reticulocyte lysate in this
study was to supply generalized functions necessary for protein
synthesis and assembly so that plant-specific requirements could be
demonstrated. The microsomes were competent to import and process
pre--lactamase, indicating a functional translocation apparatus. In
addition to the components necessary for the translocation and cleavage
of precursors, the canine microsomes may also possess membrane-associated proteins, which might have an effect on V-ATPase assembly. However, through the addition of the plant subunits we could
examine the unique requirements of the plant V-ATPase assembly.
We have demonstrated that activities contributed by canine ER, rabbit
reticulocyte lysate, and oat mRNA-encoded proteins can together
catalyze peripheral V1 assembly onto the
membrane. Although we chose the heterologous system deliberately, there
are several aspects of the in vitro-assembly assay over which we had no
control. When this study was begun, none of the plant ATPase subunits
was available from the same organism, so we used cDNAs derived from different plant species. Not all of the putative ATPase subunits from
plants have yet been cloned and sequenced. The ATPase subunits, however, are highly conserved in amino acid sequences in both the
animal and plant kingdoms. For example, the bovine- and carrot-deduced full-length A subunit sequences are 67% identical and 84% similar. Specifically relevant to this study is the fact that if one compares sequences of two Arabidopsis expressed sequence tags (accession nos.
H76218 and T14078) encoding the A subunit with the full-length carrot
cDNA (Zimniak et al., 1988), a deduced sequence (overlap of
approximately 181 amino acids, corresponding to amino acids 401-582 of
the carrot protein) shows an identity of 98% with the carrot sequence,
where the differences are extremely conservative changes (e.g. Gly for
Ala, Ser for Thr, etc.). In addition, the phenotypes of yeast mutants
defective in the A subunit (T. Wilkins, personal communication) and a
newly identified 54-kD subunit, VMA13, in yeast (Lu et al., 1997) have
been suppressed by the plant protein cognates. We believe that
heterology (between the A subunit from carrot and the B subunit from
Arabidopsis) is thus an unlikely explanation for the failure of the A
and B subunits to form a complex or to associate with the membrane when supplied from cDNAs, although the possibility of a confounding effect
of multiple isoforms of these subunits cannot be discounted.
ATPase subunits were observed to be assembled onto membranes when all
subunit RNAs were present; however, when supplied individually the A
and B ATPase peripheral subunits did not assemble on the membrane. This
is not a surprising result because current models suggest a stalk-like
arrangement of subunits that would connect the V1
sector to the membrane-associated V0. However,
when mRNAs were fractionated to remove mRNA encoding the 16-kD
proteolipid and the B subunit (such that substoichiometric amounts of
the B subunit and 16-kD proteolipid were synthesized), the A subunit was still able to assemble on the membrane. These assembly assays using
fractionated plant RNA imply a requirement for a distinct plant-encoded
protein, since the cDNA-encoded A subunit did not associate with the
membrane in vitro. The ability to assemble the A subunit in the absence
of stoichiometric amounts of the B subunit and proteolipid (Fig. 7)
implies that this subunit could be assembled in the absence of these
subunits in vivo, and that this assembly in turn could result in a
temporal discontinuity in membrane association between the A and B
subunits. In support of this, in vivo labeling (Fig. 10) of oat roots
indicated a temporary discontinuity in assembly of the A and B
subunits, with the A subunit becoming membrane associated first.
In contrast to the peripheral subunits, the 16-kD proteolipid
(V0 sector) subunit can be synthesized and
assembled on the membrane in the absence of any other ATPase subunit.
The 16-kD proteolipid associates with membranes in a posttranslational
manner very inefficiently (Fig. 3). This suggests that
cotranslational assembly and integration of the proteolipid is more
likely.
It is unlikely that the 16-kD proteolipid is necessary for the initial
A subunit assembly, because poly(A+) depleted in
16-kD proteolipid still supports the A subunit membrane assembly (Figs.
6 and 7). It is remotely possible that canine microsomes contain
unassembled proteolipid, which might allow assembly of the A subunit.
However, the proteolipid is clearly not sufficient to catalyze the A
subunit assembly (Fig. 5). These observations suggest that a
specific component or components may be necessary to attach the A
subunit to the membrane. One could speculate that this component is a
final component of the functional ATPase holoenzyme, or alternatively,
that it may be a temporary scaffolding structure that might be
dismantled after ATPase assembly. It may be that membrane association
of the A subunit occurs without any interaction with the
V1- or V0-sector
components. If a membrane-bound scaffolding apparatus is involved in
the assembly of the V1 sector onto the
V0 sector, then the A subunit may interact with a
bound scaffolding protein and therefore partition as a membrane-bound protein, as demonstrated here during the initial assembly phase. This
latter hypothesis would explain the observations we have made, in which
under both in vitro and in vivo conditions the A subunit is membrane
associated (but the B subunit is not). It remains to be determined
whether this putative receptor/scaffold is a protein homologous to the
yeast 95-kD protein necessary for membrane localization of the ATPase,
or to the 21- and 25.5-kD yeast proteins necessary for V-ATPase
targeting and assembly, or whether it is a distinct protein. Recent
evidence suggests calnexin, a membrane-associated chaperone, as a
putative scaffold, since it can be found associated with the assembled
ATPase (Li et al., 1998). Consistent with this idea is the observation
that small amounts of calnexin are present in isolated vacuoles (Seals and Randall, 1997).
The localization of the A subunit on the membrane in the absence of the
B subunit (demonstrated both in vitro [Fig. 7] and in vivo [Fig.
10]) and the localization of mRNA encoding the A subunit but not the B
subunit on the membrane (Randall and Sze, 1989) suggests the following
model. Although it may be possible to assemble the
V1 sector as a soluble complex that is later
attached to the V0 sector through stalk proteins,
as suggested for the yeast ATPase, it appears more likely that in vivo
the plant A subunit is first assembled on the membrane. This could be
accomplished through the use of a membrane-localized receptor (perhaps
a "scaffolding" protein). At a later time the B subunit (and
perhaps other subunits) is integrated into the nascent complex. One
important ramification of this model may be in the regulation of
V-ATPase activity. If the ATPase were assembled in an active form on
the ER, then it might have proton-pumping activity. An active V-ATPase,
however, has not been observed on the ER, which is consistent with our results here. Because the Golgi apparatus is acidified and an active
V-ATPase pump has been demonstrated on this organelle (Chanson and
Taiz, 1985), the ATPase must be assembled into a functional enzyme
by the time it reaches that location. In this manner temporal control of assembly could result in the spatial regulation of V-ATPase
activity.
An alternative model, differing only in the site of V-ATPase assembly,
is suggested by the results of Herman et al. (1994), in which
immunological observations in oat roots indicated the presence of the B
subunit at specific subdomains of the ER. This model would involve the
soluble B subunit (and perhaps other subunits) joining with the A
subunit, which is already membrane associated, but in this case at a
specialized region of the ER destined to be targeted to the vacuole. In
support of this model, it was also noted in this immunological study
that no B subunit was observed on the Golgi apparatus and little to no
B subunit was observed at all in cells that were already vacuolated
(cells at a distance greater than 1 cm from the root tip).
Unfortunately, the A subunit could not be visualized in this study.
Both models should be viewed as viable possibilities in light of a
number of recent reports of alternative pathways to the vacuole (Hoh et
al., 1995; Matsuoka et al., 1995; Robinson and Hinz, 1996). The work by
Herman et al. (1994) focused on very "young cells" that lacked
large central vacuoles and were likely undergoing de novo vacuolation,
whereas our work more likely describes the replenishment of existing
vacuoles. It is tempting to speculate that the pathway suggested by
Herman et al. (1994) (a more direct route from ER to vacuole) might
occur during de novo vacuole biogenesis, whereas the pathway suggested by this paper (ER to vacuole via the Golgi complex) might be the route
for the continued maintenance of existing vacuoles.
The in vitro assembly assay described here will be a useful tool in the
study of V-ATPase assembly as cDNAs encoding other V-ATPase subunits
become available for study. Further identification of the components
requisite for individual assembly events could be achieved using the
poly(A+) mRNA pool and antisense mRNAs, which
would eliminate a single mRNA species from the
poly(A+) pool. This would allow assembly studies
of initial steps lacking one or more subunits in an otherwise
functional assembly assay. When cDNAs corresponding to all subunits of
the V-ATPase have been cloned and can be transcribed together in vitro,
the minimum subunit assembly requirements and the sequence of events
leading to functional assembly can then be characterized. The role of plant proteins corresponding to the yeast 21- and 25.5-kD ER-associated proteins in V-ATPase assembly could also be examined. In addition, site-directed mutagenesis of the subunits would further our
understanding of the amino acid sequence requirements for the initial
stages of assembly of the V-ATPase holoenzyme. Finally, by generating a
cDNA library (derived from fractionated
poly[A+] RNA, as in Fig. 6, which is enriched
in the A subunit but depleted in the B subunit and the 16-kD
proteolipid), one may obtain a functional screen for cDNAs encoding
assembly factor(s) necessary for the initial steps in A-subunit
membrane association.
| |
FOOTNOTES |
1
This work was supported in part by National
Science Foundation grant no. MCB-9205052 and a faculty development
award to S.K.R.
*
Corresponding author; e-mail srandal{at}iupui.edu; fax
1-317-274-2846.
Received February 5, 1998;
accepted May 28, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DEPC, diethyl pyrocarbamate.
V-ATPase, vacuole-type H+-ATPase.
| |
ACKNOWLEDGMENTS |
The authors are grateful to Dr. Lincoln Taiz, Dr. Morris
Manolson, and Dr. Heven Sze for supplying cloned cDNAs used in this study. Thanks to Dr. Lei Yu (Indiana University Medical School, Indiana University-Purdue University at Indianapolis) for suggestions regarding the RNA fractionation. We thank Darren F. Seals for a
critical review of the manuscript.
| |
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Top
Abstract
Introduction