Pflugers Arch - Eur J Physiol (2009) 457:599–607
DOI 10.1007/s00424-007-0417-x
CELL AND MOLECULAR PHYSIOLOGY
The V-ATPase in Paramecium: functional specialization
by multiple gene isoforms
Thomas Wassmer & Ivonne M. Sehring &
Roland Kissmehl & Helmut Plattner
Received: 14 October 2007 / Revised: 23 November 2007 / Accepted: 29 November 2007 / Published online: 29 January 2008
# Springer-Verlag 2007
Abstract The vacuolar H+-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump,
is essential for numerous cellular processes in all eukaryotes
investigated so far. While structure and catalytic mechanism
are similar to the evolutionarily related F-type ATPases, the
V-ATPase’s main function is to establish an electrochemical
proton potential across membranes using ATP hydrolysis.
The holoenzyme is formed by two subcomplexes, the
transmembraneous V0 and the cytoplasmic V1 complexes.
Sequencing of the whole genome of the ciliate Paramecium
tetraurelia enabled the identification of virtually all the
genes encoding V-ATPase subunits in this organism and the
studying of the localization of the enzyme and roles in
membrane trafficking and osmoregulation. Surprisingly, the
number of V-ATPase genes in this free-living protozoan is
strikingly higher than in any other species previously
studied. Especially abundant are V0-a-subunits with as
many as 17 encoding genes. This abundance creates the
possibility of forming a large number of different V-ATPase
holoenzymes by combination and has functional consequences by differential targeting to various organelles.
Keywords V-ATPase . Paramecium . Membrane traffic .
Osmoregulation . Contractile vacuole complex . Trichocyst .
Phagocytosis . Acidosomes
T. Wassmer (*)
Department of Biochemistry, School of Medical Sciences,
University of Bristol,
Bristol BS8 1TD, UK
e-mail: twassmer@bristol.ac.uk
I. M. Sehring : R. Kissmehl : H. Plattner
Department of Biology, Universität Konstanz,
Universitätsstraße 10,
78457 Konstanz, Germany
Introduction
The V-ATPase is a proton pump that drives many essential
processes in organisms as distantly related as humans, plants,
and protozoans (for reviews, see [1–3]). It hydrolyses
adenosine triphosphate (ATP) and uses this energy to pump
protons across membranes, building up a proton gradient as
well as an electric potential. The electrochemical potential
can be used for secondary active transport by antiporter
systems for various processes. This makes the V-ATPase
crucial as an acidifier of compartments and as an energizer
of membranes.
The structural organization of the V-ATPase resembles
the evolutionarily related F-ATPase. They both have a globular cytoplasmic domain and a membrane embedded domain,
the two being connected by a central stalk [4] (Fig. 1). While
the F-ATPase has only one additional, peripheral stalk, it was
shown that V-ATPase has more than one, connecting the
cytoplasmic and transmembraneous subcomplexes [5, 6].
The cytoplasmic subcomplex is named V1 and the transmembrane subcomplex V0. The subunits forming the central
stalk belong to V1 while subunits that compose the
peripheral stalks belong to either V1 or V0.
Early insights into the genes encoding V-ATPase subunits
were gathered from the budding yeast Saccharomyces
cerevisiae (for reviews, see [7, 8]). In yeast, the transmembraneous V0 complex is composed of six different
subunits, named a, c, c′, c″, d, e, encoded by seven genes
(Table 1) [3]. The soluble, cytoplasmic V1 complex is made
of eight different subunits, termed A, B, C, D, E, F, G, H,
encoded by eight genes (Table 1). Only the V0-a-subunit is
encoded by more than one gene in S. cerevisiae (vph1 and
stv1).
The enzyme was shown to work in a way somewhat
similar to a macroscopic turbine. The hydrolysis of ATP by
DO00417; No of Pages
600
Pflugers Arch - Eur J Physiol (2009) 457:599–607
Table 1 Number of V-ATPase genes in Saccharomyces cerevisiae,
Mus musculus, and Paramecium tetraurelia
V1
A
B
C
D
E
F
G
H
V0
a
c
c′
c″
d
e
S. cerevisiae
M. musculus
P. tetraurelia
1
1
1
1
1
1
1
1
1
2
2
1
2
1
3
1
4
4
1
1
4
2
Not identified
4
2
1
1
1
1
1
4
1
0
1
2
1
17
6
0
0
3
2
Compiled from Forgac [3] and Wassmer et al. [33]. V1-subunit G
could not be identified in P. tetraurelia probably because of limited
sequence conservation but is expected to be existent.
the cytoplasmic V1 A-subunits, assisted by the noncatalytic
B-subunits, drives the rotation of the central stalk, composed of D- and F-subunits, which transmit the rotation to
the membrane-embedded V0-c-subunit ring [3, 9, 10]. Each
of the highly hydrophobic c-subunits has a glutamate
residue critical for function that can pick up a proton
delivered to this glutamate, supposedly by a hemichannel in
the V0-a-subunit that allows proton entry from the
cytoplasm [3]. The carboxy group of the glutamate is
neutralized by the proton and then can swivel into the
hydrophobic phase constituted by the membrane surrounding the V0-c-subunits. As there are five to six c-subunits
organized in a ring that is driven by the central stalk,
protons are continuously picked up from the cytoplasm.
After a turn of the c-ring, protons are thought to be released
from the c-subunits through a second hemichannel in the
a-subunit leading to the luminal side of the membrane [3, 11].
One prerequisite is that the V0-a-subunit and the V1subcomplex rest immobile with respect to the c-subunit ring
and the central stalk. This suggests that the peripheral stalks,
containing the N-terminal half of the V0-a-subunit part and
V1-subunits C, E, H, G, serve as a “stator” [10, 12]. By the
rotational coupling of ATP hydrolysis to a turbine-like ring, a
molecular pump is created, which can build up huge proton
potentials.
Several mechanisms to regulate the activity of the VATPase have been suggested. One mechanism was shown
to be reversible disulfide bond formation at the catalytic
site on the A-subunit [13, 14]. Another possibility of regulation is reversible dissociation of the V1 and the V0
complex [3, 15, 16]. A third regulation mechanism was
suggested to be the distribution of the enzyme to different
cellular membranes [17].
Cell biology of the V-ATPase
In eukaryotic cells ranging from simple, unicellular to
complex, multicellular organisms, the V-ATPase fulfills a
broad range of functions [1, 3, 18, 19].
In yeast, the V-ATPase is primarily localized in the late
Golgi and in vacuoles [20]. The difference in localization is
mediated by the different V0-a-subunits these two enzyme
complexes contain [21]. They differ in coupling efficiency,
state of dissociation/association of V1 and V0 and pump
protons with different activities [3, 4]. This is probably one
explanation for the late Golgi to be only slightly acidic
while yeast vacuoles are strongly acidified.
In yeast, knockout of any V-ATPase subunit except the
two a-subunit genes, vph1 and stv1, results in the severe
“vacuolar membrane ATPase” (vma) phenotype, which
comprises growth deficiency in neutral medium and
hypersensitivity to elevated Ca2 concentration in the
medium [22]. Growth of yeast cells displaying a vma
phenotype is only sustained at acidic pH and low Ca2+
concentration. Only the double knockout of both vph1 and
stv1 lead to the vma-phenotype, suggesting that these two
subunits can substitute for each other to some degree in
yeast [20].
In mammals, the number of genes encoding V-ATPase
subunits is strikingly higher than in yeast (compare Table 1),
and therefore the subunit composition is more complex,
with variations between cell types and tissues [3, 23]. The
enzyme is targeted to numerous organelles, among them
early endosomes, late endosomes/lysosomes, the transGolgi network, dense core secretory granules, synaptic
vesicles, and in some specialized cells, to the plasma
membrane. Those responsible for the differential targeting
seem to be the V0-a-subunits, for example, subunits a3 and
a4 targeting the enzyme to the plasma membrane of
osteoclasts and renal intercalated cells [4, 11, 24].
In mammals the V-ATPase is of crucial importance
for several membrane-trafficking events, e.g., endocytosis, endosomal sorting, endosomal maturation, and
exocytotic capacity of dense core secretory granules
and synaptic vesicles [1, 3]. Special attention has
received the fact that the mammalian V-ATPase is
important for acidification of extracellular spaces by an
enzyme pool located in the plasma membrane [25, 26].
Extracellular acidification is necessary for bone resorption
[27] and maturation of spermatozoa [28]. The V-ATPase
was shown to play a major role in the invasiveness and
metastatic potential of cancer cells [29]. It has also been
Pflugers Arch - Eur J Physiol (2009) 457:599–607
suggested to be a pH sensor on the endocytic pathway
where, depending on lumenal acidification, specific
cytosolic proteins (small GTPases and regulator proteins)
can be attached, thus governing interaction with downstream organelles [30].
Genes encoding subunits of the Paramecium V-ATPase
The first V-ATPase gene to be described in Paramecium
was the P. multimicronucleatum B-subunit [31]. Molecular
data on P. tetraurelia has recently been boosted by the
Paramecium genome project [32]. Sequence information
from a draft assembly was used to identify most of the
Paramecium V-ATPase subunits [33]. Table 1 gives an
overview on gene numbers in P. tetraurelia in comparison
with S. cerevisiae and M. musculus.
One major outcome of the P. tetraurelia genome project
is that the genome has globally and recently been
duplicated [32]. Therefore, nearly all of the P. tetraurelia
V-ATPase subunits are encoded by gene pairs that are
greater than 80% identical in deoxyribonucleic acid
sequence and nearly identical on the protein level. The
Paramecium genome also shows traces of older genome
duplications during which V-ATPase genes were duplicated
and retained in the genome. This seems to be a general
Fig. 1 Scheme of the V-ATPase. Subunits of the cytoplasmic V1
subcomplex are depicted with capital letters/gray, membrane embedded V0-subunits with small letters/white
601
phenomenon in this ciliate and could account for the large
number of about 40,000 genes present in the genome.
Because of these events, V-ATPase subunits in Paramecium
are encoded by more genes than in any other organism
investigated so far [33]. Among the most numerous genes are
the V0-a-subunit genes that form a big multigene family with
17 paralogues. In this family, 16 genes can be grouped in
pairs, while only one gene lacks a homologue from the most
recent genome duplication [34].
As the V-ATPase genes forming a pair encode nearly
identical proteins, it is difficult to imagine that there is any
functional differentiation between them. In contrast, the
presence of all the different gene pairs in the genome,
which encode similar but distinct proteins, suggests that
these differences are physiologically relevant in terms of
function, as redundant gene copies tend to be eliminated
from a genome in the course of evolution because of the
lack of selection pressure.
Localization and function of the V-ATPase
in Paramecium
Osmoregulation Fok et al. [35] were the first to establish the
localization of the V-ATPase in Paramecium on the
osmoregulatory system, also termed the “contractile vacuole
complex.” To understand the role of the V-ATPase, its
function was inhibited using concanamycin B. Thus, the VATPase has been found to be crucial for osmoregulation in
this fresh water ciliate [35, 36].
Under normal conditions, Paramecium cells possess two
contractile vacuole complexes, one being located close to the
anterior and the other one close to the posterior pole of the
cell. Each organelle is comprised of a central vacuole, the so
called contractile vacuole that undergoes a rhythmic fusion
and fission cycle with the plasma membrane. Upon fusion
with the plasma membrane, its contents (mainly water and
ions) are discharged into the medium. Closely attached to the
contractile vacuole is an array of so called “radial arms.”
Each radial arm is composed of an ampulla, which is adjacent
to the contractile vacuole, and the radial canal, which is
surrounded by two continuous complex membrane cisternae,
called the “smooth” and the “decorated spongiome.” The
current idea on the functioning of the contractile vacuole
system is that ions are concentrated in the spongiome, which is
osmotically followed by water influx [37, 38]. The water is
collected via the radial canals in the ampullae, which fuse
with and therefore pass their content on to the contractile
vacuole. When this vacuole undergoes fusion with the
plasma membrane and releases its content, one pump cycle
is completed, and the cycle starts anew. For detailed
information on the contractile vacuole complex, see the
studies of Allen and Naitoh [36] and Allen [39].
602
The main driving force for this pump cycle was shown
to be the V-ATPase [35]. The enzyme pumps protons into
the lumen of the decorated spongiome, where the V-ATPase
is located in such abundance that in electron microscopy, it
actually gives the decorated spongiome the electron dense,
hence “decorated” appearance. Most likely, the proton
potential is then used for secondary active transport of
ions, among them K+ and Ca2+, into the lumen of the
contractile vacuole complex [37, 38], probably by antiporter systems, which is followed by osmotic water influx.
In fact, if the V-ATPase is inhibited by concanamycin B,
the contractile vacuole pump cycle stops [35, 37, 38].
Localization and function of the V-ATPase in the
contractile vacuole complex could be confirmed by
studying green fluorescent protein (GFP)-tagged V-ATPase
a-, c-, or F-subunits and by ribonucleic acid (RNA)
interference with these subunits (RNAi) [33, 34]. GFPlabeled c-subunits 1, 4, and 5, representatives of all three
c-subunit pairs (c1/2, c3/4, c5/6), all localized, among
several other organelles, to the radial arms of the contractile
vacuole system, as did a GFP-tagged F-subunit. Among the
V0-a-subunits, only a2-1 specifically localized to the
osmoregulatory system [34].
RNAi aiming at the expression of c- or F-subunits led to
morphological defects in the structure of the contractile
vacuole complex, both on the light and electron microscopical level, and a heavily impaired pumping cycle [33].
No specialization with respect to the c-subunit pairs
involved in osmoregulation was detected, as RNAi with
either of the three c-subunit pairs (c1/2, c3/4, c5/6) or with
combinations of them led to similar results, suggesting that
all three pairs are important for osmoregulation but leaving
the question open, whether they occur mixed in V-ATPase
complexes or whether they compose different V-ATPase
complexes that are all necessary for the functioning of the
contractile vacuole complex. An even more drastic phenotype was observed by interfering with a-subunit 2 expression. Osmoregulation by the contractile vacuole complex
was disturbed to the point where Paramecium cells showed
massive swelling and finally died by bursting [34].
Surprisingly, the contractile vacuole complex seems to
be only mildly acidic with pH 6.4 [38] and is therefore not
stained by the pH-sensitive dye acridine orange (Fig. 2a),
despite the huge number of V-ATPase molecules it contains
in its decorated spongiome [35]. The most likely explanation is that the proton potential is coupled so tightly to
secondary active transport of ions into the lumen of the
contractile vacuole complex that no bigger proton potential
is being built up.
To date, no comprehensive study has been carried out to
dissect the role of the numerous putative antiporter systems
encoded in the Paramecium genome [32], (http://paramecium.
cgm.cnrs-gif.fr). As the major cations in the fluid expelled by
Pflugers Arch - Eur J Physiol (2009) 457:599–607
the contractile vacuole are K+ and, to a lower degree, Ca2+,
this suggests that the focus in the search for antiporter systems
connected to the funtioning of the contractile vacuole complex
should be concentrated on H+/K+ and H+/Ca2+ antiporters.
Phagocytosis and endocytosis Another readily identified
cellular function of the V-ATPase in Paramecium is
phagocytosis. Paramecium feeds on bacteria and small
eukaryotic microorganisms. In the process of phagocytosis,
bacteria are concentrated by a dense field of cilia at a region
specialized for phagocytosis, termed the “cytostome.”
There, the nascent food vacuole forms, enlarges, and
pinches off the cytostome. Within 2 to 4 min, the food
vacuole is acidified by fusing with a population of small
vesicles called “acidosomes” that transport a high number
of V-ATPase molecules [40, 41]. Acidosomes are thus
constantly delivered to the cytostome and attach themselves
to the nascent food vacuole. The pinching off process
seems to trigger the fusion of acidosomes with the food
vacuole. After the initial acidification burst, the pH rises to
nearly neutral for the rest of the lifespan of the food
vacuole. The acidosomal membrane is withdrawn from the
phagosomes and cycles back to the cytostome. This process
is paralleled by withdrawal of the V-ATPase being responsible for the strong acidification [41] and its recycling. In
Fig. 2, acidosomes at the cytostome are stained by acridine
orange and visible in the form of a horseshoe arranged
around the cytostome. For a more detailed representation of
the phagocytic cycle, see the study of Allen and Fok [42].
Recently, the observed cycle of V-ATPase delivery by
acidosomes to phagosomes and the consecutive withdrawal
from them could be correlated with molecular data on the
V-ATPase. A subunit of V0, a4, was exclusively localized
to acidosomes, the nascent food vacuole, and the freshly
formed food vacuole from which it recycled back to the
cytostome. In contrast, older food vacuoles contained the
a-subunits a5, a6, and a9 [34]. This suggests that there are
at least two cycles of V-ATPase delivery and withdrawal to
and from food vacuoles, respectively. In the first cycle, a
V-ATPase with a supposedly high activity containing
subunit a4 is delivered to phagosomes to produce a rapid
acidification burst to inactivate ingested microorganisms.
This V-ATPase is recycled back to the cytostome, while a
different V-ATPase enzyme set, containing a5, 6, and 9, is
delivered to food vacuoles, possibly by lysosomes or
lysosomal precursors. It is possible that a5, 6, and 9 are
delivered in a sequential manner depending on the age
of the food vacuole, but this has not been investigated yet.
As the older food vacuoles do contain the V-ATPase, while
they are not remarkably acidic, it is likely that the proton
potential created by the pump is used for secondary active
transport of nutrients out and to pump waste products into the
vacuole. This is another aspect that remains to be elucidated.
Pflugers Arch - Eur J Physiol (2009) 457:599–607
603
Fig. 2 Staining of acidic compartments of Paramecium using acridine
orange. Fluorescent (a, c) and corresponding phase contrast images
(b, d) show the distribution of various acidic vesicles in the cytoplasm
(all median focus plane). Note the bright labeling of acidosomes
around the cytostome (a, inset) and the punctate staining at the edge of
the anterior and posterior end of the cells (arrows), indicating early
endosomes. The surface view of a cell (e) shows the regular
arrangement of endosomes below the cell surface. The corresponding
phase contrast image (g) reveals that the osmoregulatory system is not
stained (arrow). f Detail of e as indicated, showing cortical endosomes; h same detail on median focus. Bars=10 μm
What happens to phagocytosis when the expression of
the V-ATPase is inhibited? RNA interference with the
expression of the c- or F-subunits of the V-ATPase not
only strongly reduced acidification, it also inhibited
formation of phagosomes [33]. The mechanism for this is
not clear at the moment. A possible explanation might be
that the initial acidification burst is necessary to signal
further processing of the food vacuole in terms of transport
and fusion with lysosomes. Lack of this signal might lead
to a block in downstream events and, therefore, a failure in
the retrieval of acidosomal membrane from the young food
vacuole back to the cytostome. Ultimately, lack of acidosomes at the cytostome should cause the absence of
phagocytosis.
The role of V-ATPase in endocytosis and endosomal
sorting in Paramecium has not been worked out yet. What
is established is that this acidic compartment contains the
V-ATPase with the V0-subunit a1 [34] (Figs. 2 and 3).
Dense core secretory vesicle biogenesis The third process
in Paramecium in which the V-ATPase was found to be
involved in is the formation of dense core secretory
granules, the so-called trichocysts [33]. In most organisms,
dense core secretory granules are known to be acidified by
the V-ATPase [43]. The finding that Paramecium trichocysts contain a V-ATPase was surprising, as trichocysts
were shown by two independent reports using different
methods not to be acidic compartments, neither the mature
organelle nor trichocyst precursors [44, 45]. Interference
with c-, F-, or a3-subunits of the Paramecium V-ATPase led
to malformation of these organelles [33, 34]. They do not
reach their usual carrot shape, fail to form proper tips, and
are not attached to the plasma membrane. In their
misshapen form, they resemble mutant trichocysts that are
defective in the proteolytic maturation from trichocyst
matrix precursor proteins to the correctly processed and
assembly-competent trichocyst matrix proteins [46, 47].
604
Pflugers Arch - Eur J Physiol (2009) 457:599–607
Fig. 3 Schematic distribution of V-ATPase subunits in the Paramecium
cell. The red arrows indicate transport routes during phagosomal
processing based on reviews [42, 48]. V-ATPase subunit distribution is
based on data obtained with GFP localization in vivo and with antibody
labeling [33, 34]. Blue: V0-a-subunits, black: V0-c-subunits, green: V1F-subunits; red filling: acidified vesicles/vacuoles; pink filling: mildly or
not recognizably acidic compartments but with V-ATPase subunits
detected in the membrane (presence in trichocyst precursors inferred
from findings with mature trichocysts). The scheme contains elements of
the osmoregulatory system (a ampula, cv contractile vacuole, ds
decorated spongiome, ss smooth spongiome) and of the phagosomal
apparatus (oc oral cavity, fv food vacuole, as acidosomes, cp cytoproct).
Discoidal vesicles (dv) and other recycling vesicles (rv) are responsible
for return transport of membranes. The endosomal system, composed of
parasomal sacs (ps) and early endosomes (ee), is arrayed in a regular
fashion. ci Cilia, er endoplasmatic reticulum, ga Golgi apparatus, trpc
trichocyst precursor, tr trichocysts, gh ghosts (from released trichocysts),
pm plasma membrane
Correctly assembled trichocysts fuse with the plasma
membrane upon a stimulus [48]. After membrane fusion,
extracellular calcium enters the trichocyst body, and a
major rearrangement of the paracrystaline trichocyst core
takes place. It transforms into a long and insoluble needle,
which is, thus, ejected into the medium. A possible role for
the V-ATPase in the formation of trichocysts could be to
provide an electrochemical proton potential that is exploited
by an antiporter to pump out Ca2+ for making trichocysts
competent for their exocytotic discharge, but this hypothesis remains to be tested. This would be in line with work
on dense core secretory vesicles in mammalian cells where
the V-ATPase has been shown to contribute to activation
and specific sequestration of secretory contents [49, 50].
Remarkably, besides trichocysts, there are only few dense
core secretory vesicles whose lumen is not remarkably
acidic, e.g., parotis gland granules, although their membranes contain a H+-ATPase [51].
ments to which no specific function has been attributed or
which had not been identified in any detail so far.
Further functional aspects of Hþ -ATPase activity Unfortunately, it was not possible to assign any role to the VATPase V0 part of Paramecium in exocytotic membrane
fusion [33] as had been suggested for other systems [52, 53,
54], based on experiments on homotypic yeast vacuole
membrane fusion [55].
Apart from the three major functions of the V-ATPase in
Paramecium presented here, it is likely to play many other
yet unidentified roles in cellular processes as suggested by
the presence of numerous a-subunits specific in compart-
Functional overlap between the V-ATPase
and other pumps
Beyond the V-type ATPase described in this review, the
Paramecium cell also possesses P-type ATPases that were
reported to be involved in Ca2+ metabolism. This includes a
plasmamembrane Ca2+-ATPase [56] and a SERCA-type
(sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) Ca2+
pump [57] whose biogenesis starts in the endoplasmic
reticulum and finally is enriched in the cortical Ca2+ stores,
the “alveoli” [58–60]. However, experimental analysis of the
SERCA [61] and calculations from the kinetics of these
pumps suggest that they are less powerful regulators in
maintaining a low cytoplasmic Ca2+concentration than
extrusion of Ca2+ ions via the contractile vacuole complex
[62]. From studies with concanamycin B combined with Ca2+
imaging, we know that the H+-ATPase of the osmoregulatory
system largely contributes to the re-establishment of Ca2+
homeostasis after signal transduction accompanying exocytosis stimulation (Sehring and Plattner, in preparation). This
function complements the chemiosmotic, ΔH+-based water
transport for osmoregulation, as described in the studies of
Allen and Naitoh [36] and Allen [39].
In the Paramecium genome, a group of genes with
homology to the vacuolar H+-translocating inorganic pyro-
Pflugers Arch - Eur J Physiol (2009) 457:599–607
phosphatases of plants also exists (Wassmer, unpublished
observation). Neither localization nor function of these genes
have been studied, but the presence of a whole group of
these genes suggests that not only V-type ATPases are used
for H+ pumping in Paramecium but also pyrophosphatases.
The specialists: functional diversification
of V0-a-subunits
Localization studies of the nine different a-subunit clusters
(eight gene pairs and a single gene) showed them to be
located in at least seven distinct membrane compartments
[34]. Subunit a1-1 was found to be on the endocytic
pathway, most likely on early endosomes. In many systems,
it is known that early endosomes are acidified by the VATPase. The staining of Paramecium cells with acridine
orange strongly labels early endosomes localized next to
basal bodies directly below the plasma membrane (Fig. 2),
suggesting acidification by V-ATPases containing a-subunit
pair 1. Subunit a2-1 localized exclusively to the contractile
vacuole complex, while a3-1 localized to trichocysts. a4-1
was found to shuttle between acidosomes and the nascent
food vacuole, while a5-1, a6-1, and a9-1 localized to the
membrane of older phagosomes. Subunit a7-1 is most
likely localized in endoplasmic reticulum membranes and
a8-1 in the Golgi apparatus.
Whether these a-subunits can be rerouted and replace the
function in the absence of another a-subunit was investigated by RNAi with a-subunits 2 and 3, located in the
contractile vacuole complex and trichocysts, respectively.
Knockdown of a2-1/a2-2 subunits disrupted the function of
the contractile vacuole complex and ultimately led to cell
death, demonstrating that none of the other endogenous
a-subunits could replace it. The same holds true for RNAi
with a3-1/3-2. Absence of these V0-subunits caused malformation of trichocysts, showing that no other a-subunit
can rescue this process [34].
The most likely explanation for the specificity is the
differential localization of the a-subunits. The fact that none
of the other a-subunits is rerouted in the absence of others
suggests that they contain a targeting signal that specifies
the localization of the holoenzyme, an observation previously made for a-subunits in other organisms [21, 24]. The
presence of such a targeting signal was demonstrated by
domain swap experiments and was found to lie within the
C-terminal, transmembraneous half of the V0-a-subunits in
Paramecium, although it could not be narrowed down to a
short signal peptide sequence [34]. Any domain swapping
within the C terminus of any a-subunit tested led to
abnormal targeting of the chimeric subunit accompanied
by cell division arrest and cell death. From this finding, it
was concluded that the targeting signal is complex and
605
spreads over the whole C terminus of the V0-a-subunits in
Paramecium [34]. The C-terminal localization signal in
Paramecium contrasts with observations in yeast, in which
the targeting signal was demonstrated to be in the Nterminal half of the a-subunits [21]. The difference can
possibly be explained by the evolution of the a-subunits. A
phylogenetic analysis reveals that the gene duplication in
yeast and the a-subunit multiplication in Paramecium have
occurred after these organisms have diverged from a
common ancestor [34]. It is highly unlikely that similar
signal recognition and targeting mechanisms should have
evolved independently in the two organisms to correctly
localize different a-subunits.
Establishing and comparing the two different mechanisms should prove to be an exciting task for future
work.
Combinatorial enzyme design: a V-ATPase constructed
from a building set?
The puzzling number of V-ATPase genes raises several
interesting possibilities. Are the V-ATPase molecules in the
Paramecium cell a complex mosaic of the dozens of
slightly different subunits that possibly changes over time?
What are the physiological differences between them? Does
a different subunit composition allow differential control,
i.e., pumping kinetics, lumenal acidification, and binding of
cytosolic regulators?
The fact that the same c- and F-subunits associate with
different V0-a-subunits in a compartment-specific way
clearly demonstrates the presence of a variety of different
V-ATPases in Paramecium. However, this phenomenon is
most likely not only restricted to the V0 subcomplex.
When an a-subunit chimera composed of a2-1 and a3-1
was introduced into the cell, it was correctly localized
according to the targeting signal in the C terminus of the
molecule. However, stunningly, the chimera introduced
abnormalities specifically in the organelle, to which the
V-ATPase would have been targeted, if its a-subunit had
contained the correct C terminus [34]. The a-subunit
chimera thus created a remote effect in an organelle in
which it was not present. This scenario can most easily be
explained under the assumption that the N terminus of the
chimeric a-subunit binds to V1 complexes, which, in their
unique subunit composition, “belong” to a different
organelle. The a-subunit was shown to connect V0 with
V1 as part of a peripheral stalk [63, 64]. Overexpression of
a chimera would thus deplete the cytoplasmic V 1
subcomplex pool from which a different organelle recruits
its specific V1 complexes.
This model suggests that by the combination of slightly
different V-ATPase subunits, one can create a multitude of
606
different isozymes. Paramecium thus offers an attractive
experimental system to test how an enzyme is fine tuned by
the use of different subunits in terms of function, structure,
and localization. These possibilities will be exploited in
future work.
Acknowledgement We would like to thank Jean Cohen, Janine
Beisson, and Linda Sperling (CNRS, Gif-sur-Yvette, France) for many
helpful discussions and for access to the Paramecium Genome
Database. We gratefully acknowledge the support from Genoscope,
France, for sharing sequence information with us during sequencing
and assembling the Paramecium genome.
References
1. Sun-Wada GH, Wada Y, Futai M (2004) Diverse and essential
roles of mammalian vacuolar-type proton pump ATPase: toward
the physiological understanding of inside acidic compartments.
Biochim Biophys Acta 1658:106–114
2. Schumacher K (2006) Endomembrane proton pumps: connecting
membrane and vesicle transport. Curr Opin Plant Biol 9:595–600
3. Forgac M (2007) Vacuolar ATPases: rotary proton pumps in
physiology and pathophysiology. Nat Rev Mol Cell Biol 8:917–929
4. Inoue T, Wang Y, Jefferies K, Qi J, Hinton A, Forgac M (2005)
Structure and regulation of the V-ATPases. J Bioenerg Biomembr
37:393–398
5. Wilkens S, Inoue T, Forgac M (2004) Three-dimensional structure of
the vacuolar ATPase. Localization of subunit H by difference
imaging and chemical cross-linking. J Biol Chem 279:41942–41949
6. Venzke D, Domgall I, Kocher T, Fethiere J, Fischer S, Bottcher B
(2005) Elucidation of the stator organization in the V-ATPase of
Neurospora crassa. J Mol Biol 349:659–669
7. Arata Y, Nishi T, Kawasaki-Nishi S, Shao E, Wilkens S, Forgac M
(2002) Structure, subunit function and regulation of the coated
vesicle and yeast vacuolar (H(+))-ATPases. Biochim Biophys
Acta 1555:71–74
8. Kane PM, Smardon AM (2003) Assembly and regulation of the
yeast vacuolar H+ATPase. J Bioenerg Biomembr 35:313–321
9. Yokoyama K, Nakano M, Imamura H, Yoshida M, Tamakoshi M
(2003) Rotation of the proteolipid ring in the V-ATPase. J Biol
Chem 278:24255–24258
10. Imamura H, Nakano M, Noji H, Muneyuki E, Ohkuma S, Yoshida
M, Yokoyama K (2003) Evidence for rotation of V1-ATPase. Proc
Natl Acad Sci USA 100:2312–2315
11. Nishi T, Forgac M (2002) The vacuolar (H)-ATPases—nature’s
most versatile proton pumps. Nat Rev Mol Cell Biol 3:94–103
12. Hirata T, Iwamoto-Kihara A, Sun-Wada GH, Okajima T, Wada Y,
Futai M (2003) Subunit rotation of vacuolar-type proton pumping
ATPase: relative rotation of the G and C subunits. J Biol Chem
278:23714–23719
13. Feng Y, Forgac M (1992) A novel mechanism for regulation of
vacuolar acidification. J Biol Chem 267:19769–19772
14. Feng Y, Forgac M (1994) Inhibition of vacuolar H(+)-ATPase by
disulfide bond formation between cysteine 254 and cysteine 532
in subunit A. J Biol Chem 269:13224–13230
15. Kane PM (1995) Disassembly and reassembly of the yeast
vacuolar H+ATPase in vivo. J Biol Chem 270:17025–17032
16. Qi J, Forgac M (2007) Cellular environment is important in
controlling V-ATPase dissociation and its dependence on activity.
J Biol Chem 282:24743–24751
17. Brown D, Breton S (2000) Sorting proteins to their target
membranes. Kidney Int 57:816–824
Pflugers Arch - Eur J Physiol (2009) 457:599–607
18. Mellman I (1992) The importance of being acid: the role of
acidification in intracellular membrane transport. J Exp Biol
172:39–45
19. Ijzendoorn SC (2006) Recycling endosomes. J Cell Sci 119:1679–
1681
20. Manolson MF, Wu B, Proteau D, Taillon BE, Roberts BT, Hoyt
MA, Jones EW (1994) STV1 gene encodes functional homologue
of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. J Biol
Chem 269:14064–14074
21. Kawasaki-Nishi S, Bowers K, Nishi T, Forgac M, Stevens TH
(2001) The amino-terminal domain of the vacuolar protontranslocating ATPase a subunit controls targeting and in vivo
dissociation, and the carboxyl-terminal domain affects coupling of
proton transport and ATP hydrolysis. J Biol Chem 276:47411–
47420
22. Graham LA, Flannery AR, Stevens TH (2003) Structure and
assembly of the yeast V-ATPase. J Bioenerg Biomembr 35:301–
312
23. Sun-Wada GH, Yoshimizu T, Imai-Senga Y, Wada Y, Futai M
(2003) Diversity of mouse proton-translocating ATPase: presence
of multiple isoforms of the C, d and G subunits. Gene 302:147–
153
24. Toyomura T, Oka T, Yamaguchi C, Wada Y, Futai M (2000) Three
subunit a isoforms of mouse vacuolar H(+)-ATPase. Preferential
expression of the a3 isoform during osteoclast differentiation. J
Biol Chem 275:8760–8765
25. Yao G, Feng H, Cai Y, Qi W, Kong K (2007) Characterization of
vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase
in osteoclasts. Biochem Biophys Res Commun 357:821–827
26. Breton S, Brown D (2007) New insights into the regulation of
V-ATPase-dependent proton secretion. Am J Physiol Renal
Physiol 292:F1–10
27. Ochotny N, Van Vliet A, Chan N, Yao Y, Morel M, Kartner N,
von Schroeder HP, Heersche JN, Manolson MF (2006) Effects of
human a3 and a4 mutations that result in osteopetrosis and distal
renal tubular acidosis on yeast V-ATPase expression and activity. J
Biol Chem 281:26102–26111
28. Pietrement C, Sun-Wada GH, Silva ND, McKee M, Marshansky
V, Brown D, Futai M, Breton S (2006) Distinct expression
patterns of different subunit isoforms of the V-ATPase in the rat
epididymis. Biol Reprod 74:185–194
29. Sennoune SR, Luo D, Martinez-Zaguilan R (2004) Plasmalemmal
vacuolar-type H+ATPase in cancer biology. Cell Biochem
Biophys 40:185–206
30. Hurtado-Lorenzo A, Skinner M, El Annan J, Futai M, Sun-Wada
GH, Bourgoin S, Casanova J, Wildeman A, Bechoua S, Ausiello
DA, Brown D, Marshansky V (2006) V-ATPase interacts with
ARNO and Arf6 in early endosomes and regulates the protein
degradative pathway. Nat Cell Biol 8:124–136
31. Fok AK, Yamauchi K, Ishihara A, Aihara MS, Ishida M, Allen RD
(2002) The vacuolar-atpase of Paramecium multimicronucleatum:
gene structure of the B subunit and the dynamics of the V-ATPaserich osmoregulatory membranes. J Eukaryot Microbiol 49:185–196
32. Aury JM, Jaillon O, Duret L, Noel B, Jubin C, Porcel BM,
Ségurens B, Daubin V, Anthouard V, Aiach N, Arnaiz O, Billaut
A, Beisson J, Blanc I, Bouhouche K, Câmara F, Duharcourt S,
Guigo R, Gogendeau D, Katinka M, Keller AM, Kissmehl R,
Klotz C, Koll F, Le Mouël A, Lepère G, Malinsky S, Nowacki M,
Nowak JK, Plattner H, Poulain J, Ruiz F, Serrano V, Zagulski M,
Dessen P, Bétermier M, Weissenbach J, Scarpelli C, Schächter V,
Sperling L, Meyer E, Cohen J, Wincker P (2006) Global trends of
whole-genome duplications revealed by the ciliate Paramecium
tetraurelia. Nature 444:171–178
33. Wassmer T, Froissard M, Plattner H, Kissmehl R, Cohen J (2005)
The vacuolar proton-ATPase plays a major role in several membranebounded organelles in Paramecium. J Cell Sci 118:2813–2825
Pflugers Arch - Eur J Physiol (2009) 457:599–607
34. Wassmer T, Kissmehl R, Cohen J, Plattner H (2006) Seventeen asubunit isoforms of Paramecium V-ATPase provide high specialization in localization and function. Mol Biol Cell 17:917–930
35. Fok AK, Aihara MS, Ishida M, Nolta KV, Steck TL, Allen RD
(1995) The pegs on the decorated tubules of the contractile
vacuole complex of Paramecium are proton pumps. J Cell Sci
108:3163–3170
36. Allen RD, Naitoh Y (2002) Osmoregulation and contractile
vacuoles of protozoa. Int Rev Cytol 215:351–394
37. Stock C, Gronlien HK, Allen RD, Naitoh Y (2002) Osmoregulation in Paramecium: in situ ion gradients permit water to
cascade through the cytosol to the contractile vacuole. J Cell Sci
115:2339–2348
38. Stock C, Gronlien HK, Allen RD (2002) The ionic composition of
the contractile vacuole fluid of Paramecium mirrors ion transport
across the plasma membrane. Eur J Cell Biol 81:505–515
39. Allen RD (2000) The contractile vacuole and its membrane
dynamics. Bioessays 22:1035–1042
40. Allen RD, Fok AK (1983) Nonlysosomal vesicles (acidosomes)
are involved in phagosome acidification in Paramecium. J Cell
Biol 97:566–570
41. Allen RD (1988) Cytology. In: Goertz HD (ed) Paramecium.
Springer, Berlin, pp 4–40
42. Allen RD, Fok AK (2000) Membrane trafficking and processing
in Paramecium. Int Rev Cytol 198:277–317
43. Apps DK (1997) Membrane and soluble proteins of adrenal
chromaffin granules. Semin Cell Dev Biol 8:121–131
44. Lumpert CJ, Glas-Albrecht R, Eisenmann E, Plattner H (1992)
Secretory organelles of Paramecium cells (trichocysts) are not
remarkably acidic compartments. J Histochem Cytochem 40:
153–160
45. Garreau de Loubresse N, Gautier MC, Sperling L (1994) Immature
secretory granules are not acidic in Paramecium: Implications for
sorting to the regulated pathway. Biol Cell 82:139–147
46. Gautier MC, Garreau de Loubresse N, Madeddu L, Sperling L
(1994) Evidence for defects in membrane traffic in Paramecium
secretory mutants unable to produce functional storage granules. J
Cell Biol 124:893–902
47. Madeddu L, Gautier MC, Le Caer JP, Garreau de Loubresse N,
Sperling L (1994) Protein processing and morphogenesis of
secretory granules in Paramecium. Biochimie 76:329–335
48. Plattner, Kissmehl (2003) Molecular aspects of membrane
trafficking in Paramecium. Int Rev Cytol 232:185–216
49. Taupenot L, Harper KL, O’Connor DT (2005) Role of H-ATPasemediated acidification in sorting and release of the regulated
secretory protein chromogranin A. Evidence for a vesiculogenic
function. J Biol Chem 280:3885–3897
50. Waterford SD, Kolodecik TR, Thrower EC, Gorelick FS (2005)
Vacuolar ATPase regulates zymogen activation in pancreatic acini.
J Biol Chem 280:5430–5434
51. Arvan P, Castle JD (1986) Isolated secretion granules from parotid
glands of chronically stimulated rats possess an alkaline internal
607
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
pH and inward-directed H pump activity. J Cell Biol 103:1257–
1267
Hiesinger PR, Fayyazuddin A, Metha SQ, Rosenmund T, Schulze
KL, Zhai RG, Verstreken P, Cao V, Zhou Y, Kunz J, Bellen HJ
(2005) The v-ATPase V0 subunit a1 is required for a late step in
synapic vesicle exocytosis in Drosophila. Cell 121:607–620
Liegeois S, Benedetto A, Garnier JM, Schwab Y, Labouesse M
(2006) The V0-ATPase mediates apical secretion of exosomes
containing Hedgehog-related proteins in Caenorhabditis elegans.
J Cell Biol 173:949–961
Sun-Wada GH, Toyomura T, Murata Y, Yamamoto A, Futai M,
Wada Y (2006) The a3 isoform of V-ATPase regulates insulin
secretion from pancreatic β-cells. J. Cell Sci. 119:4531–4540
Peters C, Bayer MJ, Buhler S, Andersen JS, Mann M, Mayer A
(2001) Trans-complex formation by proteolipid channels in the
terminal phase of membrane fusion. Nature 409:581–588
Wright MV, Van Houten JL (1990) Characterization of a putative
Ca2-transporting Ca2-ATPase in the pellicles of Paramecium
tetraurelia. Biochim Biophys Acta 1029:241–251
Hauser K, Pavlovic N, Kissmehl R, Plattner H (1998) Molecular
characterization of a sarco(endo)plasmic reticulum Ca2-ATPase gene
from Paramecium tetraurelia and localization of its gene product to
sub-plasmalemmal calcium stores. Biochem J 334:31–38
Kissmehl R, Huber S, Kottwitz B, Hauser K, Plattner H (1998)
Subplasmalemmal Ca-stores in Paramecium tetraurelia. Identification and characterisation of a sarco(endo)plasmic reticulum-like
Ca2-ATPase by phosphoenzyme intermediate formation and its
inhibition by caffeine. Cell Calcium 24:193–203
Plattner H, Floetenmeyer M, Kissmehl R, Pavlovic N, Hauser K,
Momayezi M, Braun N, Tack J, Bachmann L (1999) Microdomain
arrangement of the SERCA-type Ca2 pump (Ca2-ATPase) in
subplasmalemmal calcium stores of Paramecium cells. J Histochem Cytochem 47:841–854
Hauser K, Pavlovic N, Klauke N, Geissinger D, Plattner H (2000)
Green fluorescent protein-tagged sarco(endo)plasmic reticulum
Ca2-ATPase overexpression in Paramecium cells: isoforms,
subcellular localization, biogenesis of cortical calcium stores and
functional aspects. Mol Microbiol 37:773–787
Mohamed I, Husser M, Sehring I, Hentschel J, Hentschel C,
Plattner H (2003) Refilling of cortical calcium stores in
Paramecium cells: in situ analysis in correlation with storeoperated calcium influx. Cell Calcium 34:87–96
Ladenburger EM, Korn I, Kasielke N, Wassmer T, Plattner H
(2006) An Ins(1,4,5)P3 receptor in Paramecium is associated with
the osmoregulatory system. J Cell Sci 119:3705–3717
Xu T, Vasilyeva E, Forgac M (1999) Subunit interactions in the
clathrin-coated vesicle vacuolar (H(+))-ATPase complex. J Biol
Chem 274:28909–28915
Inoue T, Forgac M (2005) Cysteine-mediated cross-linking
indicates that subunit C of the V-ATPase is in close proximity to
subunits E and G of the V1 domain and subunit a of the V0
domain. J Biol Chem 280:27896–27903