Download Structural features and nucleotide-binding capability of the C subunit

Mechanisms of Bioenergetic Membrane Proteins
Structural features and nucleotide-binding
capability of the C subunit are integral to the
regulation of the eukaryotic V1Vo ATPases
G. Grüber1
Universität des Saarlandes, Fachrichtung 2.5-Biophysik, Universitätsbau 76, D-66421 Homburg, Germany
Abstract
V-ATPases (vacuolar ATPases) are responsible for acidification of intracellular compartments and, in certain
cases, proton transport across the plasma membrane of eukaryotic cells. They are composed of a catalytic
V1 sector, in which ATP hydrolysis takes place, and the Vo sector, which functions in proton conduction. The
best established mechanism for regulating the V-ATPase activity in vivo involves reversible dissociation of
the V1 and Vo domains, in which subunit C is intimately involved. In the last year, impressive progress has
been made in elucidating the structure of the C subunit and its arrangement inside the V-ATPase. Nucleotide
occupancy by subunit C, followed by conformational changes of this subunit has shed light on the mechanism
of V-ATPase regulation.
Introduction
V-ATPase (vacuolar ATPase) is an electrogenic ion pump
found in every eukaryotic cell. This enzyme harnesses the
energy derived from ATP hydrolysis to pump ions across
membranes, thereby creating an electrochemical gradient.
V-ATPases have two structural and functional parts, a
peripheral V1 complex, whose catalytic part faces the cytosol,
and a membrane-bound ion-conducting Vo part. The
eukaryotic enzyme V1 consists of eight subunits A–H,
whereas the Vo domain is composed of the different subunits
a, c, c , c , d and e [1]. ATP is hydrolysed on the V1 -headpiece,
composed of an A3 B3 hexamer, and the energy released
during that process is transmitted to the membrane-bound
Vo domain, to drive ion translocation. This energy coupling
occurs via the so-called ‘stalk’ structure, an assembly of the V1
and Vo subunits C–H and a respectively that forms the
functional and structural interface [2].
A characteristic feature of the eukaryotic V1 Vo ATPase is
the regulation by reversible disassembly of the V1 and Vo
subcomplexes [3–5], resulting in the decrease of Mg2+ dependent ATPase activity and proton pumping across the
membrane. Reassembly of both domains restores these activities. It was shown that subunits C and H are important
for inhibition of the Mg2+ -dependent ATPase activity of
dissociated V1 complexes [4]. The high-resolution structure
of the H subunit [6] and data on the gross structure of the
V1 Vo ATPase complexes suggest that, in the intact enzyme,
this subunit is involved in the formation of the peripheral stalk
region [7], despite the fact that a rearrangement within the
Key words: reversible dissociation, vacuolar-type ATPase, V1 Vo ATPase, Vma5p nucleotidebinding.
Abbreviations used: EM, electron microscopy; SAXS, small-angle X-ray scattering; V-ATPase,
vacuolar ATPase.
1
Present address: School of Biological Sciences, Nanyang Technological University, Singapore
637551 (email [email protected]).
disassembled V1 is possible [2]. Electron microscopy studies
of the disassembled V1 complex from tobacco hornworm
Manduca sexta have shown that subunit C dissociates from
the V1 subcomplex [8], although it is essential for the
reassembly of the functional V1 Vo [3,4]. Recent work on
the structure of subunit C, the relationship of nucleotide
binding of C and interactions of this stalk subunit within
the V1 ATPase is reviewed here.
Quaternary structure of the V1 ATPase
The fundamental aim of structural studies in molecular
biology is to establish a relationship between structure (or,
more precisely, structural changes) and function of biological
molecules. Over the past years, a significant amount of structural information about the V1 ATPase has been obtained
using three-dimensional EM (electron microscopy) [8,9],
SAXS (small-angle X-ray scattering) [10,11] and threedimensional crystallography [6,12]. Significant insights into
the molecular mechanism of ATP hydrolysis came from the
three-dimensional structure of the V1 ATPase without
subunit C [V1 (−C)] from M. sexta at 18 Å (1 Å = 0.1 nm)
resolution [8] (Figure 1), providing a picture of where the
three A and B subunits form a hexagon, alternating around
a central cavity, in which a seventh mass is located. This
seventh mass is not located in the centre of the cavity of
the A3 B3 hexamer, but is slightly offset to one side, thereby
strengthening the interaction between two non-neighbouring A subunits [8]. This feature is comparable with the
asymmetric location of the rotating γ subunit in the α 3 β 3
subcomplex of the related F-ATPase [13], indicating significant movements of the central mass in V-ATPases during
catalysis. In a side view, the structure shows three protuberances at the top of the A3 B3 headpiece, which might
belong to the N-termini of subunit A [8]. At the bottom side
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Figure 1 The 18 Å structure of the V1 ATPase without subunit C
Figure 2 Low-resolution structure of subunit C of the V-ATPase
from M. sexta
Modified with permission from M. Radermacher, T. Ruiz, H. Wieczorek
from S. cerevisiae derived from SAXS data [11].
and G. Grüber (2001) The structure of the V1 -ATPase determined by
three-dimensional electron microscopy of single particles. J. Struct. Biol.
c Elsevier.
135, 26–37 of the A3 B3 domain, the stalk protrudes with an angle of
approx. 7◦ with the vertical axis of the cavity. The length
of the stalk in the V1 (−C) complex is shorter than the value
determined for the complete and hydrated V1 ATPase (11 nm)
using SAXS [10]. This difference has been caused by the
separation of the C subunit in the V1 (−C) complex used in
the (EM) studies [8].
Structural features and arrangement of
subunit C and H
Previously, the structure of the C subunit (Vma5p) from
the yeast V1 Vo ATPase has been studied by SAXS [11]. The
radius of gyration Rg and the maximum dimension Dmax of
subunit C are 3.74 ± 0.03 nm and 12.5 ± 0.1 nm respectively,
suggesting that the subunit is a rather elongated particle. The
gross structure of subunit C was restored ab initio from
the scattering data, revealing that the hydrated Vma5p has an
elongated boot-shaped feature [11] (Figure 2). A recent 1.75 Å
map from X-ray diffraction studies of subunit C (Vma5p,
[12]) confirms this feature and shows that this subunit consists
of three distinct domains. An upper head domain, composed of amino acids 166–263, a large globular foot, consisting
of the N- and C-termini, and an elongated neck domain,
which connects the head and foot region. The overall length of
the stalk and structure of subunit C indicate that this subunit
might span the full stalk, thereby linking the catalytic A3 B3
domain via its head region to the Vo domain via the foot
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region. This foot region of Vma5p with approx. 5 nm in width
and 4.5 nm in height, would fit to the bottom of the stalk
domain of the hydrated V1 ATPase, determined by SAXS
[10]. These data are in agreement with two-dimensional
projections of a hydrolytic active V1 –Vma5p hybrid complex,
composed of subunit C (Vma5p) of Saccharomyces cerevisiae
V-ATPase and the C-depleted V1 from M. sexta, determined
from single particle electron microscopy [14]. The total length
of the stalk element in this hybrid complex is approx. 10.5 nm,
which exceeds the length of the central stalk in V1 complex
lacking subunit C by 4.5 nm. The EM data also suggest
that, within the hybrid complex, Vma5p is most likely to
be arranged with its long axis parallel to the stalk direction,
as shown in Figure 2. Electrostatic analysis also indicates that
the foot region of subunit C is oriented to the membrane
domain [12].
The boot-shaped structure of subunit C (Vma5p) is
remarkably similar in its overall structure to that of subunit
H (Vma13p), composed of an elongated bootleg, made up
by the N-terminal domain, and a foot, which is formed by
the C-terminal domain. The crystal structure of subunit H
shows that both domains are connected by a flexible linker [6].
The C-terminal half of subunit H is proposed to bind to the
V-ATPase subunits, whereby the long terminal shaft interacts
with proteins like Ynd1p or the Nef-binding protein-1 [6].
In comparison, the C-terminus of subunit C (Vma5p), which
includes the most highly conserved region of this subunit,
appears to be very important for the stable assembly of V1 Vo .
Mutagenesis of this region reduced ATPase activity in vitro,
because of loss of V1 subunits [4], indicating that subunit
C might also be in contact with the V1 subunits via its Cterminal domain (see below). In line with this is the fact
that binding of different maleimides to the single cysteine
residue (Cys340 ) of the C-terminal region of subunit C prevented binding of this subunit to the V1 (−C) complex [14].
Since binding of different maleimides to Vma5p prevents this
subunit from interaction with C-depleted V1 , it has been
proposed that the point of the foot, in which Cys340 is located,
Mechanisms of Bioenergetic Membrane Proteins
forms at least partially the surface for binding to the stalk
region of V1 .
Nucleotide binding and structural
alterations of subunit C
Regulation of V-ATPase function in response to physiological
stimuli is thought to be a multilevel process. It includes control of the expression of V-ATPase subunit genes, intracellular
targeting and translocation from vesicles to the plasma membrane, and reversible dissociation of the Vo and V1 domains,
entailing inactivation of the pump and decrease of MgATPase activity (reviewed in [15]). In S. cerevisiae, it has been
shown that glucose deprivation induces disassembly of the V1
and Vo section and that subunit C (Vma5p) reversibly leaves
the enzyme after removal of glucose, causing the catalytic V1
to detach from the Vo section [4]. As yet, the nature of this
signal is not clear. It has been hypothesized that subunit C
might act as a sensor for the cellular ADP:ATP level [16].
As observed independently by photoaffinity labelling and
fluorescence correlation spectroscopy, subunit C is capable
of binding ADP and ATP, whereby ATP binding is weaker
than that of ADP [16]. The C-terminal region of subunit C has
been mapped to be the nucleotide-binding site. Tryptophan
fluorescence quenching and decreased susceptibility to
tryptic digestion of subunit C after binding of different
nucleotides provides evidence for structural changes in this
subunit because of nucleotide binding [16]. It is of particular
interest that the V1 Vo disassembly is accompanied by the
dissociation of subunit C from the complex [3], and it has
been hypothesized that subunit C plays a central role in
the reversible reassembly of both domains [3,4] by binding
as an anchor protein to the actin-based cytoskeleton and
controlling the linkage of the cytoplasmic V1 complex with
the actin filaments [3]. One of the proposed actin-binding
sites of the protein is located in the C-terminal domain of
subunit C [12]. Therefore binding of the nucleotides ADP
and ATP to the C-terminus may induce structural changes in
the foot region and thereby alter the interaction with other
V1 and Vo subunits, and actin.
In summary, subunit C is bound to the catalytic domain of
V1 via the head domain and the foot region of C is oriented
to the membrane portion [12,14]. The elongated feature of
subunit C supports its role as a mediator, which facilitates
the linkage of V1 and Vo and thereby permits alterations
in the V1 Vo ATPase due to nucleotide binding. Subunit C in
V1 Vo acts not only as a stabilizer and regulator, but also as
a sensor of the cytosolic ATP/ADP ratio [16]. In addition,
the significant length of the stalk of V1 , which separates the
catalytic part from the ion-conducting part, requires conformational coupling of the stalk subunits during ATP
hydrolysis. In contrast with the related F-ATP synthases,
in which the rotary stalk subunits γ and ε bind directly to the
rotor ring in the Fo domain [17], subunits C of the V-ATPase
makes up an adapter which links catalytic site events in V1
with proton conduction in Vo , thereby implying a different
coupling mechanism to that described for F-ATP synthases.
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