A gold nanoparticle GNP technique com-bined with transmission electron microscopy TEM analysis was utilized to determine the distance between C-termini within a membrane-bound V-PPase di
Trang 1H+-pyrophosphatase determined using atomic force
microscopy and a gold nanoparticle technique
Tseng-Huang Liu1, Shen-Hsing Hsu1, Yun-Tzu Huang1, Shih-Ming Lin1, Tsu-Wei Huang2,
Tzu-Han Chuang3, Shih-Kang Fan4, Chien-Chung Fu3, Fan-Gang Tseng2,* and Rong-Long Pan1,*
1 Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua Univer-sity, Hsin Chu, Taiwan, ROC
2 Department of Engineering and System Science, National Tsing Hua University, Hsin Chu, Taiwan, ROC
3 Institute of NanoEngineering and MicroSystems, National Tsing Hua University, Hsin Chu, Taiwan, ROC
4 Institute of Nanotechnology, National Chiao Tung University, Hsin Chu, Taiwan, ROC
Introduction
Vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1)
is a homodimeric protein with a monomeric molecular
mass of 71–80 kDa [1] V-PPase catalyzes electrogenic
proton translocation from the cytosol to the vacuolar
lumen to generate an inside-acidic and inside-positive
membrane potential for the secondary transport of
ions, metabolites, and toxic substances [1–3] The cDNAs of V-PPase have been cloned from several higher plants, some protozoa, and several species of eubacteria and archeubacteria, and are highly similar (86–91% deduced amino acid identity) [1,3,4] V-PPase requires Mg2+as a cofactor, and the binding of Mg2+
Keywords
atomic force microscopy; proton
translocation; tonoplast; vacuolar
H+-pyrophosphatase; vacuole
Correspondence
R.-L Pan, Department of Life Sciences and
Institute of Bioinformatics and Structural
Biology, College of Life Sciences, National
Tsing Hua University, Hsin Chu 30013,
Taiwan, ROC
Fax: +886 3 5742688
Tel: +886 3 5742685
E-mail: rlpan@life.nthu.edu.tw
*These authors contributed equally to this
work
(Received 1 March 2009, revised 17 May
2009, accepted 10 June 2009)
doi:10.1111/j.1742-4658.2009.07146.x
Vacuolar H+-translocating inorganic pyrophosphatase [vacuolar H+ -pyro-phosphatase (V-PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cyto-sol to the vacuolar lumen, at the expense of PPihydrolysis, for the storage
of ions, sugars, and other metabolites Dimerization of V-PPase is neces-sary for full proton translocation function, although the structural details
of V-PPase within the vacuolar membrane remain uncertain The C-termi-nus presumably plays a crucial role in sustaining enzymatic and proton-translocating reactions We used atomic force microscopy to visualize V-PPases embedded in an artificial lipid bilayer under physiological condi-tions V-PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V-PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen The mean height and width of the cyto-solic V-PPase protrusions were 2.8 ± 0.3 nm and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 nm and 21.7 ± 3.6 nm, respectively Moreover, both C-termini of dimeric subunits of V-PPase are
on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6·His tags on C-terminal ends of the enzyme The distance between the V-PPase C-termi-nal ends was determined to be approximately 2.2 ± 1.4 nm Thus, our study is the first to provide structural details of a membrane-bound V-PPase dimer, revealing its adjacent C-termini
Abbreviations
AFM, atomic force microscopy; DDM, n-dodecyl-b- D -maltoside; GNP, gold nanoparticle; SD, standard deviation; V-PPase, vacuolar
H + -pyrophosphatase; TEM, transmission electron microscopy.
Trang 2can stabilize and activate the enzyme [1,5] Relatively
high concentrations of K+stimulate the
proton-trans-locating function of V-PPase, whereas excess amounts
of PPi, Ca2+, Na+and F)inhibit its enzymatic
activ-ity [6–8] It is conceivable that the V-PPase provides
specific binding domains for the substrate and the
above-mentioned ions, as well as proton translocation
Truncation of the C-terminus induces a dramatic
decline in V-PPase enzymatic activity, proton
translo-cation, and coupling efficiency [9] In addition, deletion
of the C-terminus of V-PPase increases its
susceptibil-ity to heat stress and substantially increases the
appar-ent K+ binding constant It is thus likely that the
C-terminus plays an essential role in sustaining the
physiological functions of V-PPase
Interactions between the subunits of the V-PPase
dimer have been studied [1,2,10–12] Radiation
inac-tivation analysis demonstrated that the proper
dimeric structure of V-PPase on tonoplastic
mem-branes is a prerequisite for both enzymatic activity
and PPi-supported proton translocation [2,11,12]
Further target size measurements revealed that only
one subunit of the purified dimeric complex was
suf-ficient for the enzymatic reaction of V-PPase,
although proton translocation requires the presence
of both subunits [2] Moreover, high hydrostatic
pressure was employed to inhibit V-PPase through
subunit dissociation of the enzyme, resulting in
inac-tive forms [10] The physiological substrate and
sub-strate analogs enhance the high-pressure inhibition of
V-PPase, indicating the vulnerability of the subunit–
subunit interaction [10] The above lines of evidence
illustrate explicitly the importance of dimer
forma-tion for V-PPase funcforma-tion, and suggest nonrandom
and sequestered association of V-PPase subunits
within the vacuolar membrane Furthermore, the
structures of purified V-PPases from pumpkin
(Cu-curbita sp Kurokawa Amagur) and Thermotoga
mar-itime have been examined by electron microscopy
[13,14] Notwithstanding this, structure–function
rela-tionships within this proton-translocating complex
require further study
Atomic force microscopy (AFM) is a powerful
tool used for nanoscale structural analysis of protein
complexes [15,16], and of supported lipid bilayers in
particular [17–20] For instance, AFM has provided
marvelously high-resolution images of purified
dimeric membrane proteins in 2D crystals and of
densely packed proteins in native membranes [21–23]
In the present study, we used AFM to directly
observe purified V-PPases reconstituted into planar
lipid bilayers under physiological conditions Our
images unambiguously reveal a dimeric complex for
this proton-transporting V-PPase Furthermore, the molecular volume of V-PPase calculated from AFM images suggests the presence of two identical subun-its, verifying the notion of the homodimeric V-PPase enzyme A gold nanoparticle (GNP) technique com-bined with transmission electron microscopy (TEM) analysis was utilized to determine the distance between C-termini within a membrane-bound V-PPase dimer, and indicated that the C-termini are located at the interface of subunits
Results and discussion
AFM analysis of purified V-PPase adsorbed onto mica
Recombinant DNAs for overexpression of V-PPases containing a 6·His tag at either the C-terminus or N-terminus were prepared and transformed into a yeast host Recombinant V-PPase containing a 6·His tag at the C-terminus (Fig 1C) was overexpressed in yeast and successfully purified from microsomes Unfortunately, V-PPase containing a 6·His tag at the N-terminus was poorly expressed in yeast and was therefore excluded from the study (data not shown) SDS/PAGE analysis of the purified C-termi-nal 6·His-tagged V-PPase followed by Coomassie Blue staining or western blotting showed that it was highly purified, comprising a single major band with
a molecular mass of 73 kDa (Fig 1A), as expected from the known structure of the V-PPase monomer [1,2,10] During size exclusion chromatography, V-PPase was eluted with an apparent molecular mass
of 145 kDa, similar to its native form and in agreement with previous studies suggesting a dimeric conformation [2,10–12]
The purified V-PPase was then reconstituted into liposomes by a detergent removal method using Bio-Rad SM-2 beads combined with freeze–thaw sonica-tion [13] On addisonica-tion of PPi to the proteoliposome solution containing Mg2+, a dramatic decrease in pH was generated in the interior of the liposomes (Fig 1B, lower trace) The acidic pH was eliminated by the addition of the ionophore gramicidine D (5 lgÆmL)1), indicating the integrity of the membrane (data not shown) The liposomes alone (without V-PPase) did not exhibit proton-translocating activity (Fig 1B, upper trace)
Individual V-PPase molecules were adsorbed randomly on the mica surface and exhibited a proto-typical globular structure under physiological condi-tions (Fig 2A) Figure 2B shows the heights of the adsorbed particles along the cross-section in Fig 2A
Trang 3The width of individual protein molecules was
measured at half the vertical height The mean width
and height [± standard deviation (SD), n = 21] of
purified V-PPase were 22.5 ± 3.2 nm and 1.6 ±
0.4 nm, respectively Furthermore, major peaks on
height and width histograms for the AFM images
also concurred with those parameters obtained above
for V-PPase molecules (Fig 2C,D) The flattening of
particles was presumably caused by the interaction
between the polar surface of the protein molecules
and the charged surface of the mica [24] These
images represent the first direct nanoscale observation
of V-PPase
Determination of molecular volume provides the
stoichiometry of subunit components for functional
enzymes [24] In this study, the volume of V-PPase
was calculated using Eqn (1) and determined to be
302.4 ± 40.6 nm3 (Vs) (n = 21), which was slightly
larger than the theoretical value (Vprot; 274.5 nm3) of
the protein (Table 1) This slight overestimation in
volume probably arose from the broadening effect of
the AFM tip [24] It is also likely that variations in
volume measurements might arise from distinct
inter-actions of the tip with the individual purified
V-PPase particles [25] Nonetheless, these results
unambiguously demonstrate the feasibility of this
technique for nanoscale investigation of purified
V-PPase molecules
AFM analysis of V-PPase reconstituted into liposomes
The homodimeric structure of V-PPase in a planar lipid bilayer was imaged directly by AFM (Fig 3) Purified V-PPase was first reconstituted into a sup-ported lipid bilayer, as confirmed by immunofluores-cence imaging (Fig 3A) Figure 3A1 shows a planar lipid bilayer reconstituted with V-PPases and ana-lyzed by immunofluorescence using a primary anti-body against His followed by a Cy3-conjugated secondary antibody; no fluorescence was detected in bilayers without immunofluorescence labeling of the protein (Fig 3A2) In addition, no immunofluores-cence was observed when the reconstituted sample was incubated directly with the Cy3-conjugated sec-ondary antibody in the absence of primary antibody against His (Fig 3A3) Lipid bilayers lacking V-PPases also did not exhibit detectable fluorescence (Fig 3A4) These results indicated successful incorpo-ration of V-PPase into a lipid bilayer, allowing for subsequent AFM analysis
To obtain high-resolution AFM images of individ-ual V-PPases within reconstituted membranes, the proteoliposomes prepared above were fused into a large planar lipid bilayer for direct observation The thickness of the lipid bilayer without any protein was approximately 4.6 ± 0.5 nm (n = 12), determined
Fig 1 Purification and proton transport
activity of V-PPase (A) Analysis of purified
V-PPase by western blotting (top) and SDS/
PAGE and Coomassie Blue staining
(bot-tom) Lane 1: V-PPase-enriched microsome.
Lane 2: purified V-PPase Lane 3:
reconsti-tuted V-PPase Molecular mass (kDa)
mark-ers are indicated on the left (B) PPi
-associated proton translocation of
reconsti-tuted V-PPase Proton transport was
initi-ated by adding 1.0 m M PPi At the end of
each reaction, 5 lgÆmL)1gramicidin D was
added to stop the fluorescence quenching
of acridine orange (C) Topological model of
V-PPase Cylinders 1–16 indicate
mem-brane-spanning domains.
Trang 4from a cross-section of the lipid bilayer The bilayer thickness was consistent with previous AFM mea-surements of a lipid bilayer composed of a phospha-tidylcholine/cholesterol mixture and prepared in a similar aqueous environment [26] V-PPases reconsti-tuted into the lipid bilayer protruded from the bilayer surface in a diffuse pattern with a random distribution The V-PPase images fell within two cat-egories according to the extramembranous protrusion height These height differences reflect two distinct populations of individual V-PPases facing the recon-stituted membrane surface (Fig 3D,E) Three-dimen-sional analysis of individual V-PPases randomly distributed on the membrane surface indicated that 56.7% of the protrusions were small, with a mean height of 1.2 ± 0.1 nm (n = 20) (Fig 3B, solid cir-cles), and the remainder of the protrusions (43.3%) were large, with a mean height of 2.8 ± 0.3 nm (n = 17) (Fig 3B, dotted circle) The uneven distri-bution and/or orientation of V-PPases on the mem-brane suggests that targeting of the V-PPase into the vacuolar membrane of plant cells may follow a spe-cific pattern, as previously suggested [10] Figure 3C shows a cross-section along the line in Fig 3B The widths and heights of the reconstituted V-PPase pro-trusions in Fig 3B are listed in Table 1 The AFM image of reconstituted V-PPase shows a ratio of approximately 2.40 : 1 for the height values of the cytosolic and luminal sides In addition, the theoreti-cal ratio of the total amount of amino acids on the cytosolic and luminal sides was calculated as 2.31 : 1 (data not shown), verifying the efficacy of this tech-nique
The high protein density in 2D crystals or in native membranes allows high-resolution AFM topographs and the elucidation of protein subunit organization [21–23] However, it is presently difficult to obtain V-PPase reconstituted in 2D crystals or packed at high density into a membrane (data not shown) Notwith-standing this, current AFM techniques suffice to provide unambiguous images of the dimeric structure
of V-PPase Four representative examples exhibiting minor variations are shown in Fig 4A The small differences in topography of the individual V-PPases in
A
B
D
C
Fig 2 AFM analysis of purified V-PPase (A) Three-dimensional AFM image of purified V-PPase adsorbed onto mica (B) Profile of peak heights along the cross-section shown in (A) Purified V-PPase protrudes 1.6 ± 0.4 nm (n = 21) from the mica surface (C) Histo-gram of V-PPase height determined using the AFM image in (A) (D) Histogram of V-PPase width determined using the AFM image
in (A).
Trang 5the reconstituted lipid bilayer have probably resulted
from contact with the AFM tip during scanning
Nevertheless, these AFM images are adequate for
nanoscale resolution of the structural details of V-PPase [27,28] Moreover, the resolution of the images from V-PPases in reconstituted membranes was
Table 1 Dimensions of free and membrane-bound recombinant V-PPase determined by AFM Values represent means ± SD n = number
of observations Observed and predicted volumes were determined from AFM analysis using Eqn (1) and from theoretical analysis using Eqn (2).
Volume (nm 3 )
Fig 3 Reconstitution of V-PPase into proteoliposomes (A) Immunofluorescence imaging of V-PPases reconstituted into lipid bilayers (1) Sample treated with primary and secondary antibodies (2) Sample not treated with either antibody (3) Sample treated with only secondary antibody (4) Lipid bilayer lacking V-PPases but treated with primary and secondary antibodies (B) AFM image of V-PPase extramembranous protrusions on the luminal and cytosolic sides of the membrane Solid circle, luminal side; dotted circle, cytosolic side Inset: section of a lipid bilayer with thickness of 4.6 ± 0.5 nm (n = 12) (C) Profile of protrusion heights along the cross-section shown in (B) Two populations
of V-PPase protrusions were observed: one with a mean height of 1.2 ± 0.1 nm (n = 20), and one with a mean height of 2.8 ± 0.3 nm (n = 17) (D) Histogram of V-PPase protrusion heights determined using the AFM image in (B) (E) Histogram of V-PPase peak widths deter-mined using the AFM image in (B).
Trang 6typically higher than that of those directly adsorbed
onto a mica surface
The volume of the V-PPase homodimers (Vm) in the
reconstituted membrane was estimated using the height
of the protein protrusion and the thickness of the lipid
bilayer as the parameters for the volume of a sphere
(Fig 4B) The volume of reconstituted V-PPase was
measured as 332.9 ± 46.9 nm3 (n = 17) The Vprot of
a V-PPase homodimer with a molecular mass of
145 kDa, calculated on the basis of the amino acid
composition, was determined to be 274.5 nm3 [29]
This theoretical volume correlates very well with that
measured from the AFM images Note that these
images were obtained by AFM scanning in a fluid,
and therefore probably provide an authentic
illustra-tion of V-PPase structure under physiological
condi-tions The AFM images indicate the dimeric structure
of V-PPase reconstituted in a lipid bilayer This study provides the first 3D representation of individual V-PPases protruding from the cytosolic and luminal sides of a membrane in aqueous solution
Proximity of V-PPase C-termini in reconstituted membranes
Topology studies examining heterologous V-PPase expression in yeast suggested that both the C-termini and the N-termini of each subunit are located on the lumen side and are opposite the catalytic domain on the cytosolic side of the vesicular membrane [1] Because V-PPase is homodimeric, there are two possi-ble configurations for association of the two subunits; the C-termini of both subunits may protrude from the same side or from opposite sides of the membrane
Fig 4 High-resolution AFM image of V-PPase dimers in a reconstituted membrane (A) AFM analysis of extramembranous protrusions on the cytosolic side of proteoliposomes containing V-PPase (top panels) and those on the luminal side (bottom panels) (B) Topological model of the homodimeric structure of V-PPase.
Trang 7[30] The present study demonstrated two distinct types
of protrusions randomly distributed in reconstituted
lipid bilayers If the C-termini of each V-PPase subunit
within a dimer protruded from opposite sides of the
membrane, the measured heights of these two types of
protrusion should presumably be the same The
nega-tive results above thus indicate that the C-termini of
the individual subunits of the enzyme are facing the
same side of the membrane
The relative positions and proximity of the V-PPase
C-termini on the surface of the reconstituted
mem-branes were examined using an IgG antibody against
the C-terminal His tag of the enzyme (Fig 5) The
AFM image of the immunolabeled V-PPase showed
that protrusions of different heights and widths were
randomly distributed on the lipid bilayer (Fig 5A) The
antibody could bind to V-PPase on either one or two
molecules (Fig 5B) Clearly, Fig 5B2 depicts that two antibodies bind respectively to a single V-PPase mole-cule in close vicinity AFM image analysis using spip software was used to generate histograms delineating the distribution of protrusion heights and widths (Fig 5C,D), and this revealed three major groups of protrusions: (a) lower peaks (peak 1; 1.4 ± 0.2 nm mean height, n = 10) for structures of V-PPase on the lumen side of the membrane lacking bound antibody; (b) intermediate peaks (peak 2; 2.9 ± 0.2 nm mean height, n = 20) for those on the cytosolic side of the membrane; and (c) higher peaks (peak 3; 4.2 ± 0.3 nm mean height, n = 10) for antibodies bound presumably
to the lumen side The ratio of the sum of integrals for peak 1 and peak 3 (free lumen side and antibodies bind-ing to the lumen side) to peak 2 (cytosolic side) is con-sistent with our prior results (approximately 5.6 : 4.4)
Fig 5 AFM analysis of V-PPase in a reconstituted lipid bilayer immunolabeled with an antibody against His to detect the C-terminal 6·His tag of V-PPase (A) Image of a large section of immunolabeled lipid bilayer reconstituted in the presence of V-PPase (B) High-resolution images of immunolabeled protrusions in (A) (1) Protrusion showing a single antibody bound to V-PPase (2) Protrusion showing two antibodies bound to V-PPase (C) Histogram of protrusion height determined using the AFM image in (A) (D) Histogram of protrusion width determined using the AFM image in (A).
Trang 8Previous AFM imaging studies have demonstrated
that the height of a single IgG molecule is
2.4 ± 0.1 nm [24] Taking this value into account, the
height of peak 3 protrusions (4.2 ± 0.3 nm, n = 10)
would be that of IgG molecules (2.4 ± 0.1 nm) sitting
on V-PPase at the lumen side (1.2 ± 0.1 nm, n = 20)
There were also two major groups in the histogram
representing the distribution of protrusion widths,
probably for those of the single IgG molecule (mean
width = 41.5 ± 1.8 nm, n = 12; 38.5% of
protru-sions) and those of two IgG molecules (mean width =
51.3 ± 0.8 nm, n = 20; 62.5% of protrusions) bound
to a V-PPase in the lipid bilayer, respectively
(Fig 5D) It is well established that the hinge region of
IgG links the two Fab arms to the Fc portion,
provid-ing global flexibility to the IgG The flexibility of the
IgG molecule results in Fab ‘elbow bending’, Fab ‘arm
waving’ and rotation, and Fc ‘wagging’ [31] The
observed variations in the number of IgG molecules
bound to the 6·His-tagged C-termini of V-PPase
subunits have presumably arisen from such antibody
flexibility Therefore, the space between the two
anti-body molecules could not be precisely determined
using current techniques As a result, we were also
unable to accurately determine the proximity of the
V-PPase C-termini using the antibody-binding
technique
We hence employed Ni2+–nitrilotriacetic acid GNP
labeling as an alternative technique to evaluate the
proximity of C-termini within V-PPase homodimers
Extremely small Ni2+–nitrilotriacetic acid GNPs were
bound to the 6·His tags of V-PPase C-termini
recon-stituted in lipid bilayers in aqueous solution, resulting
in two major types of protrusion as observed with
AFM: the cytosolic side of V-PPase, and the particles
bound to the lumen side of V-PPase, respectively
(Fig 6) The solid circle in Fig 6B indicates GNP
bound to V-PPase C-terminus protruding from the
surface of the lipid bilayer, whereas the dotted circle,
V-PPase protrusion at the lumen side lacking bound
GNP (Fig 6B) More than 70% of V-PPases were
covered by GNPs on the luminal side (data not
shown) The height distribution histogram indicated
that the heights of the lower V-PPase protrusions
(peak 1) were consistent with those of its cytosolic
por-tions, whereas the heights of the higher ones (peak 2)
represented those of the GNPs bound to the C-termini
of the enzyme (Fig 6C) The height of the latter
protrusions (4.9 ± 0.1 nm) reflects the sum of Ni2+–
nitrilotriacetic acid GNP heights (mean height =
2.0 ± 0.1 nm, n = 16) and V-PPase heights on the
luminal side (mean height = 1.2 ± 0.1 nm, n = 20)
In contrast, the lower V-PPase protrusions represent
those of its cytosolic sides alone Moreover, in the width distribution histogram, the higher peaks (peak 1) represent either cytosolic protrusions of V-PPase lacking GNPs (mean width = 26.3 ± 4.7 nm, n = 17)
or the luminal side containing GNP-bound C-termini (mean width = 28.2 ± 1.4 nm, n = 48) Other peaks (peaks 2 and 3) in the width distribution histogram (> 50 nm) probably reflect GNP clusters, because
20% of GNPs in solution are visualized as collec-tions after sonication (data not shown)
The number of GNPs bound to V-PPase C-termini was then predicted using a Microscope Simulator (Com-puter Integrated Systems for Microscopy and Manipula-tion, University of North Carolina, Chapel Hill, NC, USA) (Fig 6E) The width of the image for a single GNP on mica was empirically determined as 21.2 ± 1.1 nm (n = 27, data not shown); the theoreti-cal width of a single GNP on the surface of V-PPase was
24 nm (Fig 6E, solid rhombus) The mean width of GNPs on the surface of V-PPase was empirically mea-sured as 28.2 ± 1.4 nm (n = 48), suggesting that more than one GNP was present on the surface of V-PPase Because V-PPase is a homodimeric enzyme, it is conceiv-able that one GNP was bound to each C-terminus Moreover, the distance between two GNPs (reflecting that between two V-PPase C-termini) was extrapolated from a simulation plot (Fig 6E, solid circles) The solid triangle in Fig 6E reflects the mean width of GNPs on the surface of V-PPase, corresponding to a GNP dis-tance of 2.2 ± 1.4 nm Our results suggest explicitly that the two C-termini of V-PPase are in close proximity
To validate the prediction that the V-PPase C-termini are adjacent, a TEM analysis was used to directly mea-sure the distance between two GNPs bound to the C-ter-mini of purified V-PPase The TEM image displays the bound GNPs as solid spheres with a diameter of 2.0 ± 0.2 nm (n = 18) (Fig 7A) In addition, GNPs bound to V-PPase C-termini occurred in pairs (Fig 7B), indicating the dimeric structure of the enzyme The his-togram showing the distribution of distances between GNP pairs observed from the TEM image yields a mean distance of 1.9 ± 0.8 nm (Fig 7C), concurring with the result generated by AFM analysis of GNP-labeled V-PPase (Fig 6E; distance = 2.2 ± 1.4 nm) The slight fluctuation in distances between GNP pairs most likely arose from the flexibility of the V-PPase C-termini For instance, the shorter distance observed indicates two closed GNPs on the C-termini of the enzyme In con-trast, the longer distance indicates a probable extension
of the C-termini of V-PPase Verification of these possi-bilities requires further investigation
The C-terminus of V-PPase has been determined to
be relatively conserved among various plant V-PPases,
Trang 9and is presumed to be proximal to the catalytic site
[32] In addition, the importance of the V-PPase
C-terminus in sustaining enzymatic and
proton-translo-cating function and for indirect regulation of K+
binding has been demonstrated [9] Moreover,
inter-subunit interactions of V-PPase are critical for proper
enzyme function [10], suggesting that the interface
between the two subunits may participate in
enzy-matic and proton-pumping reactions In the present
study, AFM measurements and single nanoparticle
analysis using TEM further demonstrated that the two C-termini of V-PPase homodimers are approxi-mately 1.9–2.2 nm apart In conclusion, our study provides high-resolution images of single V-PPase molecules within a membrane, allowing analysis of the architecture, size and structure of V-PPase in a physiologically relevant environment We propose a working model in which the proton channel lies at the interface between the C-termini of the V-PPase homodimer (Fig 8)
Fig 6 AFM analysis of Ni 2+ –nitrilotriacetic
acid GNPs bound to the C-terminal 6·His
tag of V-PPase (A) Lipid bilayer
reconsti-tuted in the presence of Ni 2+ –nitrilotriacetic
acid GNP-bound V-PPase (B)
High-resolu-tion image of individual dimeric structures of
V-PPase labeled with GNPs in reconstituted
membrane Solid circle, GNP-bound V-PPase
protrusion on the luminal side of the
recon-stituted membrane; dotted circle, V-PPase
protrusion lacking a GNP label on the
lumi-nal side of the membrane (C) Histogram of
the protrusion heights determined using the
AFM image in (A) (D) Histogram of the
pro-trusion widths determined using the AFM
image in (A) (E) Simulation of potential
V-PPase protrusion widths based on
dis-tances between GNP pairs bound to the
V-PPase C-termini Solid rhombus, predicted
protrusion width based on a single GNP
molecule bound to the C-terminus of
V-PPase; solid circle, predicted protrusion
widths based on the distance between two
GNP molecules bound to V-PPase C-termini;
solid triangle, actual protrusion width of
GNP-bound V-PPase determined by AFM.
Data represent the mean ± SD.
Trang 10Experimental procedures
Cloning, expression, and purification
The mung bean (Vigna radiata L.) V-PPase cDNA (VPP;
accession number P21616) [33] was cloned into the yeast
expression vector pYES2 (Invitrogen, Carlsbad, CA, USA),
and the two synthesized oligonucleotides Phis (5¢-CCTCG AGCCATCATCATCATCATCATTAGGGCCGCATCAT GTAATTAGTTATGT-3¢) and PMluI (5¢-GTACACGCG TCTGATCAG-3¢) were inserted into the 3¢-end of the pYES2–VPP plasmid to generate the V-PPase–(His)6tail The pYES2–VPP–(His)6 cDNA was transferred into the Saccharomyces cerevisiae strain BJ2168 (MATa, prc-407, prb1-1122, pep4-3, leu2, trp1, ura3, GAL) according to the method described previously [34] The yeast microsomal membranes enriched in 6· His-tagged V-PPase were pre-pared as described by Kim et al [35], with minor modifica-tions Finally, V-PPase-enriched membrane fractions were resuspended in the storage buffer [10 mm Tris/HCl (pH 7.6) and 10% (w/v) glycerol] and stored at )70 C for fur-ther use The V-PPase (1 mgÆmL)1)-enriched microsomal membrane fraction was solubilized in an extraction buffer [10 mm Tris/HCl (pH 8.0), 400 mm KCl, 15% (w/v) glyc-erol, 1 mm phenylmethanesulfonyl fluoride, 0.1% (w/v) n-dodecyl-b-d-maltoside (DDM)] by adding the detergent DDM dropwise, to a final concentration of 6 mgÆmL)1, and gently stirred for 30 min on ice The solution was diluted with the extraction buffer described above three-fold to five-three-fold, and unsolubilized materials were removed
by ultracentrifugation at 75 000 g at 4C for 1 h The supernatant was incubated with Ni2+–nitrilotriacetic acid beads prewashed with the extraction buffer for 1 h The
Ni2+–nitrilotriacetic acid beads were injected into the empty column and eluted at a flow rate of 0.5 mLÆmin)1 with the elution medium [10 mm Tris/HCl (pH 8.0), 15% (w/v) glycerol, 10 mm b-mercaptoethanol, 1 mm phen-ylmethanesulfonyl fluoride, 0.1% (w/v) DDM] with a step gradient of 20, 40, 60 and 250 mm imidazole, respectively The fractions with highest PPi hydrolysis activity at
250 mm imidazole were pooled and dialyzed against med-ium containing 10 mm Tris/HCl (pH 8.0), 15% (w/v) glyc-erol, and 0.1% (w/v) Triton X-100, and then stored at )70 C for further studies
Fig 7 TEM analysis of Ni 2+ –nitrilotriacetic acid GNP-labeled
V-PPase (A) TEM image of Ni2+–nitrilotriacetic acid GNP-labeled
purified V-PPase (B) A gallery of zoomed images for the GNP pairs
labelled on purified V-PPases (C) Histogram of the distances
between GNP pairs determined using the TEM image in (A).
Fig 8 A working model of V-PPase The distance between C-termini of V-PPase is approximately 2.2 nm.