Structural Characterization of Two Aquaporins Isolated from
Native Spinach Leaf Plasma Membranes*
Dimitrios
Fotiadis
,
Paul
Jenö§,
Thierry
Mini§,
Sabine
Wirtz
,
Shirley A.
Müller
,
Laure
Fraysse¶,
Per
Kjellbom¶, and
Andreas
Engel
From the
M. E. Müller-Institute for Microscopy
and § Division of Biochemistry, Biozentrum of the University
of Basel, CH-4056 Basel, Switzerland and the ¶ Department of
Plant Biochemistry, Lund University, S-221 00 Lund, Sweden
Received for publication, October 13, 2000
 |
ABSTRACT |
Two members of the aquaporin family, PM28A and a
new one, PM28C, were isolated and shown to be the major constituents of
spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a
smaller one, were found by transmission electron microscopy of
negatively stained solubilized proteins and by atomic force microscopy
of PM28 two-dimensional crystals. The ratio of larger to smaller
particles observed by transmission electron microscopy and single
particle analysis correlated with the ratio of PM28A to PM28C
determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are
differentially expressed in spinach leaves. These findings suggest that
differential expression of the various aquaporin isoforms may regulate
the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.
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INTRODUCTION |
Water is the universal solvent and most important molecule for
life. Immense water volumes pass across the membranes of all living
cells, especially across the plasma membranes of plants (1). Simple
diffusion of water through the lipid bilayer has an activation energy
of >10 kcal/mol and cannot explain the rapid water flow through human
red cell membranes found to exhibit an activation energy of <5
kcal/mol. This led to the hypothesis that water pores must exist
(2). The discovery (3) and cloning (4) of the first known water
channel, aquaporin-1 (AQP1),1
from human erythrocytes and the demonstration of water transport in
Xenopus oocytes expressing its complementary RNA (5)
confirmed this hypothesis.
Since then, a large number of channel-forming integral proteins
homologous to AQP1 have been found in all forms of life (6). This
membrane protein family was initially named the MIP family after its
first sequenced member, the major intrinsic protein (MIP) of bovine
lens fiber cells (7). Multiple sequence alignment and phylogenetic
analysis of 164 members of the MIP family, now frequently referred to
as aquaporin super family, revealed 16 subfamilies that form two
distinct clusters, the aquaporin (AQP) cluster and the glycerol
facilitator-like cluster (8). The AQPs are highly specific for water,
whereas the glycerol facilitators allow the passage of small, nonionic
molecules such as glycerol and urea (9). In addition, ovine MIP
tetramers have been found to form a groove and tongue contact via their
extracellular surfaces, lending support to a dual function of the
protein, as a water channel and as a cell to cell adhesion molecule in
the eye lens (10).
Most members of the aquaporin super family have similar molecular
masses, ranging from 25 to 31 kDa. Based on their sequence homology all
members are predicted to comprise six hydrophobic, membrane-spanning
-helices connected by five loops of variable length and to have
cytosolic N and C termini (7, 11). Highly conserved regions are located
on the loops B and E, which contain the NPA amino acid motifs (12).
Site-directed mutagenesis experiments led to the hypothesis that loops
B and E fold back into the membrane and that the NPA boxes must be
involved in the selectivity filter of the channel (13). Other highly
conserved residues are found in the helices, revealing the
transmembrane helix-helix packing motif GXXXG (14), as well
as conserved charged buried residues that were proposed to form ion
pairs (15).
Velocity sedimentation, glutaraldehyde cross-linking, and gel
filtration (16), as well as transmission and scanning transmission electron microscopy (STEM) (17, 18), have shown that AQPs and glycerol
facilitators are tetrameric proteins. When reconstituted into lipid
bilayers, these proteins often form highly ordered two-dimensional (2D)
crystals suitable for cryo-electron and atomic force microscopy (AFM).
Consequently, a wealth of structural information is now available for
several aquaporins from different organisms, ranging from single
particle projections to the atomic model elucidated at 0.38-nm
resolution (19) and subnanometer topographical data recorded by AFM
(see Ref. 18).
Cell to cell water transport is required for many physiological
processes in plants, including the transcellular movement of water in
the transpiration stream, the circulation of water between the xylem
and the phloem, the stomatal or organ movement, and cell enlargement.
Aquaporins have been found in the plasma membrane (plasma membrane
intrinsic proteins (PIPs)) and in the vacuolar membrane (tonoplast
intrinsic proteins (TIPs)) of plants, being expressed in organ-,
tissue-, and cell type-specific manners. At the cellular level PIPs act
in water uptake and release, whereas TIPs are thought to mediate
cellular turgor maintaining the structural integrity of the cell. Plant
PIPs are further divided into two subfamilies named PIP1 and PIP2 (20).
In addition to several single amino acid residue substitutions, PIP1s
are characterized by a long N terminus and a shorter C terminus and
PIP2s by a short N terminus and a longer C terminus (21). In contrast
to PIP2, PIP1 homologs show poor water transport activity in oocytes
(20, 21). The interplay between these aquaporins is likely to be crucial in the regulation of the intracellular osmolarity. The first
plant aquaporin to exhibit water transport activity when expressed in
Xenopus oocytes similar to the human AQP1 was
-TIP from
Arabidopsis (22). Evidence for the regulation of certain plant aquaporins by reversible phosphorylation was later shown for
-TIP from seeds (23) and PM28A from spinach leaves (24) (for recent
reviews on plant aquaporins see Refs. 25 and 26).
The major integral proteins of spinach leaf plasma membranes migrate as
a single band of 28-kDa molecular mass and were thus termed
PM28. Edman degradation of endoproteinase Lys-C fragments originating from the 28-kDa band, polymerase chain reaction with oligonucleotide primers based on the obtained amino acid sequences, and
subsequent screening of a spinach leaf cDNA library yielded two
full-length clones of MIP homologs, pm28a and
pm28b. The sequence information showed that PM28A belongs to
the PIP2, whereas PM28B belongs to the PIP1 subfamily (1). In
vivo, PM28 was identified as the major phosphoprotein of the
spinach leaf plasma membrane (1). In vitro experiments using
plasma membrane vesicles demonstrated that PM28A phosphorylates in a
Ca2+-dependent manner by a plasma
membrane-associated protein kinase (1). PM28A was shown to be an
aquaporin by expression in Xenopus oocytes, and its water
channel activity is regulated by phosphorylation at two different
serine residues (24).
Here, we present the structure of PM28A and a new putative aquaporin
isoform, PM28C, whose sequence is distinct from that of PM28B but is
also a member of the PIP1 subfamily. A fast and efficient purification
protocol likely to be applicable to other plant membranes has been
developed. The oligomeric state of PM28A and PM28C particles is defined
by STEM mass analysis, and their shape, dimensions, and distribution
are defined by electron and atomic force microscopy. In addition, the
exact mass and stochiometry of the mixture containing the two isoforms
was determined by matrix-assisted laser desorption ionization-mass
spectrometry (MALDI-MS). The absence of PM28B in our preparations and
the ratio of PM28A to PM28C indicate that these PIPs are differentially
expressed in spinach leaf plasma membranes.
 |
EXPERIMENTAL PROCEDURES |
Chemicals--
Escherichia coli lipids were
purchased from Avanti Polar Lipids (Alabaster, AL),
octyl-
-D-thioglucopyranoside (OTG) was from Anatrace
(Maumee, OH), and endoproteinase Lys-C (Achromobacter protease) was from Wako Chemicals GmbH (Neuss, Germany).
Copurification of Two Plant Aquaporin Isoforms--
Preparation
of plasma membranes from spinach (Spinacia oleracea) leaves
was performed as described in Ref. 27. Proteins adhering to the plasma
membranes were removed by urea/alkali treatment (28, 29). Briefly,
membranes (protein concentration, 14-18 mg/ml) were homogenized in 100 ml of 5 mM Tris-HCl (pH 9.5), 5 mM EDTA, 5 mM EGTA, 4 M urea, 0.01% NaN3 at
4 °C and was centrifuged (40 min, 4 °C, 100,000 × g in a Kontron TFT 45.94 rotor). The pellets
were taken up in 100 ml of 20 mM NaOH (pH ~12),
homogenized at 4 °C, and centrifuged again. Finally, the pellet
obtained was homogenized with 100 ml of 5 mM Tris-HCl (pH
8), 2 mM EDTA, 2 mM EGTA, 100 mM
NaCl, 0.01% NaN3 (storage buffer) at 4 °C and was
pelleted by centrifugation. These urea/alkali-stripped plasma membranes
were resuspended at a protein concentration of 2-3 mg/ml in storage
buffer and stored frozen at
80 °C until further use.
After thawing, stripped plasma membranes were incubated with 3% OTG
(stock solution, 10% OTG in 10 mM Hepes-NaOH (pH 7.6), 0.01% NaN3) for 30 min on a shaker at room temperature.
After centrifugation (40 min, 4 °C, 117,000 × g in
a Beckman TLA 100 rotor), the supernatant was loaded onto a MiniS
column (Amersham Pharmacia Biotech) connected to a SmartTM
chromatography station (Amersham Pharmacia Biotech). The column was
equilibrated with 20 mM Bicine-NaOH (pH 8.75), 0.4% OTG,
0.008% NaN3. Proteins were desorbed from the MiniS column
using a 0 to 250 mM NaCl gradient in 20 mM
Bicine-NaOH (pH 8.75), 0.4% OTG, 0.008% NaN3 over 80 min
at a flow rate of 100 µl/min. Fractions containing PM28 were identified by SDS gel electrophoresis (30), pooled, and concentrated to
2 mg/ml in Centricon-100 cartridges (Amicon, Beverly, MA). Protein
concentrations were determined using the BCA assay (Pierce).
SDS Polyacrylamide Gel Electrophoresis and
Immunoblotting--
Solubilized or reconstituted PM28 was incubated in
sample buffer containing 1.5% SDS and 10%
-mercaptoethanol for 10 min at room temperature, run on 13% polyacrylamide gels (30), and silver-stained.
Gels were electroblotted onto Immobilon polyvinylidene difluoride
membranes (Millipore, Bedford, MA). Blots were blocked for 30 min in
2% milk powder dissolved in 10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.05% Tween 20, 0.02% NaN3
(TBST), and was subsequently incubated for 60 min with the
primary antibody (serum-diluted 1:2000 in TBST) directed against the
sequence ALGSFRSNPTN in the C-terminal region of PM28A (see Table I).
Before incubation with the anti-rabbit IgG alkaline
phosphatase-conjugated secondary antibody (Sigma; diluted 1:2000 in
TBST), the blot was washed once for 15 min in TBST containing
2% milk powder and 3 times for 10 min in TBST. Blots were washed 3 times in TBST for 10 min, once with 100 mM Tris-HCl (pH
9.5), 100 mM NaCl, 5 mM MgCl2 and were then developed with Western BlueTM (Promega) stabilized substrate for alkaline phosphatase. All washing and incubation steps of the blot
were performed at room temperature.
Two-dimensional Crystallization--
Purified protein (2 mg/ml)
was mixed with E. coli lipids solubilized in OTG (mixed
micelles stock solution, 4 mg/ml E. coli lipids in 20 mM Mes-NaOH (pH 6), 5% OTG, 0.01% NaN3) to
achieve a lipid to protein ratio of 1 (w/w). The final protein
concentration was adjusted to 1.33 mg/ml, and the final OTG content was
adjusted to 1.93%. The reconstitution mixture (60 µl) was
preincubated at room temperature for 30 min and dialyzed against 1.5 liters of 10 mM Mes-NaOH (pH 6), 100 mM NaCl,
100 mM MgCl2, 2 mM dithiothreitol, 0.01% NaN3 for 24 h at room temperature, 24 h at
37 °C, and another 24 h at room temperature.
Enzymatic Digestion and Reverse-phase
Chromatography--
Solubilized PM28 (0.3 mg/ml in 20 mM
Bicine-NaOH (pH 8.75), 150 mM NaCl, 0.4% OTG, 0.008%
NaN3) was digested with the endoproteinase Lys-C at an
enzyme to substrate ratio of 1:20 (w/w) at 37 °C for 1 h. The
reaction was stopped by adding trifluoroacetic acid to a final
concentration of 0.4%.
Separation of the peptides was performed at a flow rate of 50 µl/min
on a C18 reverse-phase column (1 × 250 mm; VYDACTM 218TP51, Hesperia, CA) connected to an Applied Biosystems 120A pump. Bound peptides were eluted with a 60-min linear gradient from 0.1%
trifluoroacetic acid (solvent A) to 80% acetonitrile containing 0.09%
trifluoroacetic acid (solvent B). Elution of protein fragments was
monitored at 214 nm.
N-terminal Peptide Sequencing--
Automated Edman degradations
were performed on an Applied Biosystems 473A protein sequencer
according to the manufacturer's recommendations.
Sequence Analysis--
Alignment between PM28 sequences were
performed using the program ClustalX v1.64b (31).
Mass Spectral Analysis--
MALDI-TOF analysis was
performed on a Bruker REFLEX III mass spectrometer (Bruker Daltonik
GmbH, Bremen, Germany). PM28 reconstituted into lipid bilayers was
solubilized for less than 20 s in 80% formic acid at room
temperature (final protein concentration, 4 mg/ml). After
solubilization of the membranes, 2 µl were immediately mixed with 1 µl of matrix solution (10 mg/ml
-cyano-4-hydroxycinnamic acid
(Aldrich) in 80% acetonitrile, 0.1% trifluoroacetic acid) and placed
on the sample plate to dry. Calibration of the instrument in the high
molecular mass range was done with bovine serum albumin (Sigma) by
using the molecular masses of the singly, doubly, and triply charged
forms of bovine serum albumin. Calibration and mass measurements of the
PM28 proteins were carried out in the linear mode. The
[M+H]+ values of PM28 isoforms shown in Fig.
7B, the ratio between the two peaks, and the corresponding
standard deviations were calculated from 13 different spectra. Mass
spectra for the endoproteinase Lys-C fragments of PM28 were acquired by
mixing 1 µl of the fractions collected by reverse-phase HPLC
with 1 µl of matrix solution and spotting the mixture onto the target
plate. For mass measurement in the low molecular range the instrument
was operated in the reflectron mode and calibrated using the
monoisotopic masses of the adrenocorticotropic hormone (fragment
18-39; Fluka), substance P (Fluka), and angiotensin (Fluka).
Scanning Transmission Electron Microscopy Mass
Measurement--
PM28 isoforms solubilized in OTG were adsorbed for 1 min to glow discharged thin carbon films supported by a thick
fenestrated carbon layer (directly after cation-exchange
chromatography). The gold-plated copper grids were then washed on 8 drops of quartz double-distilled water and were freeze-dried at
80 °C overnight in the microscope. For mass analysis, annular
dark-field images were recorded in a STEM (VG-HB5) at 80 kV and doses
of 325 ± 35 electrons/nm2. Digital acquisition of the
images and microscope parameters, system calibration, and mass analysis
were carried out as described previously (32). The total experimental
error was calculated as the standard error of the mean, plus 5% of the
measured particle mass to account for the absolute calibration uncertainty.
Transmission Electron Microscopy and Image
Processing--
Detergent-solubilized particles eluted from the
cation-exchange column were directly adsorbed for 1 min to parlodion
carbon-coated copper grids rendered hydrophilic by glow discharge at
low pressure in air. Grids were washed with 4 drops of double-distilled
water and stained with 2 drops of 0.75% uranyl formate. Images were recorded on Eastman Kodak Co. SO-163 sheet film with a Hitachi H-7000
electron microscope operated at 100 kV. Electron micrographs of single
particles adsorbed to the carbon film were digitized using a
Leafscan-45 scanner (Leaf Systems, Inc., Westborough, MA).
All image processing steps described below were performed using the
Semper image processing system (33) (Synoptics Ltd., Cambridge, United
Kingdom). For single-particle analysis PM28 complexes were extracted,
aligned, and classified. Briefly, a reference was established by
selecting a well preserved particle and symmetrizing it 20-fold
rotationally (34, 35). Cross-correlation functions of this reference
with images of digitized micrographs containing adsorbed particles of
PM28 revealed correlation peaks at the particle positions, irrespective
of their angular orientation. Using this particle picking method a
gallery of 4096 particles was created. These were submitted to a
multivariate statistical analysis (36) without alignment and were
classified into clusters of particles with similar features. To this
end, a program package kindly provided by J. P.
Bretaudière (37) was used. The various cluster averages
revealed square and round shaped particles at different angular
orientations. These averages were taken as references for subsequent
angular and translational alignment of the extracted 4096 particles.
Aligned particles were classified again, and cluster averages were
calculated. The resolution of the calculated averages was estimated
according to the Fourier ring correlation function (FRC) (38), the
phase residual (PHR) (39), and the spectral signal to noise ratio
(SSNR) (40).
Atomic Force Microscopy--
The stock solution of crystals
(1.33 mg/ml protein) was diluted 20-fold in 10 mM Tris-HCl
(pH 8.8), 150 mM KCl, 25 mM MgCl2 or 10 mM Tris-HCl (pH 8.8), 50 mM
MgCl2, depending on the experiment. A 30-µl drop of this
solution was deposited on freshly cleaved muscovite mica (Mica New York
Corp., New York, NY) prepared as described previously (41). After 15 to
30 min, the sample was gently washed with the appropriate buffer to
remove membranes that were not firmly attached to the substrate. Images
were acquired with a commercial AFM (Nanoscope III; Digital
Instruments, Santa Barbara, CA) equipped with a 120-µm scanner
(j-scanner) and a liquid cell. The liquid cell was operated without an
O-ring seal. Oxide-sharpened Si3N4 cantilevers
from Olympus (Tokyo, Japan) with a length of 100 µm and a force
constant of k = 0.1 newton/m were used. AFM images were
recorded in the contact mode with forces between 50 and 150 piconewton applied to the tip. The imaging buffer was optimized
according to Ref. 42.
 |
RESULTS |
The polypeptide pattern of spinach plasma membranes exhibits a
dominant protein band of 28-kDa molecular mass (Fig.
1A, lane 1,
arrow). In the first purification step, proteins adhering to the membranes were removed by urea/alkali treatment (Fig.
1A, lane 2). This stripping yielded a pure plasma
membrane preparation highly enriched in membrane proteins. New bands
appeared above the 42.7- and the 66.2-kDa markers, indicating
aggregation of the very dominant polypeptide otherwise running at 28 kDa (Fig. 1A, lane 2). Moreover, the lipid
bilayer not accessible to the detergent before the urea/alkali
treatment could now be solubilized by OTG and other detergents
(e.g. dodecyl-
-maltoside and
octyl-
-D-glucopyranoside; data not shown). When a
membrane preparation solubilized in 3% OTG was applied to a
cation-exchange column, a prominent peak was observed in the elution
profile at a concentration of about 55 mM NaCl (Fig.
1B). Fractions showing UV absorbance at 280 nm were
collected and subjected to SDS polyacrylamide gel electrophoresis and
Western blotting using an antibody directed against the C terminus of
PM28A. The polypeptide bands in the silver-stained SDS gel (Fig.
1C) corresponded to the bands from the Western blot (Fig.
1D) and could, therefore, be assigned to PM28A. As seen in
Table I, the peptide used to raise
the PM28A-specific antiserum is not present in the PM28B or PM28C
protein.2 The tendency of
PM28A to form higher oligomers can also clearly be seen in the Western
blot. If the cation exchange column was not overloaded by
OTG-solubilized plasma membranes, SDS polyacrylamide gel
electrophoresis of the flow-through showed complete depletion of the
band at 28 kDa, indicating full binding of the PM28 proteins to the
column matrix (data not shown).

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Fig. 1.
A, silver-stained SDS polyacrylamide gel
of spinach leaf plasma membranes before (lane 1) and after
urea/alkali treatment (lane 2). The arrow
indicates the position of the 28-kDa polypeptide band containing the
MIPs of spinach leaf plasma membranes. B, cation exchange
chromatography of stripped plasma membranes after solubilization in 3%
OTG. PM28 was eluted using a 0-250 mM NaCl gradient in 20 mM Bicine-NaOH (pH 8.75), 0.4% OTG, 0.008%
NaN3. Continuous line, elution profile monitored
at an UV absorbance of 280 nm. Broken line, NaCl gradient.
C, silver-stained SDS polyacrylamide gel of the collected fractions.
D, Western blot probed with an antibody directed against the
C terminus of PM28A.
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Table I
Alignment of the C termini of PM28A, B, and C
The peptide used to raise the PM28A-specific antiserum is depicted in
bold.
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To check the purity of isolated PM28A further, the protein pool eluted
from the cation exchanger was cleaved with the endoproteinase Lys-C,
and the digest was separated by reverse-phase HPLC. The m/z
values measured by MALDI-TOF for the fractions of the various elution
peaks are indicated in Fig. 2. Fractions
that could be identified as fragments coming from PM28A (Fig. 2,
underlined m/z values) are listed in Table
II. Fractions that could not be identified based on the measured mass were subjected to amino acid
sequencing (Fig. 2, peaks marked with an
asterisk). The fragments at m/z 2406.3 and
m/z 4165.5 yielded the N-terminal sequences a,
GFQPGPYQVGGGGSNYVHHGYTK and b, QINNWNDHWIFWVGPFIGA,
respectively. Edman degradation of the peptides at m/z
1594.5 and m/z 5526.6 yielded no amino acid sequences. Fig.
3 illustrates the high homology between
the two sequenced fragments and parts of the PM28A sequence. Accordingly, the fragment at m/z 2406.3 forms the C
loop, and that at m/z 4165.5 forms part of the E loop and
helix 6 of PM28C. The sequence of new isoform, PM28C, shows that it
belongs to the PIP1 subfamily of plasma membrane AQPs2 (see
"Experimental Procedures" for details on the sequence analysis and
assignment of the fragments to the different domains of PM28A). No
indications were found for the presence of PM28B in our
preparations.

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Fig. 2.
C18 reverse-phase chromatography of
endoproteinase Lys-C-digested PM28. The numbers above
the peaks indicate the masses of the peptides measured by
MALDI-TOF in the corresponding fractions. Masses that could be assigned
to the known PM28A isoform are underlined (for more details
see Table II). Peaks labeled with B indicate
components originating from the buffer-containing detergent (20 mM Bicine-NaOH (pH 8.75), 150 mM NaCl, 0.4%
OTG, 0.008% NaN3). The elution peaks labeled
with N yielded blank spectra in the mass spectrometer.
Elution was monitored at a UV absorbance of 214 nm. Peaks
marked with an asterisk were subjected to amino acid
sequencing. The fragments at m/z 2406.3 and m/z
4165.5 could be successfully sequenced by Edman degradation and yielded
the amino acid sequences GFQPGPYQVGGGGSNYVHHGYTK and
QINNWNDHWIFWVGPFIGA, respectively.
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Fig. 3.
Topology of PM28A and comparison with the
fragments of the new isoform PM28C. The amino acid residues shown
in reverse typeface indicate sequences that could be
identified by MALDI-MS and amino acid sequencing. The
scissors indicate the cleavage sites for endoproteinase
Lys-C. The fragments at m/z 2406.3 and m/z 4165.5 (see Fig. 2) form part of loop C and parts of loop E and helix 6, respectively, of the new aquaporin isoform isolated from spinach leaf
plasma membranes. The 6 transmembrane -helices are represented
according to Ref. 19.
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Mass measurement by STEM was used to assess the homogeneity of the
purified PM28 isoforms and determine their aggregation state. A typical
low dose dark-field image recorded from freeze-dried, unstained PM28 is
shown in Fig. 4A. The mass
analysis of 1169 particles yielded a single peak at 157 ± 56 kDa
after correction for beam-induced mass-loss. This is compatible with a
tetrameric protein embedded in a detergent micelle of about 40 kDa.
Trimeric or pentameric complexes could be excluded, because the total
experimental error of the mean amounted to ± 8 kDa (for details
see "Experimental Procedures").

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Fig. 4.
Mass analysis of the PM28 isoforms by
STEM. A, elastic dark-field STEM image of freeze-dried,
OTG solubilized PM28 particles. B, mass histogram of 1169 particles selected from images recorded at an average dose of 325 ± 35 electrons/nm2. After the data have been corrected for
beam-induced mass-loss, a mass of 157 ± 56 kDa was obtained. The
scale bar in A represents 100 nm.
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Detergent-solubilized PM28 particles were negatively stained and
examined by transmission electron microscopy. Fig.
5A shows the homogeneity of
the purified PM28 isoforms after the cation exchange chromatography
step. However, two particle types with subtle differences could be
distinguished upon close inspection of the electron micrographs, one
type being larger and rather circular (Fig. 5B) and the
other smaller and almost square (Fig. 5C). Single-particle
and multivariate statistical analysis was applied to improve the signal
to noise ratio. Top view projection averages for the two particle types
were calculated from a gallery of 4096 particles extracted from
digitized electron micrographs by automated picking. Both populations
were clearly tetramers as already predicted by STEM mass measurement,
consequently the averages were 4-fold symmetrized. The projection of
the larger and rather circular PM28 isoform (Fig. 5B,
(-) unsymmetrized and (+) 4-fold symmetrized
particles) calculated from 444 motifs of 4096 had a side length of 8.8 nm and showed less density in the center of the tetramer than at the
periphery. The smaller and almost square PM28 isoform was less abundant
(Fig. 5C, (-) unsymmetrized and (+)
4-fold symmetrized particles). The projection was calculated from 255 motifs of 4096, had a side length of 8.0 nm, and had a homogeneous
density distribution over the whole particle. The resolution of the
projection averages was determined using the following three criteria:
(i) the FRC (38), (ii) the PHR (39), and (iii) the SSNR (40). The
average of the larger and rather circular particle type (Fig.
5B) had a resolution of 1.8 (FRC), 2.4 (PHR), and 1.6 nm
(SSNR), whereas the smaller and almost square one (Fig. 5C)
had a resolution of 1.8 (FRC), 2.5 (PHR), and 2.6 nm (SSNR).

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Fig. 5.
Electron microscopy and image processing of
PM28 oligoforms. A, the homogeneity of the preparation
is reflected in the electron micrograph. However, based upon
subtle differences the particles could be separated into two
populations, one displaying larger and rather circular (gallery
B) and the other smaller and almost square projections
(gallery C). After single particle and multivariate
statistical analysis of PM28 isoforms, averages were calculated. The
unsymmetrized (-) and the 4-fold symmetrized (+)
averages are shown in B (number of particles added = 444; side length, 8.8 nm) and C (number of particles
added = 255; side length, 8.0 nm). The scale bar in
A represents 65 nm. The frame size in galleries B
and C is 16 nm.
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2D crystals formed when PM28 was reconstituted into lipid bilayers. Two
different crystal types were found; one with a p4212 symmetry and lattice vectors of a = b = 9.76 ± 0.06 nm
(Fig. 6A) and the other with a
hexagonal lattice of closely packed tetramers and unit cell dimensions
of a = b = 8.58 ± 0.18 nm (Fig. 6B). Imaging
in the AFM, which has a much higher signal to noise ratio than the
transmission electron microscope, indicated segregation of the two
isoforms into two different crystal types and confirmed the structural
features of the isoforms revealed by single particle analysis. The
tetramers in the p4212 crystal could be correlated to the
smaller and almost square PM28 isoform (Fig. 5C), whereas the particles in the hexagonal lattice corresponded to the larger and
rather circular one (Fig. 5B). Crystals containing a mixture of both particle types were not observed. The surface topograph of the
p4212 crystal (Fig. 6A and inset)
shows some similarity to the topograph of aquaporin-Z from E. coli (43). Also the unit cell dimensions of the crystal and the
side length of the particles forming it are almost identical to those
found for aquaporin-Z (44). Particles forming the hexagonal crystal
were less compact and were characterized by a deep indentation in the
center of the tetramer. This central indentation correlates with the
low density found in the larger and rather circular projection average obtained by single-particle analysis (Fig. 5B). Also in AFM
topographs, the particles displayed a less pronounced square shape,
although four corners could frequently be seen at higher magnification (Fig. 6B and inset). The tetramers seem to be
flexible, allowing the formation of a hexagonal lattice, which has
never been observed before for aquaporin crystals.

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Fig. 6.
Atomic force microscopy of PM28 isoforms
after reconstitution into lipid bilayers. The tendency of the two
isoforms to segregate into two different crystal forms is shown.
A, the topography of the less abundant crystals can be
assigned to the smaller and almost square particles found initially by
single-particle analysis of negatively stained PM28 (see Fig.
5C). This isoform crystallizes with a p4212
symmetry and lattice vectors of a = b = 9.76 ± 0.06 nm.
B, the larger and rather circular particles compatible with
those in Fig. 5B can be recognized in the more abundant
hexagonal crystal with lattice vectors of a = b = 8.58 ± 0.18 nm. The insets display well preserved particles.
Imaging buffers, 10 mM Tris-HCl (pH 8.8), 150 mM KCl, 25 mM MgCl2 (A)
and 10 mM Tris-HCl (pH 8.8), 50 mM
MgCl2 (B). Scan frequencies, 6.1 (A)
and 5.1 Hz (B). Scale bars represent 35 (A) and 25 nm (B). Frame sizes of the insets, 7.5 (A) and 10.0 nm (B). Vertical brightness ranges,
2.0 (A) and 1.0 nm (B).
|
|
Mass analysis by MALDI-TOF was performed on the reconstituted membranes
to measure the exact mass of the mature proteins and to exclude the
existence of additional isoforms, e.g. PM28B. Fig. 7A shows the MALDI spectrum of
PM28 isoforms. Two singly charged molecular ions, [M+H]+,
were recorded, one at m/z 29839 ± 11 (n = 13) and the other at m/z 30683 ± 7 (Fig. 7B, n = 13). In addition to the
[M+H]+, the doubly charged ion, [M+2H]2+,
and the singly charged dimer, [2M+H]+, of PM28A and the
new isoform were observed (Fig. 7A). No additional peaks,
which would indicate the presence of other proteins, were found. Hence,
the purification strategy described in the present report yields PM28A
and PM28C preparations of high purity.

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|
Fig. 7.
MALDI spectrum of PM28 isoforms reconstituted
into lipid bilayers. A, the singly charged molecular
ions, [M+H]+, the doubly charged ions,
[M+2H]2+, and the singly charged dimers,
[2M+H]+, of PM28A and the new isoform are shown.
B, two peaks, one at m/z 29839 ± 11 and the
other at m/z 30683 ± 7, indicate the masses of the
mature isoforms.
|
|
 |
DISCUSSION |
The urea/alkali stripping of spinach leaf plasma membranes proved
to be a crucial step in our purification protocol. Not only was the
amount of nonmembrane protein contaminants considerably reduced, but
the lipid bilayers were also made accessible to the solubilizing
detergent, enabling highly efficient solubilization. Because the plasma
membranes of various plants and their tissues can be isolated by
two-phase partitioning (27), this purification step should also
facilitate the isolation of both their aquaporins and other membrane proteins.
The aquaporin PM28A (1) from spinach leaf plasma membranes was
identified by Western blot and MALDI-MS analysis of endoproteinase Lys-C-digested PM28A peptide fragments. The latter analysis also led to
the discovery of a new isoform, PM28C, that copurifies with PM28A. The
amino acid sequences of two domains of this isoform were acquired by
Edman degradation. Comparison revealed a high homology between
corresponding domains of PM28A and PM28C, especially at the beginning
of helix 6 (see Fig. 3). The deduced amino acid sequence of the new
isoform, PM28C, has been shown to be distinct from that of PM28B.
However, both PM28B and PM28C belong to the PIP1 subfamily, whereas
PM28A belongs to the PIP2 subfamily of plasma membrane
AQPs.2 These PM28 proteins have very similar masses and
high calculated pI values (pI ~10) and should therefore copurify
together. PM28B, which was originally identified by screening of a
spinach leaf cDNA library (1) and was never isolated as a protein,
could not be detected in our preparations. This indicates that either the protein is present in very low amounts or is not located in the
plasma membrane of spinach leaves. Another explanation for this
observation could also be that PM28B is only expressed in plasma
membranes under certain, unknown stress conditions.
Both isoforms were shown to be tetramers by STEM mass analysis. The
measured mass of 157 ± 56 kDa is in good agreement with the
values determined for other detergent-solubilized MIP homologs by STEM
(17, 28, 45). The total experimental error of ± 8 kDa is
explained by counting statistics of the scattered electrons and
calibration errors. Thus, the ~3.5-kDa mass difference for the
tetramer of the two isoforms calculated from the MALDI-TOF measurement
was not resolved (Fig. 4B). However, electron microscopy of
negatively stained OTG-solubilized PM28 isoforms revealed two different
populations of particles. The most abundant form consisted of the
larger and rather circular tetramers, and the less abundant form
consisted of the smaller and almost square projections with side
lenghts of 8.8 and 8.0 nm, respectively. Because particles from
digitized electron micrographs were picked automatically (see
"Experimental Procedures"), the number of particles used to
calculate the averages in Fig. 5, B and C was a
direct indication for their distribution on the grid. The ratio of the
smaller and almost square to the larger and rather circular particles
was 0.6 as estimated from single-particle classification. This estimate is in excellent agreement with the ratio of the two peaks observed by
MALDI-MS (Fig. 7B) of 0.6 ± 0.1 (n = 13; m/z 29839:m/z 30683). Assuming similar
desorption behavior during ionization in the mass spectrometer and
similar adsorption to the carbon film of the transmission electron
microscopy grid for the two PM28 isoforms, these data indicate that the
smaller and almost square particles correspond to the isoform yielding
a peak at m/z 29839. According to the cDNA sequence,
this isoform is PM28A, which has a calculated average mass of 29931 (1).
Crystal formation was observed after reconstitution of PM28 into lipid
bilayers. Structural analysis by AFM clearly showed that the two PM28
isoforms segregate into two different crystal types; amazingly, mixed
crystals were never observed. In addition, the structural features
indicated by single-particle analysis were confirmed. The smaller and
almost square tetramers built highly ordered 2D arrays with a
p4212, whereas the larger and rather circular particles
formed less well ordered hexagonal arrays. The quality of the two
crystals is reflected by the standard deviations of the unit cell
dimensions (p4212, a = b = 9.76 ± 0.06 nm;
hexagonal lattice, a = b = 8.58 ± 0.18 nm), which was
three times higher for the hexagonal crystal. The overall shape of the
particles forming the hexagonal arrays was more round than square.
Although corners were often observed on AFM topographs recorded at high magnification (Fig. 6B and inset), there was
considerable flexibility in the shape of these particles. This
structural inhomogeneity, on the one hand, enabled the formation of a
hexagonal crystal, which is unusual for aquaporins, and on the other
hand, hindered the growth of highly ordered crystals.
MALDI-MS and more recently electrospray ionization-MS have been applied
to assess the masses of full-length membrane proteins with high
accuracy (46-48). We have used a similar approach to test the purity
of our reconstituted PM28 into 2D crystals. MALDI-MS analysis of entire
aquaporins reconstituted into lipid bilayers yielded very sharp,
symmetrical peaks and small standard deviations when the m/z
values were averaged over several spectra. Single isoforms differing by
only 844 Da could easily be resolved in the spectra. Broad,
asymmetrical peaks in the MALDI spectra arise from formylated states of
the protein. Thus, it was crucial to keep the incubation time of the
sample in formic acid as short as possible. A very interesting
perspective for the future is to demonstrate whether the resolution
achieved by this method is sufficient to detect differently
phosphorylated states of whole aquaporins or other membrane proteins.
PIP1 and PIP2 aquaporins were isolated from spinach leaf plasma
membranes and were shown to be differentially expressed. These findings
suggest that differential expression may provide a means to regulate
the water flux across the plasma membrane, in addition to the known
mechanism of regulation by phosphorylation of the PIP2 aquaporins
(24).
 |
ACKNOWLEDGEMENTS |
D. F. thanks Adine Karlsson for the
excellent introduction into plasma membrane isolation,
Dr. Christophe Reuzeau for precious help in and outside of the
laboratory during his stay in Lund, and Prof. Christer Larsson,
Dr. Ingela Johansson, Maria Karlsson, and
Dr. Cora-Ann Schönenberger for fruitful discussions.
 |
FOOTNOTES |
*
This work was supported in part by the M. E. Müller
Foundation of Switzerland, by Swiss National Foundation for Scientific Research Grant 4036-44062 (to A. E.), and by European Union-Biotech Program Grant BIO4-CT98-0024 (to A. E.). P. K. gratefully
acknowledges grants from the Swedish Council for Forestry and
Agricultural Research, the Swedish Natural Science Research Council,
European Union-Biotech Program Grant BIO4-CT98-0024, and the Swedish
Strategic Network for Plant Biotechnology.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: M. E.
Müller-Institute for Microscopy at the Biozentrum, University of
Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Tel.:
41-61-267-22-61; Fax: 41-61-267-21-09; E-mail:
Andreas.Engel@unibas.ch.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M009383200
2
L. Fraysse and P. Kjellbom, unpublished results.
 |
ABBREVIATIONS |
The abbreviations used are:
AQP, aquaporin;
[M+H]+, protonated molecular mass;
2D, two-dimensional;
AFM, atomic force microscopy;
FRC, Fourier ring
correlation function;
MALDI, matrix-assisted laser desorption
ionization;
Mes, 2-(N-morpholino)ethanesulfonic acid;
MIP, major intrinsic protein;
MS, mass spectrometry;
OTG, octyl-
-D-thioglucopyranoside;
PIP, plasma membrane
intrinsic protein;
PHR, phase residual;
HPLC, high-performance liquid
chromatography;
SSNR, spectral signal to noise ratio;
STEM, scanning
transmission electron microscopy;
TIP, tonoplast intrinsic protein;
TOF, time of flight;
Bicine, N,N-bis(2-hydroxyethyl)glycine;
TBST, Tris-buffered saline with Tween 20.
 |
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