(Received for publication, December 28, 1994; and in revised form, April 28, 1995)
From the
Homopteran insects, and especially Cicadella viridis,
display in their digestive tract a specialized epithelial
differentiation, the filter chamber (FC) acting as a water-shunting
complex. The main intrinsic membrane protein of the FC is a 25,000-Da
polypeptide (P25). In this paper we demonstrate that this P25
polypeptide is a member of the MIP family of membrane channel proteins,
and that P25 forms homotetramers in the native membranes.
Using
polymerase chain reaction, a 360-base pair cDNA, named cic,
was isolated from RNA of the FC. cic encodes a 119-amino acid
polypeptide (CIC) whose homologies with MIP26, AQP1 (CHIP), AQP2, and
We investigated the quaternary
structure of P25 in the membranes of the FC using biophysical analysis
of P25 nondenaturing detergent micelles, scanning transmission electron
microscopy, and image processing of conventional transmission electron
microscopic images. All those different approaches converged to the
conclusion that P25 exists as an homotetramer forming a regular
two-dimensional array in the membranes.
Water crosses the plasma membranes of most cells by diffusion
through the lipid bilayer. Particular cell types exhibit high water
permeability due to water selective membrane proteins(1) . Such
proteins have been recently identified and gathered in the aquaporin
family(2) : AQP1 (CHIP) in mammalian red cell membranes and
proximal renal tubules(3, 4, 5) , AQP2 in rat
renal collecting tubules(6) , AQP3 in rat renal collecting
ducts(7, 8, 9) , AQP4 in rat
brain(10, 11) , AQP5 in rat salivary
glands(12) , and
These membrane proteins belong to a
larger family of polypeptides, forming transmembrane channels and found
in bacteria, plants, and animals (14) called the MIP family,
from its archetype, MIP26, the major intrinsic protein of bovine lens
fibers(15) . The aquaporins are permeated by water, but fail to
pass protons, or other ions, or uncharged solutes. The explanation for
water-selective transport is unknown since only limited structural
information exists. The understanding of the selectivity at the
molecular level supports the quest for three-dimensional structural
information.
We previously investigated the filter chamber
(FC)
As a result of its extremely high
representation in the plasma membranes, it appears very likely that P25
takes an important part in the constitution of the regular array within
the native membranes and is involved in the water transport function of
the FC epithelia. We hypothesized that it could be a water specialized
channel and thus belongs to the MIP family as all other previously
characterized water channels do. Beside functional studies of P25, we
focused our work on cloning the cDNA encoding P25 associated with its
structural determination.
In the first part of this work we
demonstrate that the polypeptide P25 is a member of the MIP family. It
was therefore interesting to investigate the structural organization of
P25 in order to compare our observations with data relative to the
structure of two previously characterized MIP proteins: MIP26 and AQP1
(CHIP). Due to its abundance in the native membranes, P25 constitutes
two-dimensional crystals. This unique distribution for a MIP family
protein is very favorable for a structural investigation since native
membranes can be used directly for negative staining or cryoelectron
microscopy. We report, in the second part of this work, the native
structural organization of P25.
Insects, C. viridis, were harvested from
wet meadows from summer to autumn. After dissection, freshly collected
filter chambers were homogenized in 10 mM Tris-HCl, pH 7.3,
0.4 mM phenylmethylsulfonyl fluoride. Membranes were purified
over a discontinuous sucrose gradient as described(17) . The
membrane fraction was then washed 18 h at 4 °C in an alkaline
buffer (5 mM glycine, 1 mM EDTA, 5 mM
Alternatively, freshly isolated membranes were deposited on
glow-discharged carbon-coated nickel grids and incubated in the same
solutions as for sections, 1 h at room temperature. After rinsing, the
grids were fixed on a drop of 2.5% glutaraldehyde for 5 min, then
negatively stained with 2% uranyl acetate.
Total RNA was isolated by
tissue homogenization in a lithium chloride-urea solution (23) followed by phenol extraction and alcohol precipitation.
RNA was reverse transcribed using random hexamers as primers (Life
Technologies, Inc.). 100 ng of cDNA were used as template for PCR
amplification (94 °C, 1 min; 35 °C, 1 min; 72 °C, 1 min; 35
cycles) using 100 pmol of degenerated primers and 1.75 mM MgCl
Hybridization was performed
for 16 h at 50 °C. The hybridization medium was as following: 50%
formamide, 4
Linear
2-20% (w/v) sucrose density gradients were prepared from 2 and
20% stock solution of sucrose in H
Where n is Avogadro's number,
v, the partial specific
volume of detergent protein micelles was calculated from data obtained
by H
Figure 1:
Polyacrylamide gel electrophoresis and
immunoblot of the membrane proteins. A, silver-stained
polyacrylamide gel showing the protein content of purified plasma
membranes isolated from the C. viridis filter
chamber. The P25 polypeptide represents 80-90% of total proteins. B, Western blot. Purified membranes were electrophoresed as in A, blotted, and incubated with the rabbit anti-P25
serum.
Figure 2:
Immunolocalization of P25. A,
immunofluorescence staining of P25 on C. viridis cryosections incubated with anti-P25 serum. A strong labeling was
strictly limited to the FC; E, esophagus; C,
cuticula, exhibiting a strong nonspecific endogenous fluorescence.
Magnification
Figure 10:
Mass measurement by STEM of freeze-dried
unstained filter chamber purified membranes. A, dark-field
image of a membrane preparation. Higher signal intensities correspond
to double layer membranes. The scale bar represents 0.1
µm. B, similar vesicle as in A observed after
freeze-drying. A smooth and a rough surface are associated to the outer
face (OF) and inner face (IF), respectively. The scale bar represents 0.3 µm. C, histogram showing
the distribution of values of mass per unit area calculated from 89
measurements. The histogram exhibits two peaks: a single layer of 2425
Da/nm
Figure 3:
Agarose gel electrophoresis of the result
of PCR. A product of 360 base pairs is clearly seen on the lane
corresponding to the FC. Control lane without DNA (C).
Figure 4:
Amino acid sequence alignment of MIP26,
AQP2, AQP1,
Figure 5:
In situ hybridization using an antisense
probe prepared from cic. On sections of the digestive tract of Cicadella, a strong signal was observed on the filter chamber
only (fc). im, initial midgut. The scale bar represents 100 µm.
Figure 6:
Analysis of a synthetic peptide derived
from CIC. A, SDS-gel electrophoresis of chromatographically
purified P25. B, the dot blot shows an increasing signal with
increasing quantities of P25 after incubation with the antipeptide
serum.
Figure 7:
Analysis of column fractions by
SDS-polyacrylamide gel electrophoresis. Gel filtration analysis on a
Protein Pack SW300 HPLC column of filter chamber membrane proteins
extracted by 2% OG. Elutions were performed in a 2% OG buffer.
Fractions were collected and aliquots diluted in Laemmli SDS-PAGE
sample buffer. The gel was stained with silver nitrate. In fraction
number 16, P25-OG micelles are eluted in a pure
state.
Figure 8:
Physical properties of the P25-detergent
micelles. Purified C. viridis filter chamber
membranes were incubated either in 2% OG (A and C)
for 2 h or in 1% Triton X-100 for 12 h (B and D).
After 100,000
The sedimentation data together with the gel
filtration data allow the calculation of the molecular weight of the
P25-detergent complexes by the application of the formula given under
``Materials and Methods'' (Table 1). This gives a
molecular mass of 139,000 Da for P25-Triton X-100 micelles and 188,000
Da for P25-OG micelles. Assuming that v of the peptidic
portion of the micelle is 0.735 cm
Figure 9:
Electron microscopy and image analysis of
purified membranes. A, electron micrograph of isolated
membranes from the filter chamber, negatively stained with 2% uranyl
acetate. Membranes display a regular two-dimensional array of
particles. Inset shows the power spectrum calculated from this
area (scale bar, 0.1 µm). The unit cell dimensions are:
9.6
Mass per unit area were
calculated from STEM dark-field images of purified freeze dried
membranes as shown in Fig. 10A. The mass data presented
on Fig. 10C conforms to a bimodal distribution. The
first peak at 2425 ± 262 Da/nm
From the CTEM data, we measured the unit cell area to be 92.16
nm
Our interest in understanding the molecular origin of water
transport led us to characterize P25, an abundant, tissue-specific, and
conserved intrinsic protein of the MIP family found in the FC of the
digestive tract of an homopteran insect.
Based on functional
observations of FC of the sap sucking insect C. viridis, we postulated that the passive high water movement
occurring in those epithelia could be related to the presence of
specific channels. We have previously shown that the membranes of these
cells are covered with particles forming a regular array(17) ,
evidenced by the large amount of an intrinsic polypeptide: P25. These
epithelial cells were also shown to contain mRNA whose expression
induced an increase of water permeability(18) . Those data
suggested that P25 may constitute a water channel.
The water
channels are now referred to as aquaporins(2) , a subgroup of
the larger MIP family of proteins whose model is MIP26(15) .
MIP26 is a membrane channel with undefined specificity but seems to
allow the passage of small molecules except
water(15, 36, 37) .
Each protein of this
family carries in its sequence two NPA boxes. PCR performed on cDNA
prepared from C. viridis filter chamber total RNA
reveals that this tissue encodes a CIC protein which possesses the two
NPA boxes and whose sequence between the two boxes has 38% homology
with MIP26 and AQP1 (CHIP), 20% with
Having demonstrated that P25 is a member of MIP family it
appeared interesting to investigate its structure, for papers
concerning the structural organization of MIP family proteins are rare.
Zampighi et al.(38) proposed a two-dimensional
projection map of the MIP26 lens fiber protein showing a rough square
shape of the unit particle. However, the model failed to resolve the
monomers organization. Recently relevant structural information was
reported for AQP1 (CHIP)(39, 40) . The results were
obtained from proteoliposomes loaded with AQP1 which do not reproduce
the native conditions notably concerning the orientation of the protein
in the membrane.
The two-dimensional membrane crystals constituted
in the FC by an extraordinary abundant P25 allow microscopic
observations directly on native membranes. Under these conditions it is
clear that in each membrane the proteic channels are similarly oriented
in contrast with the proteoliposomes. It was therefore of interest to
use this model for a structural study of a MIP protein.
In the
present work we report studies of the quaternary structure of the
25,000-Da polypeptide. From hydrodynamic and electron microscopic
studies we conclude that this protein is organized as homotetramers in
the native membrane.
Nonionic detergents are widely known not to
disturb protein-protein
interactions(25, 41, 42) . We have used
Triton X-100 and OG for the extraction of filter chamber membrane
proteins. Such experiments have been carried out with AQP1-Triton X-100 (4) and MIP26-OG (43) and both reveal a tetrameric
structure of the solubilized proteins. The results obtained for P25
lead to the conclusion of the existence of P25 in a tetrameric native
form.
The structure of the filter chamber's membranes
constitutes an interesting field of investigation of the native
organization of P25. A dense packing of intramembrane particles was
previously reported for those membranes; it accounts for the relatively
high density (1.23) measured in sucrose gradients(17) . The
two-dimensional lattice depicted over the whole surface of the
membranes in electron microscopy was analyzed using image processing.
The basic motif of the lattice was found, from both negatively stained
and frozen-hydrated specimens, to be composed of 4 elongated bilobed
domains arranged around a central pit. Combined with the mass
informations provided by the STEM measurements, those averages were
interpreted as tetramers of P25 only. Despite the poor order
encountered in the membranes (resolution of the diffraction patterns
limited to the second order), we propose an informative two-dimensional
projection map of the P25 tetramer. Our results are consistent with the
two-dimensional structure reported for AQP1 (CHIP)(39) .
Whereas the CHIP tetramer is composed of 1 glycosylated polypeptide for
3 nonglycosylated monomers(4) , P25 bears no carbohydrate
residues.
The crystalline arrays observed in frozen-hydrated
membranes can be assumed to represent closely their native state since
no dehydration occurs during preparation and no fixatives or stains
were employed. The slightly higher resolution associated with the
frozen-hydrated specimen can be related to a refinement of the shape of
the monomer on the correlation average.
Isolated membranes of the FC
are clearly asymmetrical as observed in electron microscopy after
freeze-drying and shadowing and after freeze-fracture(17) .
This fact should reflect an asymmetry of P25 at the molecular level, as
it was shown for AQP1 (CHIP) from structure prediction and
three-dimensional reconstruction(44, 40) . For P25,
immunolabeling experiments using the anti-15-mer synthetic peptide
antibody should enable the precise mapping of the putative C
extramembraneous loop pointing in the extracellular
domain(35) .
As a conclusion, the structural investigation
of P25 in its native environment may contribute to the elucidation of
new elements concerning the conformation of the MIP family proteins.
P25 shares common structural and biochemical features especially with
AQP1 and MIP26. It also shows original particularities related to the
striking specialization of the epithelial complex constituting the
filter chamber. The functional state of P25 in vivo is
obviously a dense packing of oriented tetramers in the membranes. Its
function and specificity as a water channel are under investigation.
Whether the functional unit is a monomer or a tetramer also remains to
be elucidated.
The nucleotide sequence(s) reported in this paper has been
submitted to the GenBank®/EMBL Data Bank with accession
number(s) X77957[GenBank® Link].
We thank Dr. Katherine Le Guellec for helpful advice
and Louis Communier for photography. Electron microscopy was performed
at the Centre Commun de Microscopie Electronique Transmission of
Rennes University.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-TIP are 38, 38, 34, and 20%, respectively. Using a specific
antibody raised against a 15-amino acid peptide from the CIC sequence,
we concluded that CIC and P25 are identical entities, and hence that
P25 belongs to the MIP family.
-TIP in Arabidopsis
thaliana(13) .
(
)of some homopteran sap sucking insects.
In this highly specialized epithelial complex of the digestive tract,
the large excess of water ingested with the sap is rapidly transferred
from initial midgut to terminal midgut or Malpighian tubules down a
transepithelial osmotic gradient. We described the morphology of this
water shunting complex in Cicadella viridis(16) . We
showed that the whole surface of the plasma membranes from this highly
water permeable FC is covered by a regular array of membrane particles,
and that the major constituent of FC purified membranes is a
25,000-dalton hydrophobic polypeptide (P25) (17) . Finally, we
demonstrated that FC is highly enriched in mRNA species encoding water
channel proteins when microinjected into Xenopus oocytes(18) .
-mercaptoethanol) to eliminate the extrinsic
proteins(19) .
Electrophoresis and Immunoblotting
Electrophoretic analysis of membrane proteins were performed
on SDS-polyacrylamide gels according to Laemmli(20) . Gels were
stained with silver nitrate(21) . For Western blotting studies,
proteins were electrophoretically transferred onto nitrocellulose,
incubated with rabbit antisera, and revealed by peroxidase-conjugated
anti-rabbit IgG. Anti-P25 serum was used with a 1000-fold dilution and
antipeptide serum was used with 40- and 100-fold dilutions. Purified
P25 protein was also dotted on nitrocellulose in detergent solution and
subsequently revealed as in Western blotting.
Immunolocalization of P25
Immunofluorescence
Frozen C. viridis, stored at -70 °C were embedded in
historesine, and then plunged in liquid nitrogen-cooled isopentane.
Cryostat sections of 16 µm were obtained at -25 °C, they
were then deposited on slides treated with 2%
3-aminopropyltriethoxysilane in acetone. After 4 h, sections were fixed
for 5 min with a solution of 4% paraformaldehyde, 0.05% glutaraldehyde
in 0.1 M phosphate buffer then rinsed with the same buffer.
For labeling, sections were treated for 30 min with PAS (0.02 M phosphate buffer, pH 7.5, 0.03% saponin), then with 1% BSA in PAS.
They were incubated for 2 h at 37 °C in a solution of anti-P25
serum diluted 500-fold in BSA-PAS. Sections were then washed with PAS
and incubated for 30 min at 37 °C in a solution of
GAR/IgG/fluorescein isothiocyanate diluted 20-fold in BSA-PAS. After
washing in a solution of Evan's Blue-phosphate buffer,
preparations were mounted in a solution of Evan's Blue glycerol
phosphate buffer. Observations were carried out with a fluorescent
light microscope.
Gold Immunolabeling
Filter chambers were fixed for
4 h in a solution of 4% paraformaldehyde, 0.01% glutaraldehyde in 0.1 M phosphate buffer saline (PBS). After rinsing, samples were
dehydrated and embedded in Lowicryl according to Roth et
al.(22) . Ultrathin sections were picked up on collodion
carbon-coated nickel grids then immediately deposited on a solution of
1% BSA in PBS and incubated overnight at 4 °C. Sections were then
incubated for 2 h at room temperature with primary antibody diluted
300-fold in PBS-BSA, rinsed 3 times with PBS-BSA, and incubated 1 h at
room temperature with a 10-nm GAR-gold secondary antibody diluted
40-fold in PBS-BSA. After washing, grids were stained with 2% uranyl
acetate.
Design of Primers and PCR
A pair of degenerate primers (sense, 5`-ATC AAC CC(AGTC) GCC
GT(AGCT) ACC-3`, and antisense, 5-`CAG (AGCT)GA (GCA)CG GGC (AGCT)GG
GTT-3`) were designed according to the two highly conserved NPA boxes
found in cloned MIP family proteins.
. The PCR products were resolved on 1.8% agarose
gel and stained with ethidium bromide. The PCR products were cloned in
pBluescript (Stratagene) vector and sequenced by the double-strand
dideoxynucleotide termination method (Pharmacia kit).
In Situ Hybridization
Digestive tracts of C. viridis were frozen
in liquid nitrogen. 10-µm sections were realized with a cryostat
and mounted onto coated glass slides. Sections were dried for 2 h at
room temperature and treated for 5 min with saline buffer, 5 min with
Tris-HCl (10 mM), EDTA (1 mM), pH 7.6, and 15 min in
the same buffer with 1 µg/ml proteinase K at 37 °C. Sections
were then fixed for 5 min with a solution of 4% paraformaldehyde, 0.1%
glutaraldehyde, rinsed with PBS, dehydrated with successive gradated
alcohol baths, then dried under vacuum.
SSC, 10% dextran sulfate, 1
Denhardt's solution, 10 mM dithiothreitol, 0.5 mg/ml
yeast tRNA, and 0.1 mg/ml salmon DNA.
-
S-UTP-labeled
RNA probes were prepared after linearization by SmaI for
antisense or by HindIII for sense, of the pBluescript vector
containing the cic insert. Transcription by 2.5 units of T7
RNA polymerase for antisense probe or T3 for sense probe was performed
in 20 mM Tris-HCl, pH 8.25, 6 mM MgCl
, 2
mM spermidine, 10 mM dithiothreitol, 40 units of
RNasin, for 1 h at 37 °C. Plasmid DNA was eliminated by a 15-min
incubation in 1 unit of DNase I. Probes were precipitated and dissolved
in hybridization buffer at 2
10
cpm/ml. Following
hybridization, slides were rinsed for 1 h at room temperature with 2
SSC, 50% formamide, then 1 h in 1
SSC, 1 h at 37 °C
in 20 µg/ml RNase, 0.5
SSC for 1 h and 45 min at 45 °C,
and then 0.5
SSC for 15 min at room temperature. Slides were
then dehydrated and covered with an autoradiographic emulsion. Exposure
time was 14 days at 4 °C.
Peptide Synthesis and Antiserum Production
A peptide RVQGHSLYDESRPRC from the cic deduced amino
acid sequence was synthesized. Rabbits were immunized by a first
injection of the coupled peptide in complete Freund's adjuvant
followed by 5 boosts at 3-week intervals in incomplete Freund's
adjuvant. Preimmune or immune sera were assayed for reactivity with
homogenates of whole filter chambers or with chromatographically
purified P25 polypeptide.
Protein Detergent Extraction
Membranes were incubated in 1% Triton X-100, 10 mM Tris-HCl, 150 mM NaCl, pH 7.4, for 12 h at 4 °C or in
2% n-octyl--D-glucopyranoside (OG), 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, at room temperature for 2
h. Insoluble material was eliminated by a 105,000
g centrifugation for 1 h at 4 °C.
Hydrodynamic Studies
Stokes radius of protein-detergent complexes were obtained by
gel filtration. Aliquots of 20 µl, containing 1-2 µg of
membrane proteins were chromatographed at room temperature on a AcA 34
UltroGel column (when OG was used) or on a Protein Pack SW300 HPLC
column (with the Triton X-100) calibrated with protein markers of known
Stokes radius. Elutions were performed in a 10 mM Tris-HCl,
150 mM NaCl, pH 7.4, containing either 1% Triton X-100 or 2%
OG. Fractions were collected and their content analyzed by
SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
O or D
O (98%)
containing 10 mM Tris-HCl, 150 mM NaCl, and 1% Triton
X-100 or 2% OG. 100-µl samples were layered on top of gradients and
ultracentrifugation to equilibrium was performed for 18 h at 4 °C
at 100,000
g. Calibration curves for the determination
of the apparent sedimentation coefficient were constructed using
cytochrome c (s
= 1.17 S), bovine serum albumin (s
= 4.6 S), and IgG (s
= 9 S) as protein
markers. After centrifugation, 20 fractions were collected from the
bottom of each gradient. Protein content was analyzed by SDS-PAGE. All
calculations were performed as described by Sadler et
al.(24) . The molecular weight of the protein detergent
complexes were calculated using the following
equation:
is the viscosity of water at
20 °C (0.01002 g/(cm
s)), and
is the density of water at 20 °C (0.99823 g/ml). Stokes
radius (R
) were deduced from the
calibration of chromatography column and s
from the calibration of
H
O sucrose gradient.
O and D
O gradient sedimentation. Values of v = 0.940 and v = 0.801
cm
/g were used for Triton X-100 and OG, respectively (4, 25) . The partial specific volume of the peptide
moiety was assumed to be 0.735 cm
/g(26) .
Conventional Transmission Electron Microscopy (CTEM)
Negative Staining
Aliquots of membranes in
buffer (5 µl) were applied to freshly glow-discharged 400-mesh
collodion/carbon-coated grids and allowed to stand for 1 min. Grids
were then quickly blotted, briefly rinsed with distilled water, and
deposited on a 2% uranyl acetate drop. Excess stain was removed by
blotting with filter paper and grids were then air-dried. Grids were
observed with a Philips CM12 microscope operating at 80 kV.
Cryoelectron Microscopy
Approximately 10 µl of
membrane suspension were placed on a copper grid coated with a holey
carbon film freshly glow-discharged, blotted quickly with filter paper,
and then plunged by a quick release mechanism into liquid ethane. The
frozen grid was transferred to a Gatan cryoholder in a Philips CM12
microscope operating at 100 kV, the specimen was maintained below
-160 °C throughout the observations.
Image Recording
On-line digital recording of
pictures was carried out using a high resolution video camera CF 1500
ELCA (Sofretec, Bezons, France) linked to a microcomputer fitted with a
digital acquisition card. Images of 512 512 pixels were
integrated for 6 s on a 16 bit frame memory and saved as 256 gray
levels image files. The image sampling was of 0.8 nm on the specimen
scale and the electron dose was 25
e
/Å
. Corrections for dark current
and uneven illumination were done by software(27) .
Scanning Transmission Electron Microscopy (STEM)
Mass measurements were performed with the STEM at IGBMC in
Strasbourg (France), using a Vacuum Generator HB5 microscope operating
at 100 kV equipped with a cold-field emission gun and a dark-field
annulus detector. All the observations were made at -130 °C
using a specially designed cold stage(28) . Dark-field images
were recorded directly in digital form using the signal from the
annular dark-field detector. Drops of membrane suspension were adsorbed
to carbon film mounted on a microscope grid and allowed to stand for 2
min. Tobacco mosaic virus was added as an internal mass standard and
the grid was washed 4 times with double distilled water. The grid was
blotted to leave only a thin layer of fluid and immediately immersed in
liquid N. Freeze drying was carried out within the
microscope for 2 h at -80 °C.
Image Processing
Conventional Electron Microscopy
Images
Processing of digitized images of the particle arrays of
filter chambers membranes was achieved using the SPIDER software
system(29) , running on SUN UNIX workstations. From a raw
image, a suitable area was selected interactively on the image display
and padded into a square field of 512 512 pixels. In order to
calculate an initial reference image, the Fourier transform and power
spectrum were calculated and the diffraction pattern indexed. The
indexing was used to calculate a Fourier filter mask that was applied
to the Fourier transform to produce a filtered image. A subarea of the
filtered image was used as a reference in cross-correlation mapping of
similar areas in the raw image. Areas centered on the peaks in the
cross-correlation map were extracted from the raw image and
averaged(30, 31, 32) . Rotational correlation
coefficients were calculated for quantitative assessment of the
symmetry and the resolution was estimated by calculating the radial
correlation functions (32) and the phase
residuals(33, 34) .
STEM Mass Calculation
STEM images of membranes
vesicles were displayed on the television monitor and areas were
enclosed within squared contours. Masses were determined by integrating
the densities enclosed within each contour with appropriate background
subtraction. Recognizing that the observed membranes are flattened
vesicles and thus are very often double-sided, the corresponding
densities were divided by two and combined to densities from single
sided areas to obtain the mass per unit area. Each mass integral was
calibrated relative to corresponding integrals for tobacco mosaic
virus. 89 measurements were conducted on membrane areas having on
average a size of 10,000 nm.
P25 Is the Major Integral Polypeptide of the Filter
Chamber
When purified membranes from C. viridis filter chamber were analyzed by SDS-polyacrylamide gel
electrophoresis in the absence of -mercaptoethanol, 80% of the
protein in alkali-stripped membranes is P25. Only a second membrane
polypeptide of apparent molecular mass 150 kDa (P150) corresponding to
10-20% of the protein content was detected (Fig. 1A).
In order to demonstrate the tissue
specificity of P25, we conducted an immunofluorescence study on
cryosections over the whole insect. A strong immunofluorescence related
to the labeling of P25 by the anti-P25 serum was exclusively observed
over the filter chamber, and no immunoreactivity was detected over the
remaining parts of the insect (Fig. 2A).
40. B, immunogold localization of P25 on
ultrathin sections of the filter chamber. This image shows two
extremely thin neighboring epithelia in cross-section. Gold particles
are abundantly present on microvilli of the brush border (Mv).
A less intense labeling is obtained on the lamellar invaginations (L inv) and on tubular invaginations (T inv) at the
basal pole of the cells. The scale bar represents 1 µm. C, immunolabeling of purified membranes of the filter chamber
of C. viridis (same antibody as in B). Numerous gold
particles are located in clearly delineated zones corresponding to one
side. The scale bar represents 0.5
µm.
Subcellular
localization of P25 was carried out by immunoelectron microscopy on
ultrathin sections of filter chambers, incubated with the anti-P25
serum (whose specificity is reported in Fig. 1B) and
decorated with GAR-Gold. The epithelial cells exhibited a strong
immunoreactivity over the apical microvilli and the basal membrane
infoldings (Fig. 2B). Isolated membranes were strongly
labeled mainly on one side, thus inferring that P25 might be
asymmetrically inserted into the membranes (Fig. 2C).
This is indeed supported by the morphology of isolated freeze-dried
membranes observed in CTEM after shadowing (Fig. 10B).
and a double layer of 4551
Da/nm
.
Thus the filter chamber of C. viridis appears, in both
structure and composition, as constituted by very specialized epithelia
where P25 prevails as the major intrinsic membrane polypeptide.
In the Filter Chamber, a mRNA Population Encodes a
Protein of the MIP Family
We designed degenerate
oligonucleotides from the highly conserved two NPA boxes characteristic
of the MIP family proteins. A 360-base pair cDNA fragment was amplified
in the filter chamber of C. viridis by PCR using
these primers (Fig. 3). We called cic (from Cicadella) the cDNA fragment amplified and CIC the deduced
amino acid sequence. The homologies between members of the MIP family
and CIC are described in Fig. 4. Between the two NPA boxes,
sequence identity was 38% for CIC-MIP26 and for CIC-AQP1 (human or
rat), 34% for CIC-AQP2 (rat), 29% for CIC-bib (``big brain''
of Drosophila), and 20% for CIC-TIP (A. thaliana). Thus, the sequence of CIC is closely related to the
sequence the MIP channel family.
TIP with CIC. At each position, amino acid residues
identical with those of CIC are shaded. Accession numbers in
data libraries are P06624, D13906, M77829, M84344, and X77957,
respectively. The underlined sequence corresponds to the
synthetic peptide used for raising a rabbit
antiserum.
Sense and antisense probes were
prepared from cic and used for in situ hybridization
experiments in order to localize these mRNA, on sections of the
digestive tract of C. viridis. In all experiments
performed, hybridization was strong in the filter chamber and absent
from other parts of digestive tract such as initial or terminal midgut (Fig. 5). No significant signal was obtained after incubation of
tissue slices with the sense probe. These results indicated a selective
tissue distribution of mRNA encoding the CIC polypeptide.
P25 Belongs to the MIP Family
The hydrophobic
conserved domains identified in the sequence of MIP26 and AQP1 could
correspond to putative transmembrane segments. This could also be the
case for CIC. If so, the hydrophilic sequence RVQGHSLYDESRPRC (Fig. 4) should correspond to the ``C loop'' in the
two-dimensional model previously proposed for AQP1 by Preston et
al.(35) . As reported on Fig. 6B, a
positive signal is visualized on nitrocellulose membranes blotted with
increasing quantities of chromatographically purified P25, and
incubated with the antipeptide serum. On Western blots of whole
homogenate from filter chambers, this serum recognizes a single 25-kDa
polypeptide, while no signal was detected with the preimmune serum (not
shown).
The immunoreactivity of P25 with an antibody directed
against an hydrophilic amino acid sequence of a protein of the MIP
family (CIC), associated to their specific tissue localization in the
filter chamber, leads us to conclude to an identity of those two
polypeptides.
Physical Properties of P25 in Detergent
Solution
Further understanding of the organization of P25 in the
membranes were deduced from biophysical analysis of P25, nondenaturing
detergent micelles. We report in this section results obtained after
extraction of P25 with two distinct nondenaturing detergents,
subsequent gel filtration, and sucrose gradient sedimentation. Fig. 7shows a typical elution profile of a column loaded with OG
filter chamber membrane extracts. Following Triton X-100 or OG
extractions of filter chamber membrane proteins, only P25 and P150 were
detected by silver staining of polyacrylamide gels. As in Fig. 7, a polypeptide of apparent molecular mass of 75 kDa was
sometimes observed; its appearance corresponds to the cleavage of P150
disulfide bridge. Silver staining of electrophoresed fractions content
following gel filtration revealed that P25-OG micelles are eluted in a
single peak where no other polypeptide is coeluted. Qualitatively
identical results were obtained with Triton X-100 extracted proteins
submitted to gel filtration; in all experiments maximal elution peaks
of P25 and P150 were different. P25 is thus extracted as a single
species in micelles in a monomeric or an homo-oligomeric form.
The
columns were calibrated with proteins of known stokes radius. The K determined experimentally for P25-detergent
micelles permits extrapolation of their stokes radius from the
calibration curves. P25-OG and P25-Triton X-100 micelles have stokes
radii of 4.90 and 4.75 nm, respectively (Fig. 8, A and B). In some cases, protein extraction and gel filtration were
carried out with 0.1% SDS. The average value thus obtained for Stokes
radius of P25-SDS micelles was 2.90 nm. The differences observed for
P25 stokes radii in denaturing and nondenaturing detergent suggest that
if, as one can expect, the monomeric form of P25 is present in SDS, the
nondenaturing detergent-extracted P25 is in an oligomeric form.
g centrifugation, supernatants were
analyzed by: gel chromatography to determine the stokes radius of the
detergent-solubilized P25 (A and B), velocity
sedimentation in linear 2-20% sucrose density gradients prepared
in H
O or in D
O to determine s
and v (C and D). Following
ultracentrifugation, fractions were collected and analyzed by SDS-PAGE. Arrows indicate sedimentation position of P25-detergent
micelles.
The
hydrodynamic properties of the P25-detergent solubilized complexes were
further analyzed by sucrose gradient centrifugation to provide an
estimate of their sedimentation coefficient and molecular weight. For
that purpose, membrane extracts were subjected to ultracentrifugation
on linear 2-20% sucrose gradients made up in HO or in
D
O and calibrated with marker proteins of known
sedimentation coefficient. Representative sedimentation experiments are
shown in Fig. 8, C and D. The apparent
sedimentation coefficient (s
) deduced for P25-OG
micelles is 6.8 S and for P25-Triton X-100 micelles 5.35 S. The amount
of detergent bound to the peptide can be estimated from a determination
of the partial specific volume, v, of the complexes by the
method of Sadler et al.(24) . From the data obtained
by the sedimentation behavior of the micelles in H
O and
D
O gradients, the partial specific volumes of P25-OG and
P25-Triton X-100 complexes are 0.808 and 0.799 cm
/g,
respectively.
/g, that the v of proteins and detergent are additive, and that the native bound
lipid has been replaced by detergent(24) , the amount of
detergent bound to the peptide can be calculated to be 0.41 g/g of
protein for Triton X-100 and 0.801 g/g of protein for OG. Taking into
account the amount of detergent bound to P25, the molecular mass of the
protein part of the micelles are in both cases evaluated to 100,000 Da.
As demonstrated by gel filtration and SDS-polyacrylamide gel
electrophoresis, no other polypeptide than the 25,000 Da one
constitutes the protein part of the micelles. Thus, we can conclude
that in the two nondenaturing detergents used in this study, P25 is
extracted as homotetramer. This reflects the organization of the
peptide in the native membrane.
Structural Organization of P25 in the Membranes
A
high magnification image of a negatively stained purified membrane is
shown in Fig. 9A. Native membranes display a faint
regular square array of particles. The unit cell dimensions were
calculated from the diffraction pattern (Fig. 9A,
inset), and gave values of a = b =
9.6 nm. The presence of distinct sets of spots slightly rotated one
with respect to the other indicates that most of these sheets are
double layered. Such an image was used for correlation averaging and
680 subimages were averaged. The resulting average, after 4-fold
symmetrization, is shown in Fig. 9B. Most of the
protein mass is contained in a tetrameric core around the 4-fold
crystallographic axis which encloses a central stain-filled pit. The
tetrameric core, delineated 5.4 5.4 nm frame, has four
stain-excluding elongated domains of 4.5 nm length and 3.0 nm width.
The calculated resolution is 3.0 nm according to the radial correlation
function criterion and 3.5 nm according to the phase residual
criterion.
9.6 nm; 90°. B, two-dimensional projection map
of a membrane protein tetramer calculated by correlation averaging. The
resulting image corresponds to the average of 680 unit cells from a
single membrane crystal as in A with 4-fold symmetry imposed (scale bar, 2.5 nm). C, electron micrograph of
membranes isolated from the filter chamber and embedded in vitreous
ice. A native regular array is distinguishable (scale bar, 0.1
µm). Inset shows the calculated computer transform of this
image. D, averaged image from C obtained by
correlation averaging (n = 290). Contrast has been
inverted in order to get proteins displayed in bright (scale
bar, 2.5 nm).
In frozen-hydrated specimens, the unfixed and unstained
P25 protein appeared electron-dense and the vitreous ice surrounding
the membranes electron transparent (Fig. 9C). The
diffraction pattern reveals an intensity distribution and lattice
parameters very similar to the negatively stained preparation (lattice
constant of 9.3 nm, Fig. 9C, inset). Computer image
processing carried out on such images confirmed the tetrameric state of
the membrane particles as seen on the average of 290 subimages in Fig. 9D. Each monomer has a clear asymmetric
distribution of mass with a mass core of size 3.5 2.5 nm. The
resolution associated with this projection map have been estimated at
2.4 nm according to the radial correlation functions and 2.9 nm
according to the phase residual criterion.
(n =
40) represents a single layer membrane, the second peak 4551 ±
443 Da/nm
(n = 49) represents double
layers. The latest value corresponds to the strong signal of double
sided membranes as we observed in many freeze-dried shadowed vesicles (Fig. 10B). Consequently we divided by two this value
and averaged it with the first value, to give a resulting mass per unit
area of 2357 ± 270 Da/nm
(n = 89).
and containing 2 tetramers. Therefore with an average
mass of 2357 Da/nm
, the unit cell mass can be calculated to
be 217,221 and 27,152 Da for the monomer. This value can only fit with
one molecule of P25 (M
= 25,000 Da) associated
with 2,152 Da of lipid.
-TIP; the most conserved part
being the hydrophobic sequence where 6 bilayer-spanning domains are
predicted(5, 14) . A subsequent determination of the
3` and 5` failing region of the cDNA will permit a complete comparison
with the MIP family members. Nevertheless, it is obvious that in the
FC, a protein of the MIP family is expressed. The tissue specificity of
P25, documented by in situ hybridization (CIC being a fragment
of P25), immunoblotting, and immunocytochemistry data, can be related
to the unique function of those hyperspecialized water-shunting
epithelia.
-D-glucopyranoside; PAGE, polyacrylamide
gel electrophoresis; CTEM, conventional transmission electron
microscopy; STEM, scanning transmission electron microscopy.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.