From the Departments of Biological Chemistry and
Medicine, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205-2185, ¶ Biotechnology Institute, Akita
Prefectural University, Ogata, 010-0444, and
Microbial and
Genetic Resources Research Group, Research Institute of Biological
Resources, National Institute of Advanced Industrial Science and
Technology, Central 6, Higashi 1-1-1, Tsukuba,
Ibaraki 305-8566, Japan
Received for publication, December 5, 2002, and in revised form, January 3, 2003
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ABSTRACT |
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Researchers have described aquaporin water
channels from diverse eubacterial and eukaryotic species but not from
the third division of life, Archaea. Methanothermobacter
marburgensis is a methanogenic archaeon that thrives under
anaerobic conditions at 65 °C. After transfer to hypertonic media,
M. marburgensis sustained cytoplasmic shrinkage that could
be prevented with HgCl2. We amplified aqpM by
PCR from M. marburgensis DNA. Like known aquaporins, the
open reading frame of aqpM encodes two tandem repeats each
containing three membrane-spanning domains and a pore-forming loop with
the signature motif Asn-Pro-Ala (NPA). Unlike other known homologs, the
putative Hg2+-sensitive cysteine was found proximal to the
first NPA motif in AqpM, rather than the second. Moreover, amino acids
distinguishing water-selective homologs from glycerol-transporting
homologs were not conserved in AqpM. A fusion protein, 10-His-AqpM, was
expressed and purified from Escherichia coli. AqpM
reconstituted into proteoliposomes was shown by stopped-flow light
scattering assays to have elevated osmotic water permeability
(Pf = 57 µm·s To withstand environmental and physiological stresses, organisms
must be able to rapidly absorb and release water. Facilitated transport
of water across cell membranes must be highly selective to prevent
uncontrolled movement of other solutes, protons, and ions. Discovery of
the aquaporins provided a molecular explanation to these processes (2).
More than 200 aquaporins have now been identified, and their presence
has been established in most forms of life (3). No aquaporin from
Archaea has yet been characterized, although functional roles for a
water channel protein have been predicted in these organisms (4).
Two major protein family subsets are presently recognized,
water-selective channels (aquaporins) and glycerol-transporting homologs with varying water permeabilities (aquaglyceroporins). The
permeation selectivity of new members of the protein family may be
predicted by a small number of conserved residues (5, 6). Several
prokaryotic aquaporins and aquaglyceroporins are known. The bacterial
water channel, AqpZ, was first identified in Escherichia
coli (7, 8). Movement of water across the bacterial plasma
membrane may be part of the osmoregulatory response by which
microorganisms adjust cell turgor (9), although the regulation and
physiological role of AqpZ are being reassessed (10). AqpZ is a highly
stable tetramer with negligible permeability to glycerol. In contrast,
the glycerol permeability of the glycerol facilitator (GlpF) from
E. coli has long been recognized (11). GlpF has relatively
limited water permeability (12), and the tetrameric form has reduced
stability in some detergents (13). Atomic resolution structures have
been solved for GlpF (14) as well as human and bovine
AQP11 (15-17). These have
elucidated differential specificities and functional mechanisms
of the two sequence-related proteins.
Archaea and certain other microorganisms are able to withstand
exceptional challenges in maintaining water balance as they thrive in
extreme environments including saturated salt solutions, extreme pH,
and temperatures up to 130 °C (18). We recently recognized the
DNA sequence of AqpM, a candidate aquaporin or aquaglyceroporin
in the genome of a methanogenic thermophilic archaeon,
Methanothermobacter
marburgensis2 (19). Here
we investigate water permeability in living cells and report the
purification, functional reconstitution, and characterization of AqpM.
Materials--
Microbial growth media components were from Difco
or Bio 101, Inc. (Vista, CA). Restriction enzymes were from Takara
Biomedicals or New England Biolabs.
n-Octyl- Transmission Electron Microscopy--
M. marburgensis
cells were grown at 65 °C in 100 ml of medium as reported (20) until
exponential phase (A600 = 0.8). Culture aliquots of 10 µl were transferred to three tubes. HgCl2
was added to one tube to a final concentration of 1 mM and
gently shaken at room temperature for 30 min. The cells of the three
tubes were pelleted rapidly and resuspended in 1.0 ml of fresh media at
room temperature. A 2.5-µl drop of the cell suspension was placed
directly on a copper grid coated with a thin carbon film, upon which
osmotic challenges were performed. Osmotic up-shocks were induced by
rapidly mixing 2.5 µl of medium containing 2 M mannitol
(final mannitol concentration was 1 M after mixing). After
10 s, the cells were harvested by centrifugation and sandwiched
between 2 copper discs of 3 mm diameter, and then immediately plunged
into propane slush at liquid nitrogen temperature. The copper discs
were transferred to liquid nitrogen and separated to expose cells. The
frozen samples were freeze-substituted in acetone containing 2% osmium
tetroxide at Expression Plasmids and Strains--
The plasmid pTrc10HisAqpZ
(8) was digested with EcoRI and SalI, and
aqpM from M. marburgensis (19) was inserted in
place of AqpZ. The resulting construct, pTrc10HisAqpM (encoding
10-His-AqpM) contains the sequence of commercially available pTrc99A
(Amersham Biosciences) with the sequence
NcoI-SalI, replaced by insertion of the sequence
for a 10× His tag (MGHHHHHHHHHHSSIEGRHEF) followed by the coding
sequence for AqpM. E. coli strain BL21-CodonPlus(DE3)-RIL (Stratagene) was transformed with the expression construct. E. coli strain XL1Blue (Stratagene) transformed with pTrc10HisAqpZ was used for 10-His-AqpZ expression, and XL1Blue transformed
with pTrc10HisGlpF (13) was used for 10-His-GlpF expression. For heterologous expression of rat AQP4, the plasmid pYES2 10xHis-hAQP1 (21) was digested with EcoRI and XbaI, and the
gene encoding full-length rat AQP4 was inserted in place of hAQP1. A
pep4-deficient strain of Saccharomyces cerevisiae
with lowered proteolytic activity was transformed with the expression construct.
Expression of 10-His-tagged Aquaporins and Preparation of
Membrane Fractions--
For expression of bacterial aquaporins,
1-liter cultures of E. coli harboring the pTrc10HisAqpM,
pTrc10HisAqpZ, or pTrc10HisGlpF construct were propagated in Luria
broth containing 50 µg/ml ampicillin at 37 °C to an optical
density of about 1.5. Expression of recombinant protein was induced by
the addition of 1 mM
isopropyl- Purification of His-tagged Aquaporins--
The detergent OG was
used for AqpM, GlpF, and rAQP4 purification; the detergent DM was used
for AqpZ purification. The membrane fraction was resuspended to the
volume used for French press with solubilization buffer (3% detergent
in 100 mM K2HPO4, 10% (v/v) glycerol, 5 mM Sedimentation Analysis--
Velocity sedimentation analysis was
used to determine the oligomeric structure of purified protein.
Detergent-solubilized material (2-10 µg of purified protein in a
200-µl sample volume) was layered on top of a 4-ml continuous sucrose
gradient (20 mM Tris·HCl, 5 mM EDTA, 3% OG,
1 mM NaN3, and 5-20% sucrose, pH 8.0) and
centrifuged at 140,000 × g for 18 h in a KNOTRON
swing-out TST60.4 rotor at 20 °C. Up to 20 fractions were collected
and analyzed by SDS-PAGE to determine the migration of the protein. Pure protein was detected by Coomassie Brilliant Blue staining. The
sedimentation coefficient (s20,w) of
each species was determined by interpolation of the relative migration
versus sedimentation coefficient linear function for the
following standards: cytochrome c (1.8), carbonic anhydrase
(2.9), BSA (4.3), Functional Reconstitution--
Purified AqpM protein was
reconstituted into proteoliposomes by the dilution method, because the
dialysis method yielded non-functional proteoliposomes. E. coli total lipid extract (acetone/ether preparation, Avanti Polar
Lipids) was hydrated in 2 mM Membrane Permeability Measurements--
The osmotic behaviors of
reconstituted proteoliposomes and control liposomes were analyzed by
following the light scattering of the preparation in a stopped-flow
apparatus with a dead time of
To determine permeability to glycerol and urea, proteoliposomes were
equilibrated in assay buffer supplemented with glycerol or urea (~570
mosM). The suspensions were then rapidly mixed with a
solution in which osmolarity was compensated by a nonpermeant solute
(sucrose). Glycerol and urea permeabilities were measured at 37 °C
for 2-4 s under similar conditions as above. The external concentration of permeant solute is reduced by half (285 mosM) without a change in osmolarity, driving the efflux of
the permeant osmolyte and generating an outwardly oriented osmotic
gradient. Water efflux causes a reduction in volume and an increase in
the intensity of scattered light.
To investigate whether AqpM was sensitive to HgCl2,
proteoliposomes were incubated with 0.1 mM
HgCl2 for 30 min prior to assay; to test reversibility, 5 mM Computer Modeling of AqpM Structure--
A tetrameric derivative
of the 2.2 Å x-ray diffraction structure of bovine aquaporin-1
(Protein Data Bank code 1J4N) (17) was used as the template. By using
data from multiple sequence alignment, the amino acid sequence of AqpM
was manually threaded through the template in Swiss Protein
Database Viewer (25). The optimal tertiary structure was computed by
SWISS-MODEL. Figures were generated with VMD (26) and Raster3D
(27).
Mercury-sensitive Water Channel--
To evaluate the presence of a
water channel in archaeon, M. marburgensis cells in
exponential growth phase were subjected to hyperosmotic shock and
visualized by transmission electron microscopy. Before treatment, cells
appeared turgid (Fig. 1A). When exposed to hyperosmotic shock with 1 M mannitol, the
cells showed retraction of the cytoplasm forming plasmolytic spaces (Fig. 1B). Minimal shrinkage was observed in M. marburgensis cells pretreated with HgCl2 (Fig.
1C), suggesting the functional expression of a water channel
that can mediate osmotically driven water flux in this organism. A
similar phenomenon was observed in wild-type E. coli but not
in an AqpZ-null mutant (28).
Phylogenetic Analyses of a Candidate Aquaporin--
We recently
identified a single aquaporin-like sequence, aqpM, in the
genome of M. marburgensis (19). The deduced amino acid
sequence of AqpM was 71 and 50% identical to two candidate aquaporin
sequences from other Archaea (Archaeglobus fulgidus and
Methanosarcina barkeri) and 30-36% identical (with gaps)
to sequences from eubacteria, yeast, plants, and mammals. Multiple alignments revealed residues that are highly conserved in each of the
transmembrane domains as well as the functionally important loops B and
E (Fig. 2A). Compared with
other homologs, the archaeal sequences have a relatively long loop A
between TM1 and TM2. Information from the crystal structure of GlpF and
hydropathy analysis provided a framework for predicting the membrane
topology of AqpM (Fig. 2B). We constructed a tree with the
neighbor-joining method of phylogenetic inference (29), using sequences
from aquaporin homologs that have been functionally characterized (2,
7, 11, 30-39). The phylogenetic tree did not distinguish whether AqpM
is an aquaporin or an aquaglyceroporin (Fig. 2C). In
particular, the residues surrounding the narrowest region of the pore
(Ile-187 and Ser-196) do not conform to the corresponding residues in
either aquaporins (His-180 and Cys-189 in hAQP1) or aquaglyceroporins (Gly-191 and Phe-200 in GlpF). P2-P5 residues, which distinguish eukaryotic and eubacterial aquaporins from aquaglyceroporins, (4) did
not provide clear assignment for AqpM. The P2-P5 residues of AqpM
(Thr-203, Tyr-207, Tyr-221, and Val-222) conform with only one residue
in the aquaporins (Ser, Ala, Phe/Tyr, and Try) but with none in the
aquaglyceroporins (Asp, Arg/Lys, Pro, and Ile/Leu).
Expression and Purification of AqpM--
Xenopus
laevis oocytes injected with 5 ng of aqpM cRNA
failed to demonstrate increased water, glycerol, or urea permeabilities (data not shown) as shown for other aquaporins (37). Presumably this
reflects failure of eukaryotic oocytes to express an archaeal membrane
protein, so we attempted protein expression in bacteria for
reconstitution into proteoliposomes. The AqpM protein was predicted to
contain only short N- and C-terminal cytoplasmic domains, so the DNA
was cloned into the pTrc10HisAqpZ plasmid (8) encoding a polypeptide
with a 21-residue extension with 10 consecutive histidine residues at
the N terminus of AqpM (10-His-AqpM). E. coli cells
transformed with pTrc10HisAqpM grew normally in LB-ampicillin medium.
Addition of 1 mM
isopropyl- Oligomeric State of AqpM--
The purified 10-His-AqpM protein and
standard proteins were separately loaded on sucrose gradients. The
apparent sedimentation coefficient for the peak fraction, ~5.9 S, was
determined by comparing mobilities of 10-His-AqpM to standards ranging
from 1.8 to 11.2 S (Fig. 3A).
The value for OG-solubilized 10-His-AqpM was slightly above the values
obtained for DM-solubilized AqpZ (8) and OG-solubilized AQP1 (40),
which are known to be tetramers.
When visualized by silver staining, the purified 10-His-AqpM protein
migrated as a single molecular species of ~110 kDa in 15% acrylamide
SDS-PAGE slabs. To evaluate the molecular mass of the ~110-kDa
species, the electrophoretic behavior in SDS-PAGE was investigated at
different concentrations of acrylamide. A linear relationship was
observed between the apparent molecular mass and the acrylamide
concentration (Fig. 3B), with faster mobility in gels of
lower polymer content. This aberrant electrophoretic behavior is
characteristic of aquaporins (41). Together, the sedimentation and
electrophoretic studies suggest that 10-His-AqpM exists as a tetramer
when solubilized either in mild detergents, such as OG, or strong
detergents, such as SDS, which usually unfolds and dissociates protein subunits.
Dissociation of the tetramer was attempted under several different
conditions. Incubation of the samples with chaotropic (8 M
urea or guanidinium chloride) or hydrophilic reducing agents (500 mM Permeabilities of Reconstituted AqpM--
AqpM proteoliposomes
were prepared by dilution with E. coli lipids and
10-His-AqpM at a lipid-to-protein ratio of 75:1. AqpM proteoliposomes
and control liposomes were abruptly transferred to an outwardly
directed osmotic gradient, and the changes recorded in light scattering
were measured at
AqpM proteoliposomes consistently exhibited a transient initial phase
of glycerol permeability that was above the permeability of control
liposomes but was much less than the sustained glycerol permeability of
proteoliposomes reconstituted with the E. coli glycerol
facilitator, GlpF (Fig. 4B). AqpM proteoliposomes did not
exhibit significant urea permeability above that of control liposomes
(Fig. 4C).
Thermostability of AqpM--
Because M. marburgensis is
a thermophile, AqpM was expected to be stable at higher temperatures.
We attempted to measured water permeabilities of AqpM proteoliposomes
at elevated temperatures, but above 37 °C the control liposomes
became leaky, preventing accurate measurements (data not shown). Thus,
we pretreated AqpM proteoliposomes at temperatures up to 100 °C for
15 min and then performed stopped-flow measurements at room
temperature. AqpM proteoliposomes retained most water permeability
after pretreatments up to 80 °C but were inactive after 90 °C
(Fig. 5A). In contrast, rat
AQP4 lost most activity after pretreatments at 70 °C. Although E. coli is not a thermophile, AqpZ proteoliposomes retained
most activity after pretreatments at 90 °C but were inactive after 100 °C. Silver-stained SDS-PAGE gels of heat-treated AqpM
proteoliposomes and rat AQP4 proteoliposomes revealed evidence of
protein aggregation at the top of the lanes after pretreatments at the
inactivating temperatures (Fig. 5B).
First isolated from sewage sludge, M. marburgensis is a
methanogenic archaeon that grows optimally in an anaerobic
environment at 65 °C and utilizes carbon dioxide as a sole carbon
source (1). Hypertonic treatment of living M. marburgensis
cells revealed mercury-sensitive water permeability that led to the
archaeal aquaporin homolog, AqpM. Members of the major intrinsic
protein (MIP) family including water channels (aquaporins) and glycerol transporters (aquaglyceroporins) have been identified in diverse organisms including vertebrates, invertebrates, plants, and
microorganisms (42). This study represents the biophysical
characterization of a homolog from the third kingdom of life, Archaea.
The question of why unicellular organisms express
aquaporins remains open. The channel formed by the E. coli
water channel AqpZ has been shown to mediate large water fluxes in
response to sudden changes in extracellular osmolarity (28). A role in cell proliferation in hypotonic environments was proposed (43), but
adverse effects were not identified after disruption of aqpZ (10). There is evidence that the two aquaporin genes in S. cerevisiae, AQY1 and AQY2, may confer freeze
tolerance in industrial yeast strains (44), although both were found to
contain multiple mutations causing loss of function without clearly
adverse effects to the laboratory strains of this organism (32,
45).
Investigators (4) studying osmoadaptation in Archaea hypothesized the
existence of water channels to survive hypertonic shock following
accumulation of osmolytes. In this study, we confirmed the existence of
a water channel in one archaeal species by in vivo and
in vitro water permeability analyses. The osmotic water permeability of proteoliposomes reconstituted with purified
polyhistidine-tagged AqpM was severalfold above control liposomes but
below proteoliposomes containing E. coli AqpZ. Moreover,
AqpM proteoliposomes exhibited a transient but reproducible increase in
the initial glycerol flux, although the overall glycerol permeation was
much lower than proteoliposomes containing the E. coli
glycerol facilitator, GlpF. Our studies indicate that AqpM is a
primitive member of the large MIP family, because it functions as a
moderate water channel but a very poor glycerol transporter, so we can
only speculate about its biological function in the host organism.
Growth of Archaea in severe environments is made possible by special
plasma membranes composed of lipids that differ markedly in structure
and physicochemical properties from the glycerolipids of eubacterial
and eukaryotic cell membranes. For Archaea to maintain water balance
while growing in extreme pH environments (46), tight control of proton
and water fluxes is required (47). Unlike glycerolipids, which become
highly permeable to water and protons at elevated temperatures, the
rigid structures of archaeal lipids have particularly low permeability
to water, protons, and other ions even at high temperatures (48). AqpM
was found to retain its tertiary structure in SDS and had greater
thermostability than AQP4, a mammalian homolog (Figs. 3 and 5).
Interestingly, AqpZ from E. coli also had high
thermostability. Comparison of the amino acid sequences of aquaporins
from mesophilic species with that of thermophilic AqpM showed
considerable amino acid sequence identity (Fig. 2A) and did
not reveal an obvious explanation for the high thermostability. The
existence of AqpM provides a mechanism for thermophilic Archaea like
M. marburgensis to increase the water permeability of their
plasma membranes while remaining impermeable to protons.
Statistical sequence analyses had identified previously residues,
P2-P5, that distinguish water channels from glycerol transporters (5),
but the sequence of AqpM is ambiguous. Residues lining the narrowest
region of the pore and the P2-P5 do not completely conform to
aquaporins or aquaglyceroporins (Fig. 2, A and
B). The lower water permeability but transient glycerol
permeability observed in AqpM proteoliposomes suggests an intermediate
function. Some eubacteria such as E. coli have been shown to
possess both water-specific and glycerol-specific aquaporins, but the
Methanothermobacter genome contains only a single homologous
sequence (49). It is heuristically appealing to regard the AqpM
sequence as representative of a progenitor sequence of the more
functionally differentiated channel proteins found in other kingdoms of
life (50).
The high permeation selectivities of mammalian water channel protein
AQP1 and E. coli glycerol facilitator GlpF have been explained with atomic resolution structures (17). A hydrophilic pore-lining residue (His-180 in human AQP1) is critical for rapid water
transport but hinders passage of glycerol (17). AqpM has an aliphatic
residue at this position (Ile-187). In the three-dimensional structure
of GlpF, this position is occupied by a perpendicularly oriented
residue (Phe-200) that contributes to glycerol permeation (14). Because
of these and other differences, GlpF has a pore size that is 1 Å wider
than that of AQP1 at the point of narrowest constriction. A
computer-generated model predicts that AqpM has a pore size
intermediate between that of AQP1 and GlpF (Fig.
6). The diameter and hydrophobicity of
this aperture may be critical in providing structural clues that
determine the channel selectivities.
1 versus
12 µm·s
1 of control liposomes) that was reversibly
inhibited with HgCl2. Transient, initial glycerol
permeability was also detected. AqpM remained functional after
incubations at temperatures above 80 °C and formed SDS-stable
tetramers. Our studies of archaeal AqpM demonstrate the ubiquity of
aquaporins in nature and provide new insight into protein structure and
transport selectivity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-glucopyranoside (OG) and
n-dodecyl-
-D-maltoside (DM) were purchased
from Calbiochem. Ni-NTA-agarose was from Qiagen. E. coli
total lipid extract, acetone/ether preparation, was from Avanti Polar
Lipids. Other reagents were from Sigma or Wako Chemicals.
80 °C for 2 days, then at
20 °C for 2 h,
and 4 °C for 2 h. The samples were rinsed with fresh absolute
acetone and embedded in Spurr resin (Quetol 653). Thin sections (70-80
nm) were gathered on copper grids covered with Formvar, double-stained
with uranyl acetate and lead citrate, and examined under a transmission
electron microscope (Hitachi H-7000).
-D-thiogalactoside and incubation at 37 °C
for 2 h. Harvested cells were resuspended in 1:100 culture volume
of ice-cold lysis buffer (100 mM
K2HPO4, 1 mM MgSO4, 0.4 mg/ml lysozyme, 0.1 mg/ml DNase I, and 1 mM
phenylmethylsulfonyl fluoride) and subjected to three French press
cycles (18,000 pounds/square inch) at 4 °C. For heterologous
expression of rat AQP4, a 1-liter culture of pep4 S. cerevisiae harboring the pYES2-10xHis-rAQP4 construct was
propagated in Ura- media (27 g of Dropout Base + 0.77 g of
Complete supplement mixture minus uracil) at 30 °C to an optical
density of about 1.0 and then harvested and used to inoculate 6 liters
of YP-Gal (2% Bactopeptone, 1% yeast extract, 2% galactose). This
culture was propagated at 30 °C for about 18 h to an optical
density of around 6-7. Harvested cells were resuspended in 1/15
culture volume of 100 mM K2HPO4 and
subjected to two French press cycles (20,000 pounds/square inch) at
4 °C. For all protein preparations, unbroken cells and debris were
separated from the cell lysate by a 10-min centrifugation at 6,000 × g and discarded. The membrane fraction was recovered from
the supernatant by a 60-min centrifugation at 200,000 × g.
-mercaptoethanol, and 200 mM
NaCl, pH 8.0) and incubated on ice for 1 h. Insoluble material was
pelleted by a 45-min centrifugation at 200,000 × g and
discarded. The soluble fraction was mixed with 1-2 ml of prewashed
Ni-NTA-agarose beads and incubated with gentle agitation at 4 °C
overnight. The beads were then packed in a glass/plastic Econo column
(Bio-Rad) and washed with 100 bead volumes of wash buffer (2%
detergent, 100 mM K2HPO4, 10%
glycerol, 5 mM
-mercaptoethanol, 200 mM
NaCl, and 100 mM imidazole, pH 7.0) to remove
nonspecifically bound materials. Ni-NTA-agarose-bound material was
eluted with 0.5-1-ml amounts of elution buffer (2% detergent, 100 mM K2HPO4, 10% glycerol, 5 mM
-mercaptoethanol, 200 mM NaCl, and 1 M imidazole, pH 7.0). Protein concentrations were measured
by the Schaffner-Weissman filter protein assay method (22) with BSA as
a standard.
-amylase (8.9), and catalase (11.2).
-mercaptoethanol to a final
concentration of 50 mg/ml, incubated at room temperature for 1 h,
divided into aliquots, and frozen at
80 °C. Before use, lipids
were diluted in a borosilicate tube (16 × 125 mm) under a
nitrogen/argon atmosphere to a final concentration of 45 mg/ml in 50 mM MOPS-Na, pH 7.5, and pulsed in a bath sonicator until a
clear suspension was obtained. A reconstitution mixture was prepared in
a glass tube at room temperature by sequentially adding (to final
concentrations) 100 mM MOPS-Na, pH 7.5, 1.25% (w/v) OG,
133 µg/ml purified protein, and 10 mg/ml sonicated lipids (protein/lipid = 1:75). The reconstitution mixture was injected into 25 volumes of assay buffer under constant stirring to dilute the
detergent. Liposomes were harvested by centrifugation (45 min at
140,000 × g) and resuspended into assay buffer (50 mM MOPS and 150 mM
N-methyl-D-glucamine, pH 7.50, with HCl). Protein content was measured as described (22) with BSA as a standard.
1 ms (SF-2001; KinTek Instruments,
University Park, PA). Water permeability was measured by rapidly mixing
100 µl of a proteoliposome suspension (1 µg of protein and 75 µg
of E. coli polar phospholipids) in assay buffer (see above)
with a similar volume of hyperosmolar solution (assay buffer with 570 mosM sucrose added as an osmolyte) at 4 °C for 1 s.
The osmotic gradient (285 mosM) drives water efflux, and
the consequent reduction in vesicle volume is measured as an increase
in the intensity of scattered light (
= 600 nm). Equation 1
describes the change in volume as a function of membrane permeability
(23).
Vrel, the vesicular volume relative to
the initial volume, is proportional to the intensity of scattered light
(24) and is dimensionless. Pf is the osmotic water
permeability; S/V0 is vesicle surface
area to initial volume ratio; vw is the partial
molar volume of water (18 cm3); Ci is
the initial intravesicular osmolarity; and Co is the
external osmolarity. Single-exponential time constants (k)
were calculated by least squares fit of experimental data. A family of
simulated curves was obtained by numerical integration of Equation 1
and fitted to a single exponential. Pf was estimated
by iterative comparison of the experimental time constants with the
values obtained from the simulation by using MATHCAD software.
(Eq. 1)
-mercaptoethanol was incubated for an additional 30 min prior to assay. To determine the thermal stability of AqpM, the
proteoliposomes were incubated at temperatures from 30 to 100 °C for
15 min and gradually returned to room temperature prior to permeability
assays. For Arrhenius activation energies, water transport permeability
measurements were undertaken at temperatures from 4 to 37 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Transmission electron micrographs of
M. marburgensis cells. A,
control cells without any treatment (×39,000). B, cells
exposed to a 1 M mannitol hyperosmotic shock for 10 s
(×78,000). C, cells pretreated with 1 mM
HgCl2 for 30 min and then exposed to hyperosmotic shock
(×52,000).
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Fig. 2.
Comparative alignment, predicted membrane
topology, and phylogeny of AqpM. A, multiple sequence
alignment was performed with ClustalX 1.81, with the following
GenBankTM accession numbers: AqpM (AB055880),
A. fulgidus aquaporin homolog "AfAqp" (NP_070255),
M. barkeri aquaporin homolog "MbAqp" (ZP_00077803),
E. coli AqpZ (AAC43518), and GlpF (NP_418362), human
aquaporin-1 (NP_000376), human aquaporin-3 (NP_004916), and rat
aquaporin-4 (NP_036957). Single bold surface lines indicate
the six transmembrane domains, TM1-TM6.
Asterisks, colons, and periods
indicate perfectly, highly, and moderately conserved amino acid sites,
respectively. B, predicted membrane topology of 10-His-AqpM.
The putative transmembrane domains were assigned by hydrophobicity
analysis and manually threading the sequence of AqpM through the x-ray
crystal structure of E. coli GlpF (Protein Data Bank code
1FX8). The topology map was drawn with TeXtopo. C, an
unrooted phylogenetic tree of aquaporins was reconstructed with the
neighbor-joining method of inference. Proteins previously determined
experimentally to be aquaglyceroporins or water-selective aquaporins
are indicated.
-D-thiogalactoside arrested growth but did not
prevent protein expression. In our case, 1 liter of culture typically
yielded 3-5 mg of purified protein.
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Fig. 3.
Oligomeric state of AqpM. A,
solubilized, purified 10-His-AqpM protein was layered on top of a
5-20% continuous sucrose gradient containing 3% OG and centrifuged
at 140,000 × g for 18 h. 20 fractions were
collected and resolved by SDS-PAGE. The sedimentation coefficient
(s20,w) of AqpM was determined by
comparison with the following standards: CY, cytochrome
c (1.8); CA, carbonic anhydrase (2.9);
BSA, bovine serum albumin (4.3); AM, -amylase
(8.9); and CT, catalase (11.2). All values are mean ± S.E. B, purified 10-His-AqpM protein was resolved in
SDS-PAGE with different concentrations of acrylamide revealing apparent
sizes: ~48 kDa in 7.5% acrylamide, ~68 kDa in 10% acrylamide,
~82 kDa in 12.5% acrylamide, and ~110 kDa in 15% acrylamide.
C, purified 10-His-AqpM protein was incubated at room
temperature in 100 µl of gel loading buffer at pH values from 3.3 to
5.4 for 10 min. Samples were visualized with 15% SDS-PAGE. The
10-His-AqpM tetramer dissociated into monomers at pH <4.2.
D, purified 10-His-AqpM protein was incubated at room
temperature in 100 µl of gel loading buffer (pH 6.8) containing
50-800 mM ethanedithiol for 1 h.
-mercaptoethanol or 1000 mM
dithiothreitol) did not cause any dissociation of 10-His-AqpM, even
after 1 week (data not shown). Incubation at pH <4.0 (Fig.
3C) or with the reducing agent ethanedithiol at a
concentration of 600 mM (Fig. 3D) led to almost
complete dissociation of 10-His-AqpM to a monomer with the predicted
size of ~24 kDa.
em = 600 nm (Fig.
4A).
AqpM proteoliposomes exhibited a moderately high osmotic water
permeability of 57 ± 4 µm·s
1, whereas control
liposomes yielded much lower permeability, 12 ± 0.7 µm·s
1. The permeability of AqpM proteoliposomes was
90% inhibited by treatment with 0.1 mM HgCl2.
However, the inhibition was partially reversed with 5 mM
-mercaptoethanol. The Arrhenius activation energy was calculated
from measurements performed at various temperatures from 4 to 37 °C,
yielding a value (Ea(water) = 2.67 kcal/mol) consistent with water transport through a channel as opposed
to diffusion across the lipid bilayer as seen in control liposomes (Ea(water) = 12.9 kcal/mol).
View larger version (22K):
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Fig. 4.
Water, glycerol, and urea permeabilities of
reconstituted AqpM. A, proteoliposomes reconstituted
with purified 10-His-AqpM or control liposomes were abruptly mixed at
4 °C with a similar volume of hyperosmolar solution (reconstitution
buffer + 570 mosM sucrose). The increase in light
scattering concomitant with a reduction in vesicular volume due to
water efflux was monitored in a stopped-flow apparatus for 1 s.
The collected data were normalized between zero and unity and fitted to
an exponential rise to the maximal value curve. Indicated
proteoliposomes were pretreated at room temperature with 0.1 mM HgCl2 for 30 min or pretreated with
HgCl2 followed by incubation upon addition of 5 mM -mercaptoethanol (
-ME) for an
additional 30 min. Osmotic water permeabilities constants were
calculated as follows: Pf(liposomes) = 12 ± 0.7 µm·s
1,
Pf(AqpM proteoliposomes) = 57 ± 4 µm·s
1, Pf(AqpM + Hg2+) = 17 ± 1.3 µm·s
1,
Pf(AqpM + Hg2+ +
-mercaptoethanol) = 33 ± 4 µm·s
1. All
values are mean ± S.D. B and C, control
liposomes or proteoliposomes reconstituted with 10-His-AqpM or GlpF
were equilibrated at 37 °C with reconstitution buffer + 570 mosM glycerol or urea for 1 h. These were then
abruptly mixed at 37 °C with a similar volume of iso-osmolar
solution containing impermeant solutes (reconstitution buffer + 570 mosM
sucrose). The increase in light scattering concomitant with a reduction
in vesicular volume due to solute and water efflux was monitored in a
stopped-flow apparatus for 2-4 s, and the collected data were
normalized to fit between zero and unity. Due to a lack of fit to
single-order exponential rise to maximal value curves, solute
permeability coefficients could not be correctly calculated for AqpM
proteoliposomes; the data were fitted to double-exponential rise to
maximal curves for visual clarity.
View larger version (43K):
[in a new window]
Fig. 5.
Thermostability of reconstituted
aquaporins. A, proteoliposomes reconstituted with
10-His-AqpM, AqpZ, GlpF, or rat AQP4 and control liposomes
(lipo) were incubated for 15 min at temperatures from 30 to
100 °C and then gradually cooled to room temperature. Water
permeabilities were measured and calculated as described. B,
silver-stained 14% SDS-PAGE gels of 10-His-AqpM and rat AQP4
proteoliposomes treated at different temperatures from 30 to
100 °C.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Computer modeling of AqpM. These
figures depict the transmembrane sections of human AQP1, M. marburgensis AqpM, and E. coli GlpF, as seen from the
extracellular face through the axis of the pore. AQP1 (human residue
numbers based upon bovine AQP1) and E. coli GlpF are from
atomic resolution x-ray diffraction coordinates (Protein Data Bank
codes 1J4N and 1FX8, respectively). AqpM is computer modeled, based on
the bovine AQP1 structure. All atoms are shown as van der Waals
space-filling spheres; critical pore-lining residues are labeled and
highlighted in color (carbon atoms are turquoise, oxygen
atoms are red, nitrogen atoms are blue, and
sulfur atoms are yellow). The pore is colored bright
orange for clarity.
Mammalian water channel proteins were recognized in early studies by their reversible inhibition by mercurials (51). This may result from occlusion of the pore by covalent attachment of Hg2+ to the free sulfhydryl of a cysteine within the pore (Fig. 6, Cys-189 in AQP1) (52). Many aquaporins, including AqpZ, are not inhibited by mercurials (52), so it was surprising to find that the water permeability of AqpM was reversibly blocked by treatment with HgCl2 (Fig. 4A). Curiously, the mercury-inhibitable cysteine in AqpM does not reside proximal to the second NPA motif in loop E, as in AQP1 and some other mammalian aquaporins (52), but in the corresponding position in loop B (Fig. 2B). By site-directed mutagenesis of AQP1, this position was shown to be structurally and functionally equivalent, predicting the unique "hourglass" structure for AQP1 and other aquaporins (38).
Although disputed (53), several studies (54-56) suggested that some
aquaporins are permeated by carbon dioxide. Carbon dioxide entry into
cyanobacteria (57) and photosynthetic activity (58) were drastically
inhibited by p-chloromercuriphenylsulfonic acid and
recovered with -mercaptoethanol. M. marburgensis is an
absolute anaerobe and utilizes carbon dioxide as a sole carbon source
(20). When AqpM was expressed in E. coli strain SK46
containing disruptions of both aqpZ and glpF,
14CO2 permeability through the bacterial
membrane was increased above control E. coli.3 Thus, AqpM may
provide a model molecule for elucidation of carbon dioxide permeation
through aquaporins.
Aquaporins and aquaglyceroporins form the major intrinsic protein, MIP,
family that is believed to date back 2.5-3 billion years in
evolutionary time (42). Recognition of an aquaporin in an
archaeon suggests an even earlier origin, although it is possible that the gene was transferred horizontally from other microorganisms (59, 60). From our phylogenetic analysis, we believe
that eukaryotic members of the MIP family evolved from two basal
lineages: AqpZ-like water channels and GlpF-like glycerol facilitators.
These divergent lineages may have originated from an AqpM-like
sequence, which appears to be intermediate in sequence between the
water-selective aquaporins and the aquaglyceroporins (50). The current
abundance of sequence data together with new functional information
warrants reinvestigation of the phylogenetic origins of the ubiquitous
family of water channel proteins.
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ACKNOWLEDGEMENTS |
---|
We thank Prof. Reiner Hedderich, Max-Planck-Institut fuer Terrestrische Mikrobiologie, Germany, for kindly providing genomic DNA and M. marburgensis and Prof. Giuseppe Calamita, Dipartimento di Fisiologia Generale e Ambientale Università degli Studi di Bari via Amendola, 165/A 70126 Bari, Italy, for critical discussions. We also thank M. Odara and K. Nakahara and M. Sugawara and M. Goto for constant encouragement and technical help.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid (to Y. K.) from the Japanese Society for Promotion of Science Postdoctoral Fellowship P00208 for foreign researcher and grants from the National Institutes of Health and the Human Frontier Science Program.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.
§ Both authors contributed equally to this work.
** To whom correspondence may be addressed: Dept. Biological Chemistry, The Johns Hopkins School of Medicine, 725 N. Wolfe St., Baltimore, MD 21212-2185. Tel.: 410-955-7049; Fax: 410-955-3149; E-mail: pagre@jhmi.edu.
To whom correspondence may be addressed: Biotechnology
Institute, Akita Prefectural University, Ogata, 010-0044, Japan. Tel.: 81-185-45-3930; Fax: 81-185-45-2678; E-mail:
kitagawa@agri.akita-pu.ac.jp.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212418200
2 Methanobacterium thermoautotrophicum strain Marburg has been redesignated Methanothermobacter marburgensis Marburg (1).
3 X. Ding, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
AQP, aquaporin;
GlpF, glycerol facilitator;
MIP, major intrinsic protein;
MOPS, 3-(N-morpholino)propanesulfonic acid;
OG, n-octyl--D-glucopyranoside;
DM, n-dodecyl-
-D-maltopyranoside;
NTA, nitrilotriacetic acid;
BSA, bovine serum albumin;
TM, transmembrane.
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