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with, were, from, AqpM, water, proteoliposomes, permeability, that, protein, coli, glycerol, (Fig., sequence, aquaporins, marburgensis, cells, Agre,, control, volume, buffer, Biol., purified, have, channel, been, other, into, GlpF, aquaporin, (2001)


Functional Expression and Characterization of an
Archaeal Aquaporin
Received for publication, December 5, 2002, and in revised form, January 3, 2003
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212418200
David Kozono‡§, Xiaodong Ding§¶, Ikuko Iwasaki¶, Xianying Meng, Yoichi Kamagata,
Peter Agre‡**, and Yoshichika Kitagawa¶‡‡
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
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 poreforming loop with the signature motif Asn-Pro-Ala
(NPA). Unlike other known homologs, the putative Hg2sensitive 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 ms
1 versus 12 ms1 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.
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
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.
* 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. This 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; Email: pagre@jhmi.edu.
‡‡ To whom correspondence may be addressed: Biotechnology Institute, Akita Prefectural University, Ogata, 010-0044, Japan. Tel.: 81185-45-3930; Fax: 81-185-45-2678; E-mail: kitagawa@agri.akita-pu.
1 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.
2 Methanobacterium thermoautotrophicum strain Marburg has been
redesignated Methanothermobacter marburgensis Marburg (1).
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 12, Issue of March 21, pp. 10649–10656, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org 10649
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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--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.
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 l 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-l 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 l 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 freezesubstituted in acetone containing 2% osmium tetroxide at 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).
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
g/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-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.
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 -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-NTAagarose 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.
Sedimentation Analysis—Velocity sedimentation analysis was used
to determine the oligomeric structure of purified protein. Detergentsolubilized material (2–10 g of purified protein in a 200-l sample
volume) was layered on top of a 4-ml continuous sucrose gradient (20
mM TrisHCl, 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), -amylase (8.9), and catalase (11.2).
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 -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 g/ml purified
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).
Functional Analyses of AqpM10650
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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.
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 1 ms (SF-2001; KinTek Instruments,
University Park, PA). Water permeability was measured by rapidly
mixing 100 l of a proteoliposome suspension (1 g of protein and 75 g
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).
dVrelt/dt  PfS/V0vwCi/Vrelt  C0 (Eq. 1)
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 cm
3); 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.
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.
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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 -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.
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
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 (Thr203, 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--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.
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-soluFIG. 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 l 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 l of gel loading buffer (pH 6.8) containing
50–800 mM ethanedithiol for 1 h.
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bilized AQP1 (40), which are known to be tetramers.
When visualized by silver staining, the purified 10-HisAqpM 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 -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.
Permeabilities of Reconstituted AqpM—AqpM proteoliposomes were prepared by dilution with E. coli lipids and 10-HisAqpM 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 em  600 nm (Fig. 4A).
AqpM proteoliposomes exhibited a moderately high osmotic
water permeability of 57  4 ms1, whereas control liposomes yielded much lower permeability, 12  0.7 ms1. 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).
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. 4. Water, glycerol, and urea permeabilities of reconstituted AqpM. A, proteoliposomes reconstituted with purified 10-HisAqpM 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 ms
1, Pf (AqpM proteoliposomes) 
57  4 ms1, Pf (AqpM  Hg
2)  17  1.3 ms1, Pf (AqpM  Hg
 -mercaptoethanol)  33  4 ms1. All values are mean  S.D. B
and C, control liposomes or proteoliposomes reconstituted with 10-HisAqpM 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 doubleexponential rise to maximal curves for visual clarity.
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(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
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.
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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 glycerolspecific 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.
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 GlpFlike 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.
Acknowledgments—We thank Prof. Reiner Hedderich, Max-PlanckInstitut 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.
3 X. Ding, unpublished results.
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.
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