Báo cáo khóa học: Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen ppt

Identification of residues controlling transport through the yeast
aquaglyceroporin Fps1 using a genetic screen
Sara Karlgren
1
, Caroline Filipsson
2
, Jonathan G. L. Mullins
3
, Roslyn M. Bill
1,4
, Markus J. Tama
´
s
1
and Stefan Hohmann
1
1
Department of Cell and Molecular Biology/Microbiology, Go
¨
teborg University, Sweden;
2
Department of Biochemistry and
Biophysics, Go
¨
teborg University, Sweden;
3
Swansea Clinical School, University of Wales Swansea, UK;
4
School of Life
and Health Sciences, Aston University, Birmingham, UK
Aquaporins and aquaglyceroporins mediate the transport of

waterandsolutesacrossbiologicalmembranes.Saccharo-
myces cerevisiae Fps1 is an aquaglyceroporin that mediates
controlled glycerol export during osmoregulation. The
transport function of Fps1 is rapidly regulated by osmotic
changes in an apparently unique way and distinct regions
within the long N- and C-terminal extensions are needed for
this regulation. In order to learn more about the mechanisms
that control Fps1 we have set up a genetic screen for
hyperactive Fps1 and isolated mutations in 14 distinct resi-
dues, all facing the inside of the cell. Five of the residues lie
within the previously characterized N-terminal regulatory
domain and two mutations are located within the approach
to the first transmembrane domain. Three mutations cause
truncation of the C-terminus, confirming previous studies on
the importance of this region for channel control. Further-
more, the novel mutations identify two conserved residues in
the channel-forming B-loop as critical for channel control.
Structural modelling-based rationalization of the observed
mutations supports the notion that the N-terminal regula-
tory domain and the B-loop could interact in channel con-
trol. Our findings provide a framework for further genetic
and structural analysis to better understand the mechanism
that controls Fps1 function by osmotic changes.
Keywords: aquaglyceroporin; channel; genetic screen;
glycerol; osmoregulation.
The discovery of the aquaporins marked a breakthrough
in our understanding of water and solute transmembrane
transport [1]. Aquaporins and aquaglyceroporins [the major
intrinsic protein (MIP) family] have been found in archea,
eubacteria, fungi, plants, animals and human [2,3]. Aqua-

porins facilitate the diffusion of water across biological
membranes while the closely related aquaglyceroporins
mediate transport of water and solutes such as glycerol and
urea. These proteins are present in membranes where rapid
and controlled water or solute fluxes occur, for example, in
the mammalian kidney [2,4,5] and plant roots [6,7]. The
yeast Saccharomyces cerevisiae has four such MIP channels:
the aquaporins Aqy1 and Aqy2 and the aquaglyceroporins
Fps1 and Yfl054 [8]. Aqy1 is a strictly spore-specific
aquaporin while Aqy2 may play a role in osmoregulation
during cell growth (F. Sidoux-Walter & S. Hohmann,
unpublished observation). Possible roles in freeze tolerance
have been claimed for Aqy1 and Aqy2 [9]. The physiological
role of Yfl054 has not yet been established [8,10].
Yeast cells accumulate glycerol as a compatible solute in
osmoregulation [11]. The plasma membrane channel Fps1
mediates glycerol export and is required for survival of
a hypo-osmotic shock when glycerol has to be rapidly
exported from cells in order to prevent bursting [12,13]. On
the other hand, hyperactive Fps1 causes an inability to grow
at high external osmolarity because cells lose the glycerol
they produce [12,13]. Moreover, it has been shown that
Fps1 is required to control turgor and prevent cell lysis
during cell fusion of mating yeast cells [14]. Together, these
observations illustrate that Fps1 plays a central role in yeast
osmoregulation.
The transport function of Fps1 is controlled by osmotic
changes in order to prevent glycerol loss at high osmolarity
and to allow rapid export at low external osmolarity. The
capacity for glycerol transmembrane flux through the

yeast plasma membrane is reduced within seconds upon
a hyperosmotic shock while it increases equally fast upon a
shift to hypo-osmotic conditions. As Fps1 is responsible for
most of the glycerol transmembrane flux [10] these obser-
vations together with the phenotype caused by hyperactive
Fps1 suggest that the channel is directly controlled by
osmotic changes [12,13,15].
Aquaporins and aquaglyceroporins have six transmem-
brane spanning domains (TMD) and five connecting loops
[2,16–18]. The hydrophobic B- and E-loop, facing inside
and outside, respectively, are part of the central water/solute
pore. These a-helical loops dip into the membrane where
their highly conserved Asn-Pro-Ala (NPA) motifs form the
central pore constriction [2,16–18]. Structural analysis and
Correspondence to S. Hohmann, Department of Cell and Molecular
Biology/Microbiology, Go
¨
teborg University, Box 462, S-40530
Go
¨
teborg, Sweden. Fax: + 46 31 7732599, Tel.: + 46 31 7732595,
E-mail:
Abbreviations: MIP, major intrinsic protein; NPA, Asn-Pro-Ala;
TMD, transmembrane domains; YNB, yeast nitrogen base;
YPD, yeast peptone glucose
(Received 19 November 2003, revised 21 December 2003,
accepted 5 January 2004)
Eur. J. Biochem. 271, 771–779 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03980.x
molecular dynamic modelling as well as biophysical analy-
ses have revealed the mechanisms that ensure very rapid

transport and at the same time high selectivity of water or
glycerol transport [17–22].
Fps1 is an atypical aquaglyceroporin as the highly
conserved NPA motifs in the B- and the E-loop are NPS
(Asn-Pro-Ser) and NLA (Asn-Leu-Ala), respectively,
sequences that are also found in the Plasmodium glycerol
facilitator, although in the opposite loops [23]. While Fps1
can tolerate NPA in both positions, the Escherichia coli
homologue GlpF is inactive when its NPA motifs are
converted to NPS and NLA, suggesting a somewhat
different and more flexible arrangement of the Fps1 channel
[24]. In addition, Fps1 has unique long N- and C-terminal
domains only found in orthologues from other yeasts [15].
While large parts of these extensions can be removed
without apparent consequence, short domains close to the
first and the last TMD seem to be required for channel
control: deletions or mutations in these regions render the
channel hyperactive (K. Hedfalk, R. M. Bill, J. G. Mullins,
S. Karlgren, C. Filipsson, C. Bergstrom, M. J. Tama
´
s,
J. Rydstro
¨
m & S. Hohmann, unpublished observation)
[13,15]. The N-terminal regulatory domain may fold in a
similar way as the channel forming B- and E-loops, hence
dipping into the membrane. We suggested that this domain,
dubbed the N-loop, might directly interact with the channel
forming B-loop to control transport function [15].
As a novel approach to study the control of Fps1, we

present a random genetic screen for hyperactive Fps1.
Twenty independent mutants are reported here, represent-
ing 17 different mutations, all facing the cell interior. The
majority of the mutations are clustered in or near the
already identified N-terminal regulatory domain. Mutations
in the C-terminal domain resulted in premature termination.
Most interestingly, five different mutations hit two con-
served residues in the B-loop. The data lend support to the
notion that the N-terminal regulatory domain may interact
with the central pore and obstruct transport. Hence, this
study provides new insight into Fps1 control and opens
up for further mutational analyses this interesting aqua-
glyceroporin.
Materials and methods
Strains and plasmids
The yeast strains used in this study are YSH 642
(gpd1D::TRP1 gpd2::DURA3) [26] and YMT2 (fps1D::
HIS3) [13] in the W303-1 A background (MATa leu2-3/112
ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0)
[27]. YEpmyc-FPS1 is a 2l LEU2 plasmid expressing a
c-myc epitope-tagged Fps1 and YEpmycfps1-D1 encodes
a truncated version of Fps1 lacking the amino acids 12–231
[13].
Growth conditions
Yeast cells were grown in medium containing 2% peptone,
1% yeast extract, 2% glucose (YPD), or for selection of
transformants in yeast nitrogen base (YNB) medium [28].
Tests for hyperactive alleles of FPS1 were performed by
pregrowing the gpd1D gpd2D mutant transformed with
YEpmyc-FPS1 and derivatives thereof for two days on

YNB agar plates, resuspending them in YNB medium to an
D
600
of 0.4 and then performing 10-fold serial dilutions. Cell
suspensions (5 lL) were spotted onto agar plates supple-
mented with 1
M
xylitol, or with 0.8
M
NaCl as a negative
control, and on medium without osmoticum as a positive
control. Growth was monitored after 2–7 days at 30 °C.
For growth tests after osmotic shifts, transformants were
pregrown on YNB plates, then resuspended and spotted on
the same medium as the control. To invoke hyperosmotic
shock, cells were pregrown in medium without osmoticum
and shifted to medium with 0.8
M
NaCl. For a hypo-
osmotic shock, cells were pregrown in the presence of 0.8
M
NaCl and shifted to medium without salt. Growth was
monitored as above.
Mutagenesis and screening
Random mutations in FPS1 were introduced by transform-
ing YEpmyc-FPS1 into the E. coli strain XL1-Red from
Stratagene (La Jolla, CA, USA) following the manufac-
turer’s recommendations. Transformants were grown for
approximately 24 h yielding about 200 colonies per plate.
Colonies from each plate were pooled and grown in LB-

medium supplemented with 100 lgÆmL
)1
of ampicillin for
24 h. Plasmids were isolated (Qiagen miniprep kits) and
transformedintoagpd1D gpd2D strain using the LiAc-
method [29]. Yeast cells were spread on selective media
(YNB) to a density of approximately 200 transformants
per plate. The colonies were replica-plated onto YNB-leu
(positive control), YNB-leu plus 1
M
sorbitol, YNB-his
(negative controls) and YNB-leu plus 1
M
xylitol (selective)
to ensure that a low cell density was left on the velvet before
replicating onto selective plates. Cell densities that are too
high make distinction between growth and no growth
difficult. Cells were grown for 4–5 days and positive clones
were re-tested in growth assays on 1
M
xylitol. Plasmid was
recovered from positive transformants, checked by restric-
tion analysis, propagated in E. coli Top10 cells and then
re-transformed into the gpd1D gpd2D strain for testing on
1
M
xylitol plates. In addition, plasmids were also trans-
formed into the fps1D mutant and tested for growth after
hypo/hyper-osmotic shock. All FPS1 genes from trans-
formants that scored positive in the tests were completely

sequenced.
Western blot analysis
Cells were cultured in YNB supplemented with 2% glucose
to late log phase (typically D
600
is 0.8). The total membrane
fraction was isolated and visualized as described previously
(S.Karlgren,N.Pettersson,R.M.Bill&S.Hohmann,
unpublished observation).
Glycerol transport measurements
To determine glycerol influx following its concentration
gradient, cells were grown in liquid YNB medium to a D
600
of approximately 0.7. Cells were harvested, washed and
suspended in ice-cold Mes buffer (10 m
M
Mes, pH 6.0) to a
density of 40–60 mg cellsÆml
)1
. All subsequent steps were
performed at 4 °C. Glycerol influx in the presence or
772 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004
absence of hyperosmotic stress was measured by adding
glycerol to a final concentration of 100 m
M
ÔcoldÕ glycerol
plus 40 l
M
[
14

C]glycerol (5.9 GBqÆMmol
)1
;Amersham)in
a total volume of 250 lL [13,15]. Aliquots of 50 lLwere
collected by filtration at 0, 15, 30, 45 and 60 s, immediately
washed three times with ice-cold buffer and the radioactivity
that was retained on the filters was determined. Filters with
cells were dried at 80 °C overnight for dry weight deter-
mination. Transport experiments were performed in tripli-
cate and data are expressed in lmol per gram of dry cells.
Modelling
Models are based on previous analyses [15] using the
structural information on E. coli GlpF as a template [17].
They were generated in
MOLMOL
[31] avoiding any side
chain conflicts, and bringing the N-loop in as centrally as
possible to the pore cavity, rotating the ends of the N-loop
toward transmembrane domain 1 (the closest TMD in
sequence terms).
Results
Screen for hyperactive Fps1
In order to identify residues within Fps1 that are important
for channel control we performed a random genetic screen
for mutants that render Fps1 hyperactive. The c-myc-tagged
FPS1 gene on a YEp plasmid was randomly mutagenized
using the DNA repair-defective E. coli strain XL1-Red.
This procedure generated a series of libraries of 200 clones
each, which were individually transformed into a gpd1D
gpd2D double mutant. This strain is unable to produce

glycerol and hence cannot grow at elevated osmolarity
caused by salt [26], or by various polyols including xylitol
and even glycerol (S. Karlgren, N. Pettersson, R. M. Bill &
S. Hohmann, unpublished observation). However, growth
of the gpd1D gpd2D mutant in the presence of polyols can be
rescued when transformed with a plasmid encoding hyper-
active Fps1 (FPS1-D1) that alleviates the osmotic dis-
equilibrium by permitting solute influx (S. Karlgren,
N. Pettersson, R. M. Bill & S. Hohmann, unpublished
observation) [12,13] (Fig. 1). Although hyperactive Fps1
can rescue growth of the gpd1D gpd2D mutant through
influx of various polyols (S. Karlgren, N. Pettersson, R. M.
Bill & S. Hohmann, unpublished observation) we chose
xylitol for the screen as it gave the clearest phenotype. We
note that we actually screen for hyperactive xylitol uptake
while the objective is to obtain mutants that fail to retain
internally produced glycerol under hyperosmotic stress, an
aspect that will be discussed when interpreting the muta-
tions obtained.
Transformants with mutagenized FPS1 in the gpd1D
gpd2D strain were grown to colonies on YNB and then
replica-plated onto plates supplemented with 1
M
xylitol.
Approximately 5000 colonies were screened and 31 grew on
xylitol plates. These were re-tested for growth on 1
M
xylitol.
Plasmids were isolated from these positive yeast colonies,
amplified in E. coli, checked by restriction analysis and

retransformed into the gpd1D gpd2D mutant. Those trans-
formants were again tested for growth on 1
M
xylitol (Fig. 1)
leaving a total of 20 different clones for further analysis.
Mutations obtained
Sequence analysis of the entire FPS1 gene from these 20
plasmids revealed 19 mutants with single amino acid
replacements (Table 1) that clustered in a characteristic
manner (Fig. 2). The mutation P236L was represented five
times, leaving a total of 15 unique single mutations. One
mutant had a point mutation in position S246P in the
approach to the first TMD as well as a stop codon in
Fig. 1. Growth on plates. Cells were dropped
in a 1 : 10 dilution series on synthetic YNB
medium with the indicated osmotica. A
hyperactive Fps1 function is indicated by
growth on xylitol (or sorbitol) in the gpd1D
gpd2D mutant and by poor growth on NaCl in
the fps1D mutant. The hypo-osmotic test
(shift from 0.8
M
NaCl to medium without
salt) is a test for function: poor growth
indicates no or reduced function.
Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 773
approximately the middle of the C-terminal extension
(Q592stop). Six mutations fall within the previously char-
acterized regulatory domain, which, according to our
previous mutational analysis and sequence conservation

among yeast Fps1 orthologues, encompasses the stretch
from Met219 until about Ser248. A nine amino acid linker
follows this domain to the first TMD, which is predicted
to start at Leu257. Figure 2B provides an overview of the
relevant mutations from previous [15] and present analyses
within this region. One mutation was found in the approach
to the first TMD in a lysine (K250E) that is conserved
among yeast Fps1 orthologues [15]. Importantly, two
residues within the channel forming B-loop, which are both
highly conserved throughout the MIP family, were affected
by multiple exchanges. All three mutations occurring in the
C-terminal extension caused premature translation termin-
ation, either by generating a stop codon or a frame shift
leading to a stop some codons further downstream. As the
newly isolated mutations do not hit all residues that we
previously found to be critical for Fps1 control, while at the
same time we identified new relevant residues, the present
genetic screen is not saturated. A significant larger number
of mutations, probably more than 100, will be needed for a
fully comprehensive mutational map of channel control.
The genetic screen employed selected for mutated
versions of Fps1 that retain function, hence the proteins
Table 1. Summary of mutations obtained.
Mutation Nucleotide change Location
K223E AAGfiGAG In front of the N-terminal regulatory domain
Q227R CAGfiCGG Within the N-terminal regulatory domain
T231A ACAfiGCA Within the N-terminal regulatory domain
P232S CCTfiTCT Within the N-terminal regulatory domain
P236L (found five times) CCCfiCTC Within the N-terminal regulatory domain
S246P + Q592 stop TCTfiCCT + CAAfiTAA Between the N-terminal regulatory domain and TMD1

K250E AAAfiGAA Between the N-terminal regulatory domain and TMD1
G348D GGTfiGAT Loop B
G348R GGTfiCGT Loop B
G348S GGTfiAGT Loop B
H350L CATfiCTT Loop B
H350Y CATfiTAT Loop B
L451W TTGfiTGG Loop D
I531FIRVMNLQSTG T insertion at 1595 C-terminus
S537QLVFTSL 1613CAGTC1617 deleted C-terminus
W541stop TGGfiTAG C-terminus
B
(219)MVKPKTLYQNPQTPTVLPSTYHPINKWSS(248)
L225A
Q227A
N228A
P229A
Q230A
T231A
P231A
T232A
P236A
K223E
Q227R
T231A
P231S
P236L
S246P
K250E
K223E
Q227R

T231A
P323S
P236L
S246P +
Q592stop
G348D
G348R
G348S
H350L
H350Y
L451W
Q592stop + S246P
B-loop
E-loop
N-terminus
C-terminus
W541stop
+
H
3
N
I531FIRVMNLQSTG
S537QLVFTSL
-
OOC
A
Fig. 2. Summary of mutations obtained. (A)
Topology map of Fps1 indicating mutations
found in the genetic screen reported here. For
clarity the N- and the C-termini are not shown

according to scale. (B) Summary of mutations
affecting the function of the N-terminal regu-
latory domain. Data shown above the
sequence is from a previous study [15]. Data
shown below the sequence is from this study.
Underlined mutations cause particularly
strong osmosensitivity.
774 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004
should be expressed and localized to the plasma membrane.
However, differences in expression levels could be relevant
for the interpretation of the results. For this reason we
performed Western blot analysis, making use of the
C-terminal c-myc-tag (Fig. 3). This was only possible for
mutants that retained a complete C-terminus. Some of the
Fps1 mutants were apparently less abundant in the plasma
membrane, in particular those in His350. However, this
does not seem to affect their function because they mediate
strong growth on xylitol and fully complement the hypo-
osmosensitivity of an fps1D mutant (Fig. 1, see below). Also
in previous mutational analyses we have observed that the
apparent protein abundance of Fps1 does not affect
performance in functional assays over a wide range [15].
One explanation may be that mutations cause different
detectability in immuno blots. Another possibility is that
Fps1 is present in excess such that even much lower levels
can perform full function. None of the mutants was more
abundant than wild type Fps1, excluding simple expression
changes as a cause for the observed gain of function.
Phenotypic characteristics of mutants obtained
All mutations obtained rescued growth on 1

M
xylitol to the
gpd1D gpd2D mutant, albeit clearly to different extents
(Fig. 1). The three most upstream mutations as well as the
C-terminal truncations conferred the weakest growth on
xylitol. The double mutant, S246P plus stop at 591,
conferred particularly robust growth on xylitol and in
contrast to all other mutations even allowed growth in the
presence of 1
M
of the C6 polyol
D
-sorbitol. As all other
mutations in the C-terminal extension occur much further
upstream and caused much weaker growth on xylitol and
because we did not observe any effects conferred by
truncations that far downstream in the C-terminus in a
different study (K. Hedfalk, R. M. Bill, J. G. Mullins,
S. Karlgren, C. Filipsson, C. Bergstrom, M. J. Tama
´
s,
J. Rydstro
¨
m & S. Hohmann, unpublished observation), we
believe that the effect in the double mutant is mainly due to
the S246P mutation, although we can not fully exclude a
synergistic effect of both mutations.
As we had isolated the mutants based on their ability to
mediate xylitol influx we wished to test if the novel
Fps1 derivates were still fully able to exert their normal

physiological function in mediating efflux of glycerol upon a
hypo-osmotic shock. This is most conveniently tested by
monitoring the survival after a hypo-osmotic shock, as a
smaller proportion of mutants lacking Fps1 survive, and
survivors start growth more poorly. We have previously
shown these tests to be very reproducible and to correctly
reflect the ability of Fps1 to mediate glycerol release [13,15].
All plasmids were transformed into an fps1D strain, which
was wild type for GPD1 and GPD2 and therefore capable
of producing glycerol. The ability of the novel alleles to
complement the lack of FPS1 was tested by shifting
transformants from high osmolarity medium (0.8
M
NaCl)
to medium without osmoticum (Fig. 1). Most of the
transformants grew like wild type but in some instances
the mutation reduced survival due to hypo-osmotic shock,
indicating impaired glycerol export function under these
conditions. This effect was most pronounced for C-terminal
truncations, some of which conferred sensitivity almost like
deletion of FPS1 (Fig. 1). In addition, mutations at G348 in
the B-loop reduced survival upon hypo-osmotic shock.
Hyperactive Fps1-D1 confers both hyperosmosensitivity
in cells competent of producing glycerol and ability to grow
on 1
M
xylitol to a strain unable to produce glycerol. The
mutants isolated here were selected for the latter phenotype
and hence we wished to test if they were also affected for
retention of internally produced glycerol. We have shown

previously that this ability is well reflected by growth
characteristics in the presence of high external osmolarity
[12,13,15].
The different mutants showed varying degrees of osmo-
sensitivity (Fig. 1). The strongest effects were observed for
mutations within the N-terminal regulatory domain and in
particular for mutations of the two prolines P232 and P236,
the double mutant (S246P plus stop at Q591), K250E in
the linker to the first TMD and mutations in G348 in the
B-loop. Interestingly, different exchanges of the highly con-
served G348 caused different effects, with G348D causing
strongest osmosensitivity. The two different exchanges of
the conserved His350 caused similar effects.
Two residues in the N-terminal regulatory domain had
previously been studied by alanine-scanning mutagenesis:
Gln227 and Pro236 [13]. In order to compare the effects of
the different exchanges directly they were tested side-by-side
(Fig. 4). Q227A and Q227R caused similar strong osmo-
sensitivity indicating that the two exchanges, although
chemically very different, affected channel control in a
similar way. While exchange of Pro236 with alanine only
Fig. 3. Western blot analysis of the whole membrane fraction probed
with an anti c-myc Ig against the C-terminal c-myc-tag of Fps1.
Amounts of protein loaded from left to right: 50, 20, 30, 50, 50, 30, 50,
50, 30, 50, 50, 100, 50 and 50 lg. The lower double band is probably a
degradation product.
Fig. 4. Growth on plates. Cells were pregrown on YNB and dropped in
a 1 : 10 dilution series on the same medium with or without NaCl.
Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 775
caused moderate osmosensitivity, exchange with the some-

what larger but chemically similar leucine seemed to affect
channel control much more dramatically.
If the ability of the Fps1 alleles for mediating influx of
xylitol and efflux of glycerol were equally affected by the
novel mutations we would expect that good growth on 1
M
xylitol would correlate with poor growth on 0.8
M
NaCl.
This was the case for most of the mutations with some
exceptions. G348D, which caused poorest growth on 0.8
M
NaCl only mediated moderate growth on 1
M
xylitol.
L451W, on the other hand, which mediated robust growth
on 1
M
xylitol, caused almost no sensitivity to 0.8
M
NaCl.
Also mutations that truncated the C-terminus caused either
no or poor sensitivity to 0.8
M
NaCl while permitting
growth on xylitol. These observations indicate that the two
functions tested here, xylitol influx and glycerol efflux, share
common determinants while at the same time they can be
distinguished through specific mutations.
High glycerol transport capacity through the Fps1

mutant proteins
We have previously shown that the ability of Fps1
derivatives to mediate glycerol transport and to down-
regulate glycerol transport upon a hyperosmotic shock
can be monitored by measuring the influx of radiolabelled
glycerol following its concentration gradient in unstressed
cells and in cells exposed to 0.8
M
NaCl. We selected some
mutations from the new collection that represented different
characteristics including P236L, G348D, H350L and
L451W. As observed previously, glycerol transport through
hyperactive Fps1 is higher than that through wild type
under all conditions and is down-regulated by hyperosmotic
shock. This is also the case for the mutants studied here
(Fig. 5). All of them, however, maintained a much higher
glycerol transport capacity then wild type even after
hyperosmotic shock. The apparent rate of glycerol transport
is lower for H350L than for the other mutants, which is
consistent with the fact that it only conferred moderate
osmosensitivity, a measure for glycerol loss (Fig. 1). The
three other mutants conferred similar glycerol influx while
they caused different degrees of osmosensitivity. Glycerol
uptake rates correlated better with the ability to grow on
xylitol, suggesting that in some of the mutants isolated
influx may be enhanced to a higher degree than efflux.
Discussion
The transmembrane transport of glycerol in yeast is rapidly
controlled by osmotic changes to ensure glycerol accumu-
lation under hyperosmotic stress and fast glycerol release

upon a hypo-osmotic shock. Fps1, an aquaglyceroporin,
mediates most of the glycerol transport through the plasma
membrane. Its importance is illustrated by the fact that
hyperactive Fps1 causes glycerol loss and sensitivity to
hyperosmotic conditions while inactivation of Fps1 causes
inability to release glycerol upon a hypo-osmotic shock and
poor survival. We have observed previously that even in
cells expressing hyperactive Fps1 a hyperosmotic shock
mediates substantial down-regulation of glycerol transmem-
brane transport. One simple explanation for this observa-
tion is that cells shrink after hyperosmotic shock, which
means that both their cell surface and volume rapidly
diminish, which may reduce the capacity for uptake. We
can, however, not exclude that even hyperactive Fps1 still
retains some regulatory capacity. It also appears that hyper-
active Fps1 mediates higher glycerol flux under normal as
well as hypertonic conditions. This might indicate that only
a subset of all Fps1 molecules is active at any given time
under normal conditions while such mutations fully activate
all channels. Hence, Fps1 is likely to constantly switch
between open and closed conformations, and osmotic
conditions alter the probability for either conformation.
Mutations that render Fps1 hyperactive may therefore
increase the ÔopenÕ probability.
In this work we have used a novel genetic approach to
identify intramolecular determinants of Fps1 control. The
genetic screen we employed is based on the observation that
hyperactive Fps1 allows mutants unable to produce glycerol
(gpd1D gpd2D) to grow in the presence of 1
M

xylitol. Hence
we screen for enhanced uptakeof glycerol, while the
physiological role is glycerol export. Most, although not
all, mutants we obtained in this way also conferred
osmosensitivity (and hence enhanced glycerol loss under
these conditions). Moreover, we obtained several mutations
in residues that we previously identified as important by
targeted mutagenesis. These observations confirm the
validity of the approach.
Although we screened for enhanced uptake, all mutations
faced the inside of the cell. Recently we screened for
suppressors of truncated, hyperactive Fps1 and obtained
mutations that reduced transport. The four mutants char-
acterized faced the outside of the cell [15]. Structural analysis
of AQP1 and GlpF suggested that these channels are largely
symmetric (except for the tails facing the inside) [17,32].
While our mutational analysis may not yet be representa-
tive, the distribution of mutations may suggest that the
Fig. 5. Uptake profile of 100 m
M
radiolabelled glycerol by the strains
indicated, before and after an osmotic shift to 0.8
M
NaCl.
776 S. Karlgren et al. (Eur. J. Biochem. 271) Ó FEBS 2004
outside face is mainly important for transport and the inside
face for control, at least in the case of the somewhat unusual
Fps1.
Some mutations, such as L451W and C-terminal trun-
cations but also alterations in His350 allow solid growth in

the presence of xylitol while causing only moderate osmo-
sensitivity (i.e. moderate glycerol loss). We have observed
previously that certain mutations affected glycerol transport
in one direction more than in the other [24]. The phenotype
of the mutants described here suggests that it might be
possible to partly dissect uptake and efflux functions using
specific genetic screens.
The results of our genetic screen confirm and extend
previous analyses. Mutations identified in and around the
previously characterized N-terminal regulatory domain
confirm its importance but at the same time also indicate
that residues involved in channel control are located
between this domain and the first TMD. We also confirm
previous observations on the importance of the C-terminus
(K. Hedfalk, R. M. Bill, J. G. Mullins, S. Karlgren,
C. Filipsson, C. Bergstrom, M. J. Tama
´
s, J. Rydstro
¨
m
& S. Hohmann, unpublished observation), although we
note that the truncations obtained here only cause moderate
if any osmosensitivity (indicative of glycerol loss during high
external osmolarity). More mutations need to be charac-
terized to judge if those obtained here, which all confer
premature translational stop rather then specific amino acid
replacements, are truly representative.
A particularly significant finding of this study is that even
mutations in highly conserved residues of the channel
forming B-loop can mediate hyperactive transport. So far,

important residues were identified on the basis of mutations
that block transport. Hence, the approach used here, which
is novel as it screens for gain of function, leads to completely
novel insight into the structure-function relationship of MIP
channels.
Based on the structure of GlpF [17] and previous
modelling [15] we have attempted to rationalize the
mutations obtained in this study. We have shown previously
that the N-terminal regulatory domain, dubbed the N-loop,
has sequence and predicted structural similarity with the
channel forming B- and E-loop. We suggested that B- and
N-loops may interact.
In the models shown (Fig. 6), the 226–236 region of the
N-loop and 347–356 of the B-loop are in close proximity.
Residues affected by mutation are then located for the most
part in the functionally critical pore region. These include
Lys223 (violet), Gln227 (white), Thr231 (brown), Pro232
and Pro236 (grey), and on the B-loop they include Gly348
(orange) and His350 (blue). Lys451 (black) is the only
mutation to be clearly located away from the pore. Ser246,
Lys250, Ile531 and Ser537 lie in regions of the protein that
are not currently structurally modelled.
K223E (violet) and K250E (not modelled) result in a
charge reversal, which is likely to introduce electrostatic
interactions with other nearby lysine residues. This, in turn,
is likely to reduce the flexibility of the section linking the
N-terminal regulatory domain with TMD1, thereby holding
the pore more permanently open.
Q227R (white) lies directly adjacent to the NPQ (Asn-
Pro-Gln) region of the regulatory motif (which is similar to

NPA of the B-loop [15]). Even slight changes in the nature
of amino acids in this sensitive region are liable to affect the
regulatory domain. Likewise, T231A (brown), immediately
on the other side of the NPQ motif, disrupts the regulation
of the pore, but more markedly so than Q227R. This is most
likely due not only to its closeness to the NPQ motif but also
to the major role of threonine residues in hydrogen bonding.
This mutation is consistent with our previous findings
regarding T231, as well the general importance of the role of
threonine residues in the pore region of Fps1. T256 on the
B-loop is conserved across the whole MIP family.
Fig. 6. Structural modelling. The B-loop is shown in yellow and the N-loop in green. The residues involved in random mutations are found to be
located for the most part in the functionally critical pore region. On the N-loop, these include Lys223 (violet), Gln227 (white), Thr231 (brown),
Pro232 and Pro236 (grey), and on the B-loop they include Gly348 (orange) and His350 (blue). Leu451 (black) is the only mutation to be clearly
located away from the pore. Ser246, Lys249, Ile531 and Ser537 lie in regions of the protein that are not currently structurally modelled.
Ó FEBS 2004 Control of aquaglyceroporin Fps1 (Eur. J. Biochem. 271) 777
The random mutations involving proline residues of the
regulatory domain, P232S and P236L (both shown in grey
at either end of the N-loop a-helix), are in line with our
previous inferences regarding the importance of the pro-
nounced secondary structure in this region. Mutation of
either proline could result in reduction or increase in the
length of the helical secondary structure of the exiting
N-loop, affecting flexibility and function. Similarly, the
introduction of a proline residue, as at S246P (not modelled)
could result in marked changes in local secondary structure.
Mutations involving Gly348 (orange) on loop B, con-
served across the MIP family, to larger charged or polar
residues, G348D, G348R, G348S could have several effects.
Substitution by a larger residue could disrupt the close

arrangement with nearby residues, notably Q227 (white) on
the N-loop and His350 (blue) on the B-loop. The mutation of
this glycine residue could also disrupt the capacity of the
B-loop for membrane insertion, as it is clearly located in
the interfacial environment between the membrane face and
the cytosolic compartment (the orange residue, best viewed
in the side-on view of the model). Indeed, the mutations to
charged or polar residues have the most capacity for disrupt-
ing membrane insertion. A striking finding is the greater
effect observed with G348D than with G348R. This suggests
that the region surrounding Gly348 requires some flexibility
and distancing from His350, and perhaps to lie more
intimately with Gln227 on the N-loop to regulate normally.
In the model, where the beginning and end of the N-loop
region are aligned so that they face TMD1 as much as
possible, and with the NPQ turn of the B-loop maximally
immersed in the pore cavity, there is also a notably close
arrangement between Gln227 of the N-loop (shown in
white) and His350 on the B-loop (coloured blue). Both these
residues are highly polar, and have complementary charges
for interaction with each other.
The mutations of His350 (blue) caused marked effects
despite changing to similarly large residues. Hence, the
positive charge of His350 appears to be critical for normal
function. In this regard, its proximity in the model to the
negative dipole of Gln227 is of significant interest. His350 is
also conserved across the MIP family. Indeed, the random
mutations reported here regarding Gly348 and His350 are
likely to be of more general relevance for the understanding
of the structure-function relationship of the MIP family of

channel proteins.
Acknowledgements
This work was supported by the European Commission contract
QLR3-CT2000-00778 and the Human Frontier Science Foundation.
S.H. holds a research position of the Swedish Research Council.
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