From the Department of Cell and Molecular
Biology/Microbiology, Göteborg University, Box 462, 40530 Göteborg, Sweden, the § Laboratorium voor Moleculaire
Celbiologie, Katholieke Universiteit Leuven and Departement Moleculaire
Microbiologie, Vlaams Interuniversitaire Instituut voor Biotechnologie,
3001 Leuven, Belgium, the ¶ Department of Molecular Biotechnology,
Chalmers University of Technology, 40530 Göteborg, Sweden, the
§§ Faculty of Health and Social Sciences,
University of Luton, Park Square, Luton, Bedforshire LU1 3JU, United
Kingdom, and the
Department of Biochemistry
and Biophysics, 40530 Göteborg University, Sweden
Received for publication, September 24, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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The controlled export of solutes is crucial for
cellular adaptation to hypotonic conditions. In the yeast
Saccharomyces cerevisiae glycerol export is mediated by
Fps1p, a member of the major intrinsic protein (MIP) family of channel
proteins. Here we describe a short regulatory domain that restricts
glycerol transport through Fps1p. This domain is required for retention
of cellular glycerol under hypertonic stress and hence acquisition of
osmotolerance. It is located in the N-terminal cytoplasmic extension
close to the first transmembrane domain. Several residues within that
domain and its precise position are critical for channel control while
the proximal residues 13-215 of the N-terminal extension are not
required. The sequence of the regulatory domain and its position are
perfectly conserved in orthologs from other yeast species. The
regulatory domain has an amphiphilic character, and structural
predictions indicate that it could fold back into the membrane bilayer.
Remarkably, this domain has structural similarity to the channel
forming loops B and E of Fps1p and other glycerol facilitators.
Intragenic second-site suppressor mutations of the sensitivity to high
osmolarity conferred by truncation of the regulatory domain caused
diminished glycerol transport, confirming that elevated channel
activity is the cause of the osmosensitive phenotype.
Accumulation of osmolytes is a ubiquitous strategy of cellular
osmoadaptation (1). Cells produce or actively take up osmolytes in
order to increase their solute content and thereby maintain turgor and
volume under hypertonic conditions (high extracellular osmolarity).
Upon shift to hypotonic conditions, i.e. when the extracellular osmolarity drops, cells export solutes to prevent excessive swelling or bursting in a process termed regulated volume decrease (2). While proteins that mediate solute export from mammalian
cells have not been identified yet, such channels have been described
in bacteria and yeast (3-6). The MscL channel from Escherichia
coli has been particularly well studied, and the structural basis
for gating by osmotic changes has been discussed in detail (5,
7-11).
Proliferating yeast (Saccharomyces cerevisiae) cells employ
glycerol as osmolyte, which they produce in two steps from the glycolytic intermediate dihydroxyacetonephosphate (12). We have previously demonstrated that Fps1p mediates export of glycerol across
the yeast plasma membrane (13-16). Mutants lacking Fps1p are unable to
rapidly export glycerol upon hypo-osmotic shock and only a fraction of
such cells survive under these conditions (14, 15). We have also
provided evidence that transmembrane glycerol flux is regulated by
osmotic changes. Glycerol flux is diminished within seconds after a
shift to high osmolarity and it increases again at an apparently
similar time scale when cells are shifted to low osmolarity (15). The
N-terminal extension of Fps1p appears to be required for controlling
glycerol transport as its deletion renders the channel hyperactive.
Yeast cells expressing such a hyperactive channel are sensitive to high
osmolarity because they need much longer to build up high intracellular
glycerol levels due to a higher level of glycerol leakage (15).
Fps1p belongs to the MIP1
(major intrinsic protein) family of channel proteins. Members of this
ancient family have been identified in organisms ranging from Archea to
human (17-20). MIP channels comprise water channels (aquaporins) and
glycerol facilitators (aquaglyceroporins) (17). Water channels are
highly specific to water (17, 21, 22), although transport of ions has
also been reported (23, 24). Glycerol facilitators commonly transport small polyols and a range of other uncharged molecules (25), and
apparently even metalloid ions (26, 27). The three-dimensional structure of human aquaporin AQP1 and of the E. coli
glycerol facilitator GlpF have been determined (21, 22, 25, 28). MIP
channels consist of six transmembrane domains (TMDs) comprised of an
internal repeat of three TMDs. Loops B and E form two half TMDs that
interact within the membrane and are part of the selective pore. These
two loops contain the canonical NPA (asparagine-proline-alanine) motifs
that are part of the MIP channel signature sequence, which is conserved
in almost all of the presently known 300 MIP channels (17, 29, 30).
Fps1p is an unusual MIP channel. The NPA motifs in loops B and E are
replaced by NLA and NPS, respectively. This observation together with
mutational analyses suggests that the channel architecture of Fps1p
differs from that of other glycerol facilitators (31). In addition,
Fps1p has unusually long N- and C-terminal extensions of 255 and 139 amino acids, respectively. These extensions have no obvious similarity
to other proteins in the databases.
In this study we identified a regulatory domain within the N-terminal
extension. It is located close to the first TMD and restricts
transmembrane glycerol flux both under high osmolarity and under normal
growth conditions. The regulatory domain shows structural similarity to
the channel forming loops B and E suggesting that it might itself dip
into the membrane. We discuss implications for the mechanisms by which
this domain could be involved in controlling channel function.
Strains and Growth Conditions--
The S. cerevisiae
strains used in this study were wild-type W303-1A (MATa
leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0)
(32) and an isogenic fps1
Plate growth assays were performed by pregrowing cells either in medium
without salt (for hyperosmotic shock) or in medium supplemented with 1 M sorbitol (for hypo-osmotic shock). Cells were suspended
in the same medium to an OD600 of 1.0. Five microliters of
each dilution of 10-fold serial dilutions were spotted onto agar plates
supplemented with 2% glucose and 0.8 M NaCl (hyperosmotic shock) or lacking osmoticum (hypo-osmotic shock). Growth was monitored after 2-3 days at 30 °C.
Construction of FPS1 Mutants--
YEpmyc-FPS1 is a
2µ LEU2 plasmid expressing a c-myc
epitope-tagged Fps1p (15). The FPS1 alleles containing
larger truncations (constructs fps1-
FPS1 alleles containing alanine or phenylalanine point
mutations were constructed using the megaprimer polymerase method (34) with YEpmyc-FPS1 as template and the mutagenesis and
flanking (M5 and M6) primers listed in Table
I. The resultant PCR products were
co-transformed into S. cerevisiae with KpnI- and
CspI-digested YEpmyc-FPS1. The resulting
gap-repaired plasmids (35) were propagated in E. coli TOP
10F' and confirmed by sequencing. All other molecular biological
manipulations were performed using standard techniques (36).
Glycerol Levels and Glycerol Transmembrane
Flux--
Intracellular glycerol levels were determined essentially as
described previously (15). To determine the proportion of produced glycerol that is retained, cells were grown in liquid YNB medium to an
OD600 of 0.5-1.0, sedimented, and resuspended in medium containing 0.8 M NaCl, and samples were collected by filtration.
To determine glycerol influx following its concentration gradient,
cells were grown in liquid YNB medium to an OD600 of
~2.0. Cells were harvested, washed, and suspended in ice-cold MES
buffer (10 mM MES, pH 6.0) to a density of 40-60 mg of
cells ml Membrane Preparation and Western Analysis--
Yeast membranes
were prepared as previously described (15). 10 µg of total protein
were separated by SDS-PAGE and blotted onto nitrocellulose filters. The
filters were probed with mouse monoclonal anti-c-myc (9E10,
Santa Cruz Biotechnology) as primary antibody and alkaline
phosphatase-conjugated anti-mouse IgG as secondary antibody. For
detection, the membrane filters were incubated with 50 mg of
5-bromo-4-chloro-3-indolyl phosphate and 75 mg of nitroblue tetrazolium
salts per ml. Protein was quantified using the method of Bradford (37)
with bovine serum albumin as standard.
Structural Predictions--
Sequence alignments were carried out
using ClustalW (38). Mean values for hydrophobicity were obtained as
described (39, 40). Propensity for Deletion Analysis of the N-terminal Extension Reveals a Domain
Necessary for Control of Fps1p--
The predicted Fps1 protein
consists of a hydrophilic N-terminal extension (amino acids 1-255),
the core of six transmembrane domains (TMDs 1-6) with their connecting
loops A-E (amino acids 256-530), and a hydrophilic C-terminal
extension (amino acids 531-669) (13, 31). Previously we have shown
that deletion of the N-terminal extension between positions 13 and 230 renders Fps1p hyperactive (15). Deletion of residues 76-230 and
151-230 also increased transport through Fps1p while truncation
upstream of position 145 did not cause any obvious effect. These data
indicated that the segment important for control of Fps1p is located
between positions 150 and 231 (15).
To define this regulatory domain in more detail we generated a further
six deletions (Fig. 1A). Those
deletions were constructed in an FPS1 gene cloned under
control of its own promoter into a multicopy plasmid and then expressed
in an fps1
We then tested the functionality of the Fps1p constructs. The
fps1
To test if channel function was controlled normally we tested
transformants for hyperosmosensitivity on plates with 0.8 M NaCl. Cells expressing FPS1 alleles 6-9 grew on high
osmolarity plates like transformants carrying wild type FPS1
(Fig. 1B; see also left panel in Fig.
1D). This indicated efficient retention of glycerol produced
by the cell and successful adaptation to high osmolarity and hence
normal restriction of Fps1p-mediated glycerol export. The largest such
truncation, FPS1- Mutagenesis Defines Amino Acids Required for Restricting Glycerol
Transport--
To further analyze this regulatory domain we scanned
the sequence between 217 and 244 (except for residues Ser-237 and
Thr-238) by pairwise replacement of amino acids with threonine and
serine (Fig. 2). We chose this
combination of amino acids because they are fairly neutral in their
effect on secondary structure while we expected their insertion to
affect the amphiphilic character of the region. All mutated genes were
again expressed from the endogenous FPS1 promoter on a
multicopy plasmid. The mutated proteins were detected in the plasma
membrane fraction, they were all functional glycerol exporters as
judged from their ability to complement the hypo-osmosensitivity of the
fps1
Several of the mutations conferred strong osmosensitivity (Fig. 2);
some did not have any effect at all while others resulted in
intermediate sensitivity. Again, we interpret hyperosmosensitivity as
an inability to restrict channel function under high osmolarity stress.
Mutation of amino acids Pro-217, Ile-218, Met-219, Val-220 caused
moderate osmosensitivity while mutation of the subsequent two amino
acids caused strong osmosensitivity, indicative of a hyperactive
channel. While exchange of amino acids 223 and 224 again did not seem
to affect channel control, all mutations in the region 225-232 caused
strong osmosensitivity, identifying this area as being of crucial
importance. Note that 231 and 232 are threonine-proline and hence
mutagenesis only changed Pro-232. Exchanges at positions 233-236
resulted in partial osmosensitivity suggesting that these residues also
contribute to the function of the regulatory domain. Residues 233 and
234 are threonine-valine and hence mutagenesis only affected
Val-234.
Based on these observations we decided to focus further mutagenesis on
the region between residues 222 and 238. We exchanged individual amino
acids to alanine (with the exception of Tyr-226, which was replaced by
phenylalanine) and expressed and analyzed the mutant alleles in the
same way as described above. All mutated proteins were detected in the
plasma membrane fraction, complemented the hypo-osmosensitivity of the
fps1
We noted that the region between 217 and 244 contains a total of six
proline residues, which could be of structural importance. Hence we
first focused on the prolines in positions 222, 229, 232, and 236. Replacement of Pro-232 caused strong osmosensitivity, confirming the
observation from the TS-scanning mutagenesis. However, replacement of
Pro-222 had no effect. Since TS replacement of residues Lys-221 and
Pro-222 caused strong osmosensitivity it appears that either Lys-221 is
critical for channel control and/or that position Pro-222 tolerates
alanine but not serine. Replacement of Pro-229 and Pro-236 caused
moderate osmosensitivity, suggesting that the exchange with alanine
affected channel control only to a certain extent.
We further noted that the region under study contained phosphorylatable
amino acids. Previous attempts to link different signaling pathways and
protein kinases to channel control had not provided any evidence for a
role of phosphorylation of Fps1p in channel regulation (15) but could
not exclude such a possibility. We replaced all four threonine residues
and the serine residue by alanine and the tyrosine residue by
phenylalanine. Only replacement of Thr-231 and Thr-233 caused
intermediate or moderate osmosensitivity. These two residues
immediately flank the critical Pro-232. Finally, replacement of
residues Asn-228, Gln-230, and Leu-225 resulted in different degrees of
osmosensitivity. Taken together, it appears that residues Leu-225,
Asn-228, Gln-230, Thr-231, and Pro-232 are of particular importance for
channel control.
We then chose a subset of the mutants representing different types of
mutations for a more detailed analysis of the effects on glycerol
transport: the shortest truncation (Fig. 1) that caused osmosensitivity
(FPS1-
The influx of glycerol is significantly reduced after hyperosmotic
shock, as reported previously (14, 15). Some reduction was even
observed in fps1 The Position of the Regulatory Domain Is Important for
Function--
Pro-236, which is the most proximal amino acid to TMD1
apparently important for channel control, is located only 20 residues from the first TMD. We therefore asked if the exact position of the
regulatory domain was important for its function. To this end, we
inserted TS, TSL, and TSLS behind Ile-242, thereby increasing the
distance between the regulatory domain and TMD1 by 2, 3, and 4 amino
acids (Fig. 5). Increasing the distance
between the regulatory domain and TMD1 reduced its apparent
functionality as it resulted in progressively stronger sensitivity to
0.8 M NaCl. We also deleted amino acids 239-244 and
replaced them by threonine and serine thereby diminishing the distance
by four amino acids. Also this manipulation resulted in osmosensitivity
and hence poorer function of the regulatory domain (Fig. 5). All four
FPS1 alleles encoded functional glycerol export channels and
were expressed in the plasma membrane (data not shown). Taken together,
it appears that the exact position of the regulatory domain is
important for restricting glycerol export.
The Regulatory Domain and Its Exact Position Are Conserved--
We
searched for sequence data of Fps1p orthologs from other yeasts in the
Saccharomyces Genome data base SGD at
genome-www.stanford.edu/Saccharomyces/, the Washington University
Genome Center at www.genome.wustl.edu/blast/yeast_client.cgi and from
the Genolevure data (45) at cbi.labri.fr/Genolevures/index.php. For a comparison we used the complete Fps1p sequences from
Saccharomyces bayanus (90% identical to ScFps1p and of
similar size; 661 versus 669 amino acids) and
Saccharomyces kluyveri (56% identical, 647 amino acids) as
well as partial sequences from Kluyveromyces lactis and
Kluyveromyces marxianus. The K. lactis and
K. marxianus protein fragments are 72% identical to each
other and 46 and 42% identical, respectively, to Fps1p from S. cerevisiae.
There is high sequence identity over the six TMDs and four of the five
loops (Fig. 6 and data not shown). Loop A
is much longer in the yeast glycerol facilitators as compared with that
of the bacterial GlpFs (19), and it seems to be poorly conserved among the different yeast proteins. We note that KlFps1p, KmFps1p, and SkFps1p have the typical MIP family NPA motifs in loop B, whereas both
ScFps1p and SbFps1p have NLA. In loop E, three version of the NPA motif
exist: NPA (KlFps1p), NLA (ScFps1 and SbFps1), and NMA (SkFps1p).
Proximal to TMD1, the regulatory domain identified in this work is
highly conserved and represents, together with TMD1 and loop B, the
most highly conserved segments. Also the distance between the
regulatory domain and TMD1 is conserved. Proximal of the regulatory
domain there is little apparent sequence conservation except for about
25 amino acids around position 110 (ScFps1p; not shown); the function
of this domain is not known.
The Regulatory Domain Is Predicted to Have Structural Similarity to
Loops B and E and May Dip into the Membrane--
Surprisingly, the
N-terminal regulatory domain of Fps1p exhibits significant structural
similarity to loops B and E of Fps1p and GlpF. Those loops
dip from both sides into the membrane forming two bell-shaped half
TMDs, which, together with genuine TMDs, form the channel and its
central constriction (22, 25, 28).
The sequence alignment (Fig.
7A) centers around conserved
NP motifs (NL in Fps1p loop E). The loop E sequences both have a methionine residue 9 amino acids before the NP motif, which is also
found in the regulatory domain (here called loop N). There is also a
threonine residue conserved (although not positionally) on the approach
to the NP motif. Two amino acids before NP the loop B sequences have a
histidine, and the regulatory domain has a tyrosine in that position;
both residues are bulky and polar. Furthermore, threonine residues
occupy similar positions after the NP motif in both the loop B and loop
N sequences. Threonine is frequently involved in hydrogen bond
interactions between transmembrane regions.
There are two main requirements that a loop dipping into the membrane
bilayer must satisfy. Firstly, it must be sufficiently amphiphilic to
allow it to enter the membrane, but not go all the way through. The NP
motif is inherently hydrophilic and for this reason there must be a
series of relatively hydrophobic amino acids at the beginning and the
end of the dipping domain to allow the central section to remain in the
membrane. Secondly, it must contain amino acids that allow it to adopt
a suitable conformation, usually a
In order to test those characteristics for the regulatory domain we
employed a range of prediction methods. Using the Kyte and Doolittle
scale and Eisenberg scale (in parentheses), the average hydrophobicity
of the regulatory domain is
We then analyzed the propensity for
We have modeled the region around the regulatory domain
(Val-216-Trp-245), using the GlpF dipping loop B (Ile-56-Lys-85) as a
template of known structure (Fig. 7C). The majority of the
regulatory domain comodels with GlpF loop B but some differences in
conformation are indicated. In the regulatory loop N, the Intragenic Suppressors Confirm that the Hyperosmosensitive
Phenotype of Fps1p Truncations Is Due to Hyperactive
Transport--
Truncation or mutation of the N-terminal regulatory
domain caused poor growth on high osmolarity plates (Figs. 1-3). In an
attempt to obtain more information on the structural basis of Fps1p
regulation we isolated and characterized suppressors of this
osmosensitive phenotype. We chose for the suppressor screen the two
shortest deletions within the N terminus that gave such a phenotype.
Fps1
Hence, the observed suppression of the sensitivity to increased
osmolarity conferred by the truncated FPS1 alleles is
probably due to a strongly reduced glycerol export rate. Interestingly, all four mutations mapped to regions of Fps1p facing to the outside of
the cell and were located in loops C and E and in TMDs 4 and 5 (Fig.
8C). Ala-410 is conserved in E. coli GlpF and
replaced in the Fps1p orthologs by small hydrophilic residues; hence
glutamic acid may affect the structure of this loop. The position of
Ser-429 at the beginning of TMD5 is commonly taken by small amino acids and hence the more bulky phenylalanine may disturb this helix. Ala-469
is conserved in GlpF and the Kluyveromyces Fps1p orthologs and the change to proline is expected to alter secondary structure of
loop E, which is crucial for channel function. Arg-483 is located immediately distal to the important NPA (NLA) motif. Its replacement in
human AQP2 renders this protein non-functional and causes a pathological phenotype (46).
In this work we have defined a short segment in the N-terminal
extension of Fps1p that is required for restricting glycerol transport
through that MIP channel. Taking deletion and mutagenesis analysis as
well as sequence comparison together, we conclude that the regulatory
domain is located between residues 219 and 239 with its core between
225 and 236.
The role of this regulatory domain is to restrict the transport through
Fps1p. There are several lines of evidence that support this notion.
First, yeast cells expressing Fps1p that lacks the regulatory domain or
carries specific mutations in this domain show a strongly enhanced
glycerol transport both under standard conditions as well as under high
osmolarity (Refs. 14-16, 31, and this work). Second, cells expressing
such FPS1 alleles adjust poorly to high osmolarity
conditions, overproduce glycerol and leak glycerol into the growth
medium (Refs. 14-16, 31, and this work). Finally, mutations that
suppress the sensitivity to high osmolarity of cells expressing
hyperactive Fps1p map to FPS1 and mediate strongly reduced
flux through Fps1p. This observation directly demonstrates that
hyperactive glycerol transport through Fps1p is the cause of the
observed osmosensitivity.
The regulatory domain seems to perform its restricting function both
under standard conditions as well as under high osmolarity; truncation
or mutation of the domain causes higher glycerol uptake both in the
presence and the absence of hyperosmotic stress (Fig. 4). We have
previously shown that glycerol flux through the yeast plasma membrane
is rapidly regulated by altered osmolarity: it is decreased upon a
shift to high osmolarity to ensure internal accumulation of glycerol
produced by the cell and it is increased upon a hypo-osmotic shock to
allow rapid glycerol export (14, 15). Part of the reduction of glycerol
flux upon a hyperosmotic shock is independent of Fps1p, as is also
observed in cells lacking FPS1 (Fig. 4). This could be due
to other transport proteins or passive diffusion through the lipid
bilayer, which could be affected by altered membrane tension. It also
appears that the Fps1p-dependent glycerol flux through the
hyperactive channel is partially diminished upon hyperosmotic shock
(see for instance fps1- One way to interpret these observations is that the proposed gating
mechanism operates in a stepwise manner as has been shown for the
mechanosensitive MscL channel from bacteria. MscL, which mediates
unspecific solute export upon severe hypo-osmotic shock, seems to exist
in a closed, extended and an open state dependent on the degree of cell
swelling (8-10). While the physiological roles of Fps1p and MscL
appear to be similar there is no evidence that the mechanisms
underlying their control are similar too. However, based on the
analysis of the role of the N-terminal regulatory domain one might
speculate that the regulatory loop N plays a role in the transition
from one state to another, such as between a fully open state and an
expanded state. Such a stepwise opening/closing mechanism may make
sense given the various roles Fps1p plays in yeast cells: it is not
only needed for bulk, rapid glycerol export upon hypo-osmotic shock but
also for moderate, sustained glycerol export during growth under
anaerobic conditions (15) and for turgor control during cell fusion of
mating yeast cells (47).
Unexpectedly, the regulatory domain is predicted to exhibit structural
similarity to the channel forming loops B and E of Fps1p and GlpF and
has the potential to form a loop that dips into the membrane; loops B
and E have been shown to do so based on structural analysis of GlpF and
AQP1 (22, 25, 28). With the regulatory loop N, the four amino acids
preceding the motif are of similar hydrophobicity to loops B and E (due
in large part to Leu-225) and would be well suited to enter the
membrane. In this regard, it is interesting that Leu-225 and Tyr-226
seem to be critical as their simultaneous mutation to TS abolished
channel control. Replacement of Leu-225 by alanine also affects the
function of the regulatory domain (Fig. 3). Position 225 being very
close to the membrane face could be critical for membrane insertion, and it is feasible that this is affected even by replacement with alanine. This is particularly salient in light of the hydrophilicity of
the amino acids surrounding Leu-225 on each side (KT and YQ), rendering
the section dependent on a highly hydrophobic residue at its center in
order to maintain a sufficiently hydrophobic nature for membrane
insertion. Mutation of Tyr-226 to the more hydrophobic phenylalanine
has no obvious effect (Figs. 2 and 3).
How could such a membrane dipping domain control channel function? In
one possible scenario it may assist in orienting the transmembrane
domains and dipping loops B and E into a conformation that diminishes
glycerol flux (such as in an expanded, partly open conformation). In
that case it could be amendable to changes in membrane conformation
upon osmotic changes and participate in a gating mechanism by altering
the relative orientation of the TMDs and dipping loops involved in
transport. While such a mechanism might be analogous to that for MscL
it should be noted that there are limitations to the comparison between
Fps1p and MscL. While MscL probably was designed as a gated efflux
channel Fps1p is part of a large family of channel proteins where only AQP6 has been reported to be controlled by gating (23), although not by
osmotic changes; hence Fps1p may have adopted a control mechanism as a
secondary event in evolution.
In a different scenario the regulatory domain could directly interact
with loop B and hence with the transmembrane pore. Such direct
interactions would require the spacing between the regulatory loop N
and the TMDs to be particularly important, as we have shown here to be
the case. Some observations from structural modeling could also support
such an idea. If membrane inserted, the intramembrane portion of the N
loop is likely to be shorter than those of loops B and E, starting from
around Thr-224. This raises the possibility that the N loop might lie
buried in the bilayer close to the extramembrane face, with either of
the glutamine residues possibly in interaction with His-350 of the B
loop. This could bring the two NP motifs of loops B and N into close
proximity. Perhaps the regulatory loop N pushes against the pore,
thereby altering its open/closed probability. Upon membrane stretch
such pushing may be increased or decreased thereby either increasing
the open or the closed probability. However, more work is needed to
understand the underlying mechanisms. Crucial aspects of ongoing work
encompass further mutational studies and the purification of the
protein such that it can be reconstituted for in vitro
studies and to open the possibility for structural analyses.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
::HIS3 (YMT2). Yeast cells were routinely grown on a rotary shaker at 30 °C in YNB (yeast
nitrogen base) medium (33) containing 2% glucose as a carbon source.
6 to
fps1-
11), pairwise amino acid replacements (constructs
fps1-12 to fps1-24) or insertions/deletions
(constructs fps1-25 to fps1-28) were constructed
by completely amplifying YEpmyc-FPS1 except for the region
to be deleted or altered (15). The primers used contained either a
SacII site (constructs fps1-
6 to
fps1-
11) or a SpeI site (constructs
fps1-12 to fps1-28) and three additional
nucleotides at their 5'-end. Ligation results in the insertion of a
proline and an arginine residue (SacII) or a threonine and a
serine residue (SpeI) at the site of the deletion. All
constructs were confirmed by sequencing.
Primers used for mutagenesis
1. Glycerol influx in the presence or absence of
hyperosmotic stress was measured by adding glycerol to a final
concentration of 100 mM "cold" glycerol plus 40 µM [14C]glycerol (160mCi/mmol; Amersham
Biosciences) in a total volume of 250 µl (15). Aliquots of 50 µl
were collected by filtration and washed twice, and the radioactivity
retained on the filters was determined. Filters with cells were dried
at 80 °C overnight for dry weight determination. Transport
experiments were performed in triplicate.
and
conformation was
predicted by a sliding window calculation of the cumulative index for
three successive residues, using previously reported scales (41). The
models of the putative membrane dipping regions were generated by
extraction of the C
atom coordinates of loop B of E. coli
GlpF from the Brookhaven Protein Data bank (PDB) file, 1FX8, using
appropriate commands in RASMOL (42), followed by replacement with
corresponding amino acid residues of the aligned Fps1p regulatory
domain and construction of the loop using the MaxSprout algorithm (43). Predicted secondary structure was corroborated by analysis of the
modeled structures using TMAlpha (44).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant. All constructs carried a C-terminal
c-myc tag. By Western blot analysis of membrane preparations
we confirmed that all constructs were expressed and that the gene
products were located in fractions containing the plasma membrane (Fig.
1C). The amount of protein located in the plasma membrane
seemed to differ between different alleles. Since we did not observe
any correlation between the apparent amounts of membrane-localized
Fps1p and functionality we assumed that the different protein levels
did not affect the interpretation of the experiments. Similar
observations were reported previously (31). For unknown reasons in some
samples two bands appeared. Neither evidence for phosphorylation (15)
nor glycosylation2 of the
protein, which could possibly explain the nature of the two bands, has
been obtained so far.
View larger version (61K):
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Fig. 1.
Deletion analysis of the N-terminal extension
of Fps1p. A, sketch of the N-terminal extension
indicating the endpoints of deletions. TMD1, first
transmembrane domain. B, growth phenotypes of the
fps1 mutant transformed with plasmids carrying the
different deletion constructs. Serial 1:10 dilutions starting with a
cell suspension of OD600 nm = 1 were spotted on synthetic
YNB medium with and without 0.8 M NaCl. Failure to grow on
salt medium indicates expression of an unregulated channel.
C, Western blot analysis of cell membrane preparations
probed with an anti-c-myc antibody raised against the
C-terminal c-myc tag attached to each deletion construct.
All constructs are expressed and membrane localized, albeit at
different levels. D, growth phenotype upon shift from high
(1 M sorbitol) to low (no sorbitol) osmolarity of
transformants expressing the different deletion constructs. Poor growth
after the hypo-osmotic shift or a lower proportion of surviving cells
indicates a non-functional Fps1p, as in the fps1
control
strain.
mutant is sensitive to hypo-osmotic shock because it
is unable to rapidly release accumulated glycerol (15). In order to
test if the constructs could complement the hypo-osmosensitivity of the
fps1
mutant cells were pregrown in medium containing 1 M sorbitol and then plated in serial dilutions on medium
lacking sorbitol (Fig. 1D). It appeared that all constructs
could complement the hypo-osmosensitivity of the fps1
mutant and hence encoded functional glycerol export channels located in
the plasma membrane.
9, lacks amino acids 13-215, which
hence seemed to be dispensable for channel control. However, cells
expressing constructs 10 and 11 grew only poorly on high osmolarity
plates (Fig. 1, B and D), very much like we
observed previously for cells expressing Fps1p lacking amino acids
13-230 (15). The poor growth on high osmolarity plates was associated
with a pronounced delay in building up high intracellular glycerol
levels (data not shown). This is in line with previous observations
with other N-terminal truncations (15) and indicated an inability to
restrict glycerol transport under hyperosmotic stress. Alleles 10 and
11 lack a segment immediately upstream of position 230 and the smallest
truncation, FPS1-
11, lacks amino acids 217-230 but
retains all 216 amino acids upstream. Hence, amino acids relevant for
channel control are contained within the region covered by residues
217-230. All sequences upstream of this position seem to be
dispensable for this control, and their function remains unknown for
the moment.
mutant, and they grew like wild type on control
plates lacking osmoticum (data not shown).
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Fig. 2.
Mutagenesis of the regulatory domain by
pairwise replacements. Two adjacent amino acid residues were
replaced simultaneously by TS. Poor growth on 0.8 M NaCl
indicates expression of a hyperactive channel.
mutant and grew like wild type on control plates
without osmoticum (data not shown).
11, residues 217-230), the longest truncation that
did not cause osmosensitivity (FPS1-
9, residues 13-215) and alanine mutations (Fig. 3) that
caused different degrees of osmosensitivity: L225A, N228A, T231A, and
P232A. We observed that the alleles that conferred osmosensitivity
could less well retain the glycerol they produced, suggesting that they
had a higher capacity of glycerol transmembrane flux (data not shown).
To monitor this effect more directly, we determined the influx of
radiolabelled glycerol following its concentration gradient in
unstressed cells and during the first minute after shifting cells to
0.8 M NaCl. The different transformants displayed very
different profiles of glycerol influx (Fig.
4; note that the two graphs have
different scales). Truncation of the regulatory domain
(FPS1-
11) as well as mutations of Thr-231 and Pro-232
caused the highest levels of glycerol influx while mutation of Leu-225
caused intermediately high glycerol influx. These data correlate well
with the degree of sensitivity to 0.8 M NaCl of cells
expressing the same alleles (Figs. 1 and 3). The simplest
interpretation of those data is that the regulatory domain controls and
restricts glycerol flux through Fps1p.
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Fig. 3.
Alanine-scanning mutagenesis of the
regulatory domain. Different residues within the regulatory domain
were replaced by alanine (or phenylalanine in case of Tyr-226) and
expressed in an fps1 mutant. Failure to grow like wild
type on medium with 0.8 M NaCl indicates a hyperactive
channel.
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Fig. 4.
Glycerol influx. Influx of glycerol into
yeast fps1 cells expressing different FPS1
alleles as a measure for Fps1p channel activity. Cells were incubated
in the presence of a total of 100 mM glycerol containing 40 µM radiolabeled glycerol. Error bars
indicating S.D. of three independent experiments are too short to
display.
cells and hence is independent of Fps1p. Furthermore, a significant hyperosmotic shock-induced reduction of
glycerol influx was also observed in cells expressing hyperactive Fps1p
alleles. However, glycerol influx after hyperosmotic shock is still
much higher in these cells than in cells expressing wild type Fps1p.
This suggests that even hyperactive Fps1p lacking the N-terminal
regulatory domain retains an ability to reduce channel activity upon
hyperosmotic shock albeit not sufficiently to support normal glycerol
retention and growth in high osmolarity medium.
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Fig. 5.
Moving the regulatory domain results in
hyperactive channel. The regulatory domain was moved 2, 3, and 4 amino acids further away from the first TMD or was moved four amino
acids closer by deletion of four amino acids. Poor growth on 0.8 M NaCl indicates a hyperactive channel.
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Fig. 6.
Sequence comparison. The predicted
sequence of S. cerevisiae Fps1p is compared with those from
S. bayanus, S. kluyveri, K. lactis, and K. marxianus. X stands for any amino acid that could not
be predicted due to DNA sequence ambiguity. An asterisk
indicates where all sequences are identical, a colon where
the sequence is conserved. Only the sequences around the regulatory
domain (black with white letters) and the transmembrane
domains (TMDs) 1 and 2 (gray) as well as loops A
and B (with the underlined NPA motif) are shown.
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Fig. 7.
The regulatory domain shows similarity to
loops B and E from GlpF and Fps1p. A, alignment
of the regulatory domain of Fps1p and loops B and E from GlpF
and Fps1p. Conserved residues discussed in the text are presented in
bold, and residues shown to be particularly sensitive to
exchange against alanine are underlined. B,
Deleage and Roux plots of conformational parameters for -helix and
-turn conformations for the N-terminal regulatory domain and
averaged loops B and E from Fps1p and GlpF. C, model of the
structure of the N-terminal regulatory domain of Fps1p based on x-ray
crystallography data of GlpF loop B. The constituent amino acids are
shown beneath each loop.
-turn that is sufficiently
hydrophobic to enter the membrane and a strong
-helix to ensure the
positioning of the NP motif and a spatially precise exit.
0.52 (0.103). This is within the range of
those of the B and E loops of Fps1p (0.667 (0.333) and
0.75 (0.01)
respectively), and GlpF (1.1 (0.5) and
0.224 (0.221) respectively).
Therefore the regulatory domain appears sufficiently hydrophobic to
reside in the membrane.
and
conformation of loops B
and E and the regulatory domain. The Deleage and Roux (Fig.
7B), and also Levitt scales (not shown) indicate a strong
propensity (and a weak
propensity) immediately preceding the NP
motif. This is in line with the predictions for membrane dipping loops
B and E and in accordance with the known structure of GlpF (25).
structure
before the motif is more extensive, due to the disruption of any
preceding
structure by the frequent proline residues. Similarly,
the
structure following the NP motif runs for only 2.5 turns,
compared with 4 turns for GlpF loop B. This fits with the predicted
extramembrane location of the region between the regulatory domain and
TMD1. In contrast loop B of GlpF is preparing to reenter the membrane at Lys-85 for TMD3. Interestingly, according to this model the residues
shown to be particularly important for channel control, Thr-231 and
Pro-232, are on the same side of the helix as Asn-228 and likely to be
involved in stabilizing hydrogen bond interactions with that residue.
mutant cells expressing FPS1-
10 or
FPS1-
11 were plated on selective medium (SC; lacking
leucine for plasmid selection) containing 0.8 M NaCl and
spontaneous osmoresistant colonies were obtained at a frequency of
about 1-2 × 10
5. We reasoned that intragenic
suppressors should either restore channel control or abolish transport
function. In order to eliminate mutants in which Fps1p was fully
inactivated we tested for survival of a hypo-osmotic shock. Indeed, the
majority of the osmoresistant clones carried FPS1 alleles
that proved non-functional in this test (data not shown). We obtained
13 mutant clones out of an initial 33 selected that conferred both
resistance to high osmolarity while retaining the ability to complement
the hypo-osmosensitivity of the fps1
mutant. The 13 clones corresponded to just four point mutations probably because they
were clonal repeats. Those mutants carried exchanges of A410E, S429F,
A469P, and R483H. All alleles were expressed at similar levels (not
shown), they conferred almost wild type resistance to high osmolarity
(Fig. 8A) and accumulated the
glycerol they produced like wild type cells (data not shown). The four
mutant alleles complemented the fps1
mutant for tolerance to a hypo-osmotic shock to different degree (Fig. 8B).
Transformation with wild type FPS1 and allele S429F resulted
in an about 100-fold higher proportion of cells surviving a
hypo-osmotic shock as observed with control fps1
cells.
Alleles A410E, A469P, and R483H conferred an intermediate sensitivity,
i.e. increased the proportion of surviving cells by about a
factor of ten. These observations correlated well with the glycerol
export capacity within the first 5 min after a hypo-osmotic shock.
While wild type Fps1p mediated the release of 65% of the intracellular
glycerol, cells lacking Fps1p released less than 5% of their glycerol.
The S429F allele conferred the highest glycerol release of the four
mutants (26%). The other three alleles conferred low level of glycerol
export (around 13%; A410E, about 19%), which, however, was
consistently higher than that observed in fps1
cells.
These low levels are apparently just sufficient to allow a 10-fold
increase in survival as compared with control cells lacking Fps1p.
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Fig. 8.
Intragenic suppressor mutations of the
osmosensitivity conferred by hyperactive Fps1p. A,
growth phenotypes on 0.8 M NaCl of fps1
mutants expressing the four different mutant alleles. The ability to
grow in the presence of salt indicates suppression of the osmosensitive
phenotype of fps1-
10 or fps1-
11.
B, growth phenotypes of the same set of strains after shift
from 1 M sorbitol to medium without sorbitol. The ability
to survive and normally grow after such an osmotic downshift indicates
presence of a functional channel; the fps1
mutant is
sensitive to such conditions. C, sketch of the Fps1 protein
showing the six TMDs, the NPS and NLA motifs in loops B and E,
respectively, the regulatory domain loop N identified in this work, and
the four mutations that suppress the sensitivity to high osmolarity
conferred by truncation of the regulatory domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
11 in Fig. 4) but remains at a
much higher level than that observed for wild type Fps1p. While these
data are in line with the notion that Fps1p is controlled by gating, it
appears that the regulatory domain studied here is not the only
determinant for a possible gating mechanism. It rather seems as if the
regulatory loop N keeps Fps1p in a conformation that allows it to
readily restrict glycerol transport both in the presence and absence of
osmotic stress. In the absence of this domain transmembrane glycerol
flux is too high to allow efficient accumulation of glycerol produced by the cell and hence sensitivity to high osmolarity.
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FOOTNOTES |
---|
* This work was supported by Grant QLK3-CT2000-00778 from the European Commission and the Human Frontier Science Organization (to S. H.) as well as by grants from the Fund for Scientific Research, Flanders and the Research Fund of the Katholieke Universiteit Leuven (Concerted Research Actions) (to J. M. T.).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.
Present address: School of Life and Health Sciences, Aston
University, Aston Triangle, Birmingham B4 7ET, UK.
** Present address: Instituto Sperimentale per la Cerealicoltura, Bergamo, Italy.
¶¶ Holds the position of a special researcher of the Swedish Research Council. To whom correspondence should be addressed: Dept. of Cell and Molecular Biology/Microbiology, Göteborg University, Box 462, 40530 Göteborg, Sweden. Tel.: 46-31-773-2595; Fax: 46-31-773-2599; E-mail: hohmann@gmm.gu.se.
Published, JBC Papers in Press, December 16, 2002, DOI 10.1074/jbc.M209792200
2 K. Hedfalk, Roslyn M. Bill, J. Rydström, and S. Hohmann, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MIP, major intrinsic protein; MES, 4-morpholineethanesulfonic acid; TMD, transmembrane domain.
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REFERENCES |
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