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

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 bur

Identification of residues controlling transport through the yeast aquaglyceroporin Fps1 using a genetic screen

Sara Karlgren1, Caroline Filipsson2, Jonathan G L Mullins3, Roslyn M Bill1,4, Markus J Tama´s1

and Stefan Hohmann1

1

Department of Cell and Molecular Biology/Microbiology, Go¨teborg University, Sweden;2Department of Biochemistry and Biophysics, Go¨teborg University, Sweden;3Swansea Clinical School, University of Wales Swansea, UK;4School of Life

and HealthSciences, Aston University, Birmingham, UK

Aquaporins and aquaglyceroporins mediate the transport of

water and solutes across biological membranes

Saccharo-myces cerevisiaeFps1 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: hohmann@gmm.gu.se

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)

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 facrepresent-ing 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

D600of 0.4 and then performing 10-fold serial dilutions Cell suspensions (5 lL) were spotted onto agar plates supple-mented with 1Mxylitol, or with 0.8MNaCl as a negative control, and on medium without osmoticum as a positive control Growth was monitored after 2–7 days at 30C 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 0.8M NaCl For a hypo-osmotic shock, cells were pregrown in the presence of 0.8M 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)1of ampicillin for

24 h Plasmids were isolated (Qiagen miniprep kits) and transformed into a gpd1D 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 1M sorbitol, YNB-his (negative controls) and YNB-leu plus 1Mxylitol (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 1Mxylitol 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

1M 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 D600is 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 D600

of approximately 0.7 Cells were harvested, washed and suspended in ice-cold Mes buffer (10 mMMes, pH 6.0) to a density of 40–60 mg cellsÆml)1 All subsequent steps were performed at 4C Glycerol influx in the presence or

absence of hyperosmotic stress was measured by adding

glycerol to a final concentration of 100 mMcold glycerol

plus 40 lM[14C]glycerol (5.9 GBqÆMmol)1; Amersham) in

a total volume of 250 lL [13,15] Aliquots of 50 lL were

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 80C 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

FPS1gene 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 1M xylitol Approximately 5000 colonies were screened and 31 grew on xylitol plates These were re-tested for growth on 1Mxylitol 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 1Mxylitol (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.

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

I531FIRVMNLQSTG T insertion at 1595 C-terminus

S537QLVFTSL 1613CAGTC1617 deleted 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 +

G348R G348S

H350L H350Y

L451W

Q592stop + S246P

B-loop

E-loop

W541stop

+ H3N

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.

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 1Mxylitol 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 1Mof 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 (0.8MNaCl)

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 1Mxylitol 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.

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 1M

xylitol would correlate with poor growth on 0.8MNaCl

This was the case for most of the mutations with some

exceptions G348D, which caused poorest growth on 0.8M

NaCl only mediated moderate growth on 1M xylitol

L451W, on the other hand, which mediated robust growth

on 1Mxylitol, caused almost no sensitivity to 0.8MNaCl

Also mutations that truncated the C-terminus caused either

no or poor sensitivity to 0.8M 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 0.8MNaCl 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 open 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 1Mxylitol 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 NaCl.

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.

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 strikdisrupt-ing finddisrupt-ing 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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