| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 REQUIMTE, Dept Química, FCT-UNL, 2829-516 Caparica, Portugal
2 Faculdade de Farmácia, Universidade de Lisboa, 1649-003 Lisboa, Portugal
3 Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal
4 Centro de Engenharia Biológica, FC-UL, C2, 1749-016 Lisboa, Portugal
5 Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, Katholieke Universiteit Leuven and Department of Molecular Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB), Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium
Correspondence
Graça Soveral
soveral{at}dq.fct.unl.pt
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Macey et al., 1972; Paganelli & Solomon, 1957; Vieira et al., 1970) and renal epithelia (Verkman, 1992), and was based mainly on measures of high osmotic water permeability (inhibited by mercurial reagents) and of low activation energy for water transport. In 1992 Agre and co-workers revealed that a 28 kDa integral membrane protein was the erythrocyte water channel and that the same protein was also present in renal epithelia (Preston et al., 1992). This protein is now known as aquaporin 1 (Aqp1) and it belongs to a large family of proteins that are widely distributed in nature. In mammals and plants they have important functions in water homeostasis and osmoregulation of individual cells and whole organisms. In micro-organisms, aquaporins were also recognized but their physiological role is yet being explored.
Analysis of the Saccharomyces cerevisiae genome revealed four ORFs encoding members of the major intrinsic protein (MIP) family (Bonhivers et al., 1998): FPS1 and YFL054c related to glycerol facilitator homologues (genetically close to the aquaglyceroporins), and AQY1 and AQY2, more closely related to aquaporin water channels. The functionality of aquaporins in S. cerevisiae has been debated in the literature. To study the polymorphism of aquaporins in yeast, a survey on 52 strains was performed, revealing that all strains carry an allele encoding a functional Aqy1p, but in none of these strains did the Aqy2p seem to be functional, with the exception of strain 
1278 (Laizé et al., 2000). The authors considered the hypothesis that selective pressure kept an active Aqy1p, but not Aqy2p, in yeast.
Expression of AQY1 in Xenopus oocytes showed an active Aqy1p, mediating water transport (Bonhivers et al., 1998), while the expression of AQY2 from strain 1278 was not detectable. Nevertheless, evidence that Aqy2p was expressed and functional in vesicles derived from strain 1278 cells has been presented, while the expression of Aqy1p was not detectable (Meyrial et al., 2001). By immunocytochemistry and biochemical subcellular fractionation it was found that Aqy2p was located in the endoplasmic reticulum and in the plasma membrane. Under the experimental conditions tested, AQY2 was expressed only in rich medium, during exponential growth, when glucose was the energy source. The expression of AQY2 was repressed by ethanol or galactose in rich medium, by minimal medium, hyperosmolar medium and by sporulating conditions.
Recently, it was shown that Aqy1p was involved in sporulation (Sidoux-Walter et al., 2004) and that this could be its only function. The authors claimed that Aqy1p was detectable exclusively in spore membranes under ambient conditions and that the lack of Aqy1p reduced spore fitness.
An important role for aquaporins was found in the increase of freeze tolerance in baker's yeast (Tanghe et al., 2002, 2004). Deletion of the genes AQY1 and AQY2 rendered a laboratory strain more sensitive to freezing, while overexpression of these genes improved freeze tolerance. In these transformants growth and fermentation characteristics were not affected as compared with the parental strains, but the improvement of freeze–thaw resistance was so notorious that a transformant was selected based on this characteristic (Tanghe et al., 2002). This phenotype was observed only when freezing occurred rapidly (Tanghe et al., 2004).
Surprisingly, it was found that when spores are rapidly frozen, those that lack Aqy1p survive better, providing for a functional test of active spore water channels (Sidoux-Walter et al., 2004). Comparing all these recent works (Sidoux-Walter et al., 2004; Tanghe et al., 2002, 2004), the amazing conclusion is that water channels improve freeze tolerance in vegetative cells and reduce freeze tolerance in spores.
The purpose of our work was to evaluate the role of aquaporins in plasma membrane water permeability of yeast strains for which the effect of aquaporins on freeze tolerance has been documented. We approached this problem by measuring activation energies of osmotic water transport in protoplasts prepared from S. cerevisiae 1278b-derived parental strain 10560-6B, and from three derivatives of this strain, one where AQY1 and AQY2 were deleted and two others where either AQY1 or AQY2 was overexpressed. These protoplast populations were submitted to hypo- and hyperosmotic shocks with an impermeant solute at different temperatures; the time dependence for volume equilibration was followed and water permeability coefficients evaluated.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
leu2 : : hisG trp1 : : hisG his3 : : hisG ura3-52; 10560-6B/pYX012 KanMX (=strain ANT29, indicated as parental); 10560-6B/pYX012 KanMX AQY1-1 (=strain ANT27, indicated as overexpressing AQY1); 10560-6B/pYX012 KanMX AQY2-1 (=strain ANT26, indicated as overexpressing AQY2); 10560-6B aqy1 : : KanMX4 aqy2 : : HIS3 (=strain YSH 1172, indicated as double deleted).
Integrative plasmid pYX012 (Novagen) was modified with a dominant marker gene by cloning the EcoRV–PvuII fragment containing the loxP-KanMX4-loxP cassette from pUG6 (Güldener et al., 1996) in the URA3 marker (StuI restriction site), resulting in plasmid pYX012 KanMX. The aquaporin ORF AQY1-1 was PCR-amplified using primers AQY1-FP (5'-GCGAATTCTTAACTATAACATGTCTTCGAACG-3'; EcoRI site underlined) and AQY1-RP (5'-CACCCGGGAAAAACACTAATTACCTCAGTAG-3'; XmaI site underlined) on genomic DNA of strain 10560-6B. The resulting fragments were cloned into pYX012 KanMX downstream of the constitutive TPI1 promoter using EcoRI and XmaI restriction sites. The aquaporin ORF AQY2-1 was subcloned from pYX242/AQY2-1 (Meyrial et al., 2001) in pYX012 KanMX using EcoRI and BamHI restriction sites. Integration of SphI-linearized pYX012 KanMX/AQY1-1 or AQY2-1 at the TPI1 locus resulted in geneticin-resistant strains of 10560-6B, overexpressing AQY1-1 and AQY2-1 respectively.
Preparation of yeast protoplasts.
Cells were grown in YPD medium [1 % (w/v) peptone, 0·5 % (w/v) yeast extract, 2 % (w/v) glucose], with orbital shaking, at 28 °C. Protoplasts were prepared essentially as described by Faye et al. (1974). Cells were harvested in the exponential phase (OD640
1) by centrifugation (18 000 g) for 5 min at 4 °C, and washed once with 1·2 M sorbitol, after which 10 g (wet wt) was resuspended in 30 ml digestion buffer and incubated in an orbital shaking water bath at 30 °C. To prepare the digestion buffer, 30 ml 2 M sorbitol was mixed with 3 ml 1 M potassium phosphate buffer, pH 7·5; 0·1 ml 0·5 M EDTA; 1·25 ml 0·25 mM dithiothreitol; 15·25 ml water; and 2 units zymolase 100T ml–1. When the digestion was complete, 150 ml of a 1·2 M sorbitol solution was added to the suspension, which was centrifuged at 3000 g for 10 min (4 °C). The pellet was then washed twice with 150 ml 1·2 M sorbitol. The protoplasts were resuspended in 1·2 M sorbitol. The concentration of 1·2 M sorbitol was chosen throughout the preparation of protoplasts, because disruption of the protoplasts was observed when they were suspended in lower concentrations of sorbitol. In 0·8 M sorbitol many disrupted membranes were observed under the microscope and only at 1 M did most protoplasts remain intact. The concentration of 1·2 M guarantees that protoplasts will not burst during swelling experiments.
For fluorescence experiments, protoplasts were pre-loaded for 10 min at 30 °C with the non-fluorescent precursor 5-(and 6)-carboxyfluorescein diacetate (CFDA, 1 mM in iso-osmotic solution) prior to the osmotic challenges in the stopped-flow apparatus. To avoid pH interference in fluorescence, cell suspensions and osmotic solutions were prepared in 50 mM K+-citrate buffer, pH 5.
Protoplast size determination.
Protoplast size (initial equilibrium volume) of all the preparations tested was determined by quasi-elastic light scatter (QELS) using a particle sizer (BI-90 Brookhaven Instruments). An application of this technique in the determination of vesicular sizes has been published (Perevucnik et al., 1985).
The protoplast preparations were homogeneous and protoplasts were spherical in shape, as observed under light microscopy. The QELS technique for measuring the initial diameter has shown unimodal populations for all the preparations with a mean value of 1·9±0·2 µm.
Stopped-flow experiments
Experiments were performed on a HI-TECH Scientific PQ/SF-53 stopped-flow apparatus, which has a 2 ms dead time and is temperature controlled, interfaced with an IBM PC/AT compatible 80386 microcomputer. Experiments were performed at temperatures ranging from 7 to 38 °C. Three runs were usually stored and analysed in each experimental condition. In each run 0·1 ml protoplast suspension [1 : 20 dilution in preparation buffer, initial osmolarity (osmout)0=1·2 M] was mixed with an equal amount of iso-, hypo- or hyperosmotic sorbitol solutions to reach different out or inwardly directed gradients of solute. The time-course of volume change was followed by light scatter and/or fluorescence of a trapped fluorophore.
Light-scatter experiments.
The time-course of 90° scattered light intensity at 400 nm was followed until a stable light-scattered signal was obtained (1024 data points were acquired for periods ranging from 15 to 90 s).
Fluorescence experiments.
A concentration-dependent self-quenching fluorophore was used to monitor cell volume changes. Carboxyfluorescein (CF) was excited using a 470 nm interference filter and detected using a >530 nm cut-on filter. Cells were subjected to hyper- and hypo-osmotic shocks following the same protocol applied for the light-scatter experiments, and changes in CF fluorescence due to fluorescence quenching were followed until a stable signal was obtained.
Signals were recorded on a computer interfaced directly with the Hi-Tech SF-53 using dedicated software (Hi-Tech Scientific) and were fitted to a single exponential from where the total signal amplitude and the rate constant K were evaluated.
Pf evaluation from the rate constant of volume equilibration.
The osmotic water permeability coefficient Pf can be estimated from the linear relationship between Pf and K (van Heeswijk & van Os, 1986), Pf=K(V0/A)(1/Vw(osmout)
), where Vw is the molar volume of water, V0/A is the initial volume to area ratio of the protoplast population, and (osmout) is the final medium osmolarity after the osmotic shock.
The activation energy (Ea) of water transport was evaluated from the slope of the Arrhenius plot (lnPf as a function of 1/T).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Water permeability measurements
Fig. 1 shows records of a typical stopped-flow experiment (obtained with AQY2 overexpressing strain) where the light-scatter intensity from a protoplast suspension suddenly exposed to three different solutions (iso-, hyper- and hypo-osmotic) was followed for 20 s. The three curves are identified by the tonicity of the shock (
), defined as the ratio of the final to initial osmolarity of the outside medium, i.e. =(osmout)/(osmout)0. Fig. 1 insert shows the linear dependence of the signal amplitude on 1/, obtained from these stopped-flow traces. For all protoplast preparations, similar traces were obtained and linear relations between the total amplitude and 1/ were also obtained for all the temperatures tested. The time-dependent volume changes at 23 °C for the different strains subjected to the same osmotic shocks (=0·75, or =1 for the baseline) are shown in Fig. 2. These traces show similar time constants for the deletion and parental strains, while strains harbouring overexpression constructs for AQY1 or AQY2 show higher rate constants. Equivalent symmetrical traces were obtained for hypertonic shocks of =1·25 (data not shown). Each strain presented equivalent Pf values for both hypo- and hypertonic shocks at all temperatures tested. The mean Pf (x104 cm s–1) values (±SD) at 23 °C were 7·28±1·07 and 5·13±0·59 for the strains overexpressing AQY1 and AQY2, respectively, and 1·88±0·25 and 2·02±0·20 for the parental and deletion strain, respectively.
|
|
); the concentration-dependent self-quenching properties of the fluorophore enable cell volume changes to be recorded as changes in fluorescence.
Applying in these fluorescence experiments the same osmotic shocks used in the light-scatter experiments (=0·75, 1 and 1·25), it was observed that, at all the temperatures tested, the rate constants for volume equilibration obtained by this approach were similar to the rate constants obtained by light scatter, giving similar Pf values. Table 1 compares the Pf values obtained at 23 °C by the two different methods and for the lower and higher temperature tested. These results demonstrate that for small perturbations, the light scatter is an adequate methodology to follow protoplast volume changes and perform measurements of osmotic water permeability in yeasts.
|
shows the plots obtained for the four strains for swelling and shrinking respectively when Pf was evaluated using the light-scatter technique. The deletion strain and the strain with overexpression of AQY1 show a linear plot with a lower Ea value for the overexpressing AQY1 strain (confirmed in fluorescence experiments, data not shown). The shrinking and swelling experiments for the parental strain showed different behaviours. A single straight line could only be fitted to the shrinking data points with Ea value equivalent to the deletion strain (Fig. 3).
|
).
|
presents the Pf values for all the strains obtained for the lowest and highest temperatures tested (7 and 38 °C). For both temperatures the lowest value was obtained for the deletion strain. The table also shows the permeabilities for each strain relative to the deletion strain, calculated as the ratio 
. This ratio shows that the increase in permeability due to the presence of aquaporins becomes more relevant at low temperatures.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
All our experiments were performed in protoplasts derived from strains with different levels of expression of aquaporins and they responded osmotically to the different tonicity shocks (hypo- and hypertonic, Fig. 1
and Fig. 2), rendering these protoplasts a useful model to study water transport in yeast. For small volume perturbations, the light-scatter technique has proved to be an adequate method to evaluate the osmotic permeability coefficient as the use of an independent technique (concentration-dependent quenching fluorescence) applied to the same AQY1 strain gave equivalent values of Pf for all the temperatures tested and, consequently, an equivalent Ea value.
A similar methodology was previously used (Laizé et al., 1999) when working with strain 1278b presenting Pf values much higher that those we measured. We speculate that the differences observed may be explained by different initial concentrations of sorbitol used. In the work by Laizé et al. (1999), the initial concentration was 0·5 M, but in our experiments we observed that an important fraction of the protoplasts was disrupted when suspended in 0·8 M sorbitol and only at 1 M did most protoplasts remain intact. We chose 1·2 M to guarantee that disruption would not occur during swelling experiments.
Our results from both hypo- and hypertonic experiments (protoplast swelling and shrinking) show that, in general, the Pf values for all the temperatures tested were higher for the AQY1 followed by the AQY2 overexpressing strains. Since both genes are expressed behind the same promoter, this result suggests that Aqy1p may be slightly more active than Aqy2p, although the hypothesis that this difference is due to different levels of expression cannot be discarded.
For the lower temperature range (7–23 °C) the parental strain showed higher Pf values than the deletion strain, representing clear evidence that at least one of the aquaporins was expressed in the parental strain under the growth conditions used for the experiments (Table 3). Nevertheless, the values for both these strains were similar for the higher temperature range tested (23–38 °C). This may indicate that the presence of aquaporins is of major importance at lower temperatures and is in agreement with the aquaporin-mediated improvement of freeze tolerance as suggested (Tanghe et al., 2002). Although the concept of cold-induced improvement of freeze tolerance is highly controversial, the most recent studies on this topic are all indicative of an adaptation of yeast cells to cold stress (Alves-Araujo et al., 2004; Rodriguez-Vargas et al., 2002; Sahara et al., 2002).
The activation energy values obtained for the deletion strain were high and compatible with water flowing mainly through the lipid bilayers as expected from the genotype of this strain. For the strain overexpressing AQY1, much lower values were found for the Ea, indicating the presence of active water channels, under all the temperatures tested (Table 2).
Interestingly, however, the Arrhenius plots for the parental (swelling experiments) and overexpressing AQY2 strains (swelling and shrinking experiments) were not linear, presenting a break point with a change in slope around 23 °C. The calculated Ea for the higher temperature range (23–38 °C) was high and only for temperatures below 23 °C was the value low (Table 2, Fig. 3). This behaviour indicates that below this temperature the water fluxes through the water channels become important.
The question is, why were the water fluxes across the channels not visible at higher temperatures? Were the water channels less active at higher temperatures or was their activity masked by the flux across the bilayer at higher temperatures? If this latter explanation is true then at the very low temperatures the contribution of the lipid pathway may even become insignificant and the activation energy could become much lower. On the other hand, and according to this hypothesis, if we could increase the temperature range to much higher values (above 40 °C), we should probably also detect a break in the AQY1 plot.
A numerical simulation of this hypothesis is shown in the Arrhenius plot of Fig. 4. The two dashed straight lines represent the typical behaviour for the lipid pathway with an activation energy of 18 kcal mol–1 (75·3 kJ mol–1; line with greater slope) and the channel pathway with an Ea of 4 kcal mol–1 (16·7 kJ mol–1; line with lesser slope). These lines will cross at a certain point corresponding to the temperature below which the channel pathway overcomes the lipid pathway (right side of the graph). This temperature can vary depending on the number of active channels present. The sum of these two contributions for the total permeability will give rise to a non-linear Arrhenius plot (solid curve). In this example, if the temperature is sufficiently high (left side of the graph) the total permeability will equal the lipid permeability with an Ea of 18 kcal mol–1 (75·3 kJ mol–1) while, if the temperature is sufficiently low (right side of the graph), the total permeability will be equivalent to the channel permeability with an Ea of 4 kcal mol–1 (16·7 kJ mol–1). In the middle temperature range, the slope varies from 18 to 4 kcal mol–1 (75·3 to 16·7 kJ mol–1). Within our experimental temperatures the four strains behaved differently, showing a diverse contribution of both pathways. The deletion strain had no contribution of the channel pathway, as expected. The overexpressing AQY1 was the strain with the highest contribution of the channel pathway at all temperatures tested. The overexpressing AQY2 and parental strains showed a mixed behaviour, since at high temperatures they behaved like lipid membranes but as the temperature decreased the channel pathway became visible and the activation energy decreased accordingly.
|
; Tanghe et al., 2002, 2004). If the lipid membrane is more rigid and less permeable it makes sense that water channels become important during water volume increase and crystal formation at low temperature, preventing cell bursting (Tanghe et al., 2003).
Cold stress responsiveness in plants has also been associated with aquaporins. It was shown that cold promotes the expression of the water channel 
TIP in Tulipa gesneriana associated with the stalk elongation (Balk & de Boer, 1999). In maize, a contribution of aquaporins to the recovery of root hydraulic conductance after chill injury was reported (Aroca et al., 2005). The hypothesis that aquaporins may be involved in the cryopreservation of animal cells was also considered. Data indicate that the regulation of aquaporins can attenuate cold-induced osmotic damage in rat kidney cells (Wang & Ben, 2004).
In summary, and following our experimental results, we propose that yeast aquaporins may have a relevant function restricted to low temperature.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aroca, R., Amodeo, G., Fernandez-Illescas, S., Herman, E. M., Chaumont, F. & Chrispeels, M. J. (2005). The role of aquaporins and membrane damage in chilling and hydrogen peroxide induced changes in the hydraulic conductance of maize roots. Plant Physiol 137, 341–353.
Balk, P. A. & de Boer, A. D. (1999). Rapid stalk elongation in tulip (Tulipa gesneriana L. cv. Apeldoorn) and the combined action of cold-induced invertase and the water-channel protein gammaTIP. Planta 209, 346–354.[CrossRef][Medline]
Bonhivers, M., Carbrey, J. M., Gould, S. J. & Agre, P. (1998). Aquaporins in Saccharomyces. Genetic and functional distinctions between laboratory and wild-type strains. J Biol Chem 273, 27565–27572.
Chen, R. F. & Knutson, J. R. (1988). Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers. Anal Biochem 172, 61–77.[CrossRef][Medline]
Farmer, R. E. & Macey, R. I. (1970). Perturbation of red cell volume: rectification of osmotic flow. Biochim Biophys Acta 196, 53–65.[Medline]
Faye, G., Kujawa, C. & Fukuhara, H. (1974). Physical and genetic organization of petite and grande yeast mitochondrial DNA. IV. In vivo transcription products of mitochondrial DNA and localization of 23 S ribosomal RNA in petite mutants of Saccharomyces cerevisiae. J Mol Biol 88, 185–203.[CrossRef][Medline]
Güldener, U., Heck, S., Fielder, T., Beinhauer, J. & Hegenann, J. H. (1996). A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 24, 2519–2524.
Laizé, V., Gobin, R., Rousselet, G., Badier, C., Hohmann, S., Ripoche, P. & Tacnet, F. (1999). Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domain. Biochem Biophys Res Commun 257, 139–144.[CrossRef][Medline]
Laizé, V., Tacnet, F., Ripoche, P. & Hohmann, S. (2000). Polymorphism of Saccharomyces cerevisiae aquaporins. Yeast 16, 897–903.[CrossRef][Medline]
Macey, R. I., Karan, D. M. & Farmer, R. E. (1972). Properties of water channels in human red cells. Biomembranes 3, 331–340.[Medline]
Meyrial, V., Laizé, V., Gobin, R., Ripoche, P., Hohmann, S. & Tacnet, F. (2001). Existence of a tightly regulated water channel in Saccharomyces cerevisiae. Eur J Biochem 268, 334–343.[Medline]
Paganelli, C. V. & Solomon, A. K. (1957). The rate of exchange of tritiated water across the human red cell membrane. J Gen Physiol 41, 259–277.
Perevucnik, G., Schurtenberger, P., Lasic, D. D. & Hauser, H. (1985). Size analysis of biological membrane vesicles by gel filtration, dynamic light scattering and electron microscopy. Biochim Biophys Acta 821, 169–173.[Medline]
Preston, G. M., Carroll, T. P., Guggino, W. B. & Agre, P. (1992). Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256, 385–387.
Rodriguez-Vargas, S., Estruch, F. & Randez-Gil, F. (2002). Gene expression analysis of cold and freeze stress in Baker's yeast. Appl Environ Microbiol 68, 3024–3030.
Sahara, T., Goda, T. & Ohgiya, S. (2002). Comprehensive expression analysis of time-dependent genetic responses in yeast cells to low temperature. J Biol Chem 277, 50015–50021.
Sidoux-Walter, F., Pettersson, N. & Hohmann, S. (2004). The Saccharomyces cerevisiae aquaporin Aqy1 is involved in sporulation. Proc Natl Acad Sci U S A 101, 17422–17427.
Tanghe, A., Van Dijck, P., Dumortier, F., Teunissen, A., Hohmann, S. & Thevelein, J. M. (2002). Aquaporin expression correlates with freeze tolerance in baker's yeast, and overexpression improves freeze tolerance in industrial strains. Appl Environ Microbiol 68, 5981–5989.
Tanghe, A., Van Dijck, P. & Thevelein, J. M. (2003). Determinants of freeze tolerance in microorganisms, physiological importance, and biotechnological applications. Adv Appl Microbiol 53, 129–176.[Medline]
Tanghe, A., Van Dijck, P., Colavizza, D. & Thevelein, J. M. (2004). Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions. Appl Environ Microbiol 70, 3377–3382.
van Heeswijk, M. P. & van Os, C. H. (1986). Osmotic water permeabilities of brush border and basolateral membrane vesicles from rat renal cortex and small intestine. J Membr Biol 92, 183–193.[CrossRef][Medline]
Verkman, A. S. (1992). Water channels in cell membranes. Annu Rev Physiol 54, 97–108.[CrossRef][Medline]
Vieira, F. L., Sha'afi, R. I. & Solomon, A. K. (1970). The state of water in human and dog red cell membranes. J Gen Physiol 55, 451–466.
Wang, W. & Ben, R. N. (2004). Upregulation and protein trafficking of aquaporin-2 attenuate cold-induced osmotic damage during cryopreservation. In Vitro Cell Dev Biol Anim 40, 67–70.[CrossRef][Medline]
Received 9 November 2005;
revised 20 January 2006;
accepted 3 February 2006.
This article has been cited by other articles:
|
|
 |
|
  A. Minami, M. Fujiwara, A. Furuto, Y. Fukao, T. Yamashita, M. Kamo, Y. Kawamura, and M. Uemura Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation Plant Cell Physiol., February 1, 2009; 50(2): 341 - 359. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.-W. Li, M.-H. Zhang, W.-M. Cai, W.-N. Sun, and W.-A. Su Characterization of OsPIP2;7, a Water Channel Protein in Rice Plant Cell Physiol., December 1, 2008; 49(12): 1851 - 1858. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Soveral, A. Madeira, M. C. Loureiro-Dias, and T. F. Moura Water Transport in Intact Yeast Cells as Assessed by Fluorescence Self-Quenching Appl. Envir. Microbiol., April 1, 2007; 73(7): 2341 - 2343. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
, AQY1; 
, AQY2; 
, parental; 
, deletion. (a) Hypotonic conditions, swelling experiments; (b) hypertonic conditions, shrinking experiments. All the experimental points are presented as mean values±SD of triplicates.
).