Department of Physiology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore MD 21205, USA1
Author for correspondence: Rajini Rao. Tel: +1 410 955 4732. Fax: +1 410 955 0461. e-mail: rrao{at}jhmi.edu
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ABSTRACT |
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Keywords: Na+/H+ exchanger, osmotolerance, hypertonic shock, vacuole, Saccharomyces cerevisiae
Abbreviations: GPDH, NADH-dependent glycerol-3-phosphate dehydrogenase; HOG, high-osmolarity glycerol; MAP, mitogen-activated protein
a Present address: Center for Molecular Neuroscience and Department of Pharmacology, Vanderbilt University School of Medicine, Nashville TN 37232, USA.
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INTRODUCTION |
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The yeast vacuole is involved in osmoregulation (Matile, 1978 ; Banta et al., 1988
) and may play a role in protecting cells from a sudden osmotic challenge. Many vacuole mutants that have altered morphology, or have defects in protein sorting, are also sensitive to osmotic stress (Banta et al., 1988
; Latterich & Watson, 1991
, 1993
). Thus a yeast mutant defective in vacuole biogenesis loses viability within 10 s exposure to 1·5 M NaCl (Latterich & Watson, 1993
).
Tolerance to osmotic shock is dependent on the physiological state of the cells. Exponential-phase cells are much more sensitive to osmotic shock relative to the slow-growing postdiauxic-phase cells or non-growing stationary-phase cells (Brown et al., 1986 ; Mackenzie et al., 1986
; Blomberg et al., 1988
; Mager & Varela, 1993
; Werner-Washburne et al., 1993
). Although this phenomenon remains largely unexplained, the non-reducing disaccharide trehalose may partially contribute toward osmotolerance; thus osmoresistance in yeast correlates with an increase in the intracellular content of this sugar, which accumulates during stationary phase (Crowe et al., 1984
; Hottiger et al., 1987
). However, trehalose levels alone may not be sufficient to counter the large increase in osmotolerance (Andre et al., 1991
).
Higher eukaryotes regulate intracellular ion concentrations in response to an immediate osmotic challenge. The vacuole in plant cells is involved in regulating turgor pressure (Zimmermann, 1978 ; Boller & Wiemken, 1986
). In halophytes, Na+ accumulation (with Cl- and water) in the vacuoles of the leaf protects the cells from the detrimental effects of high salt and osmolarity (Flowers et al., 1986
). The transport of Na+ into the tonoplast is believed to be mediated via a Na+/H+ antiporter, which is driven by the H+ gradient generated by the vacuolar H+-ATPase (Blumwald & Poole, 1987
; Garbarino & DuPont, 1988
; Barkla & Blumwald, 1992
).
In mammalian cells, plasma membrane Na+/H+ exchangers are activated in response to both short- and long-term hyperosmotic stress (Grinstein et al., 1989 ; Soleimani et al., 1995
; Wakabayashi et al., 1997
). Although the exact molecular mechanisms involved in exchanger activation are unknown, the exchanger is believed to become activated in response to cell shrinkage (Donowitz et al., 1996
; Wakabayashi et al., 1997
). The decrease in the cell volume causes the exchanger to become more sensitive to intracellular protons, and the activation of the exchanger causes an increased influx of Na+ (and Cl-). With the obligatory influx of water, cell volume is restored (Grinstein et al., 1985
, 1992
; Hoffmann & Simonsen, 1989
).
We have recently identified a novel intracellular Na+/H+ exchanger in yeast, Nhx1, which contributes to cellular Na+ homeostasis (Nass et al., 1997 ). We have localized the exchanger to the late endosome/prevacuolar compartment (PVC) and have proposed that it may be involved in Na+ transport, water movement and vesicle volume regulation (Nass & Rao, 1998
). Consistent with these functions, we have shown that the exchanger co-localizes with Gef1, the yeast homologue of the CLC family of chloride channels, and is required for salt amelioration conferred by overexpression of an Arabidopsis vacuolar H+ pyrophosphatase in yeast (Gaxiola et al., 1999
). Considering the important role of Na+/H+ exchangers in the osmoregulatory response of plants and mammalian cells, and the similarities in ion homeostasis in plants and yeast, we asked whether Nhx1 contributes to osmotolerance. Our results indicate that Nhx1 plays a role in the recovery of cell growth following exposure to hyperosmotic media. Furthermore, we show that the osmotolerance conferred by Nhx1 contributes to the postdiauxic-phase resistance to osmotic stress, and allows for the continued growth of cells until acquired osmotolerance can occur.
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METHODS |
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Growth assays.
Seed cultures of K601 and R100 were grown at 30 °C in APG medium, adjusted to pH 4 with dilute acetic acid. The cultures were diluted tenfold and grown for 72 h in fresh medium. Postdiauxic-phase cells were then diluted 100-fold in APG medium, pH 4, and growth for 60 h on a shaker at 290 r.p.m. in the absence or presence of 1 M sorbitol was monitored at 600 nm. Where indicated, cultures were grown to the appropriate growth phase and then equivalent numbers of cells were inoculated into 1 ml medium containing a range of sorbitol concentrations in a multiwell plate. Growth was monitored by measuring OD600 after culturing for the indicated times at 30 °C. Growth assays shown are representative of one of at least three independent experiments. Relative growth is the OD600 at any sorbitol concentration, divided by the OD600 of the culture grown in the absence of sorbitol, expressed as percentage.
Confocal imaging.
Cultures of K601 and R100 were grown to late exponential phase, loaded with the styryl dye FM 4-64 and examined by confocal microscopy as described previously (Nass & Rao, 1998 ). Cells were collected by centrifugation for 20 s at 5000 r.p.m., resuspended in APG medium, pH 4, containing 2 M sorbitol and incubated at 23 °C for the indicated times.
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RESULTS |
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Nhx1 plays a role in the short-term response to hyperosmotic shock but not in long-term acquired osmotolerance
To determine whether Nhx1 may play a role in protecting cells from sudden exposure to osmotic dehydration, we followed duplicate growth curves in APG, pH 4, and monitored growth at or before 40 h (short-term) and at 90 h (long-term). The inoculum contained postdiauxic-phase cells. As shown earlier (Fig. 1), there was a difference in cell density of the
nhx1 mutant relative to wild-type after 40 h. However, this difference in growth was completely eliminated after 90 h, indicating that the absence of Nhx1 does not affect viability or long-term acquired osmotolerance (data not shown). For example, in cultures containing 1·4 M sorbitol, the cell density of the mutant was threefold lower than wild-type after 40 h of growth, but was identical to wild-type after 90 h. We conclude that Nhx1 plays a significant role in the events following sudden exposure to hyperosmotic media.
Nhx1 contributes to vacuolar shrinkage upon exposure to hypertonic stress
Yeast cells can alter their cell volume in response to osmotic challenge, decreasing volume in response to hypertonic stress and increasing volume in the presence of hypotonic stress (Niedermeyer et al., 1977 ; Morris et al., 1986
; Blomberg & Adler, 1992
). These responses are reversible, and in the case of dehydration, occur within a minute (Morris et al., 1986
). The vacuolar size also decreases following exposure to hypertonic solutions (Morris et al., 1986
; Blomberg & Adler, 1992
). To determine if Nhx1 contributes to changes in vacuolar volume, we directly visualized vacuole size in vivo, by confocal microscopy, after acute exposure to hyperosmotic medium. Vacuoles in living cells were labelled with the styryl dye FM 4-64, which intercalates into the plasma membrane and is internalized by endocytosis, accumulating in the acidic vacuolar compartment (Vida & Emr, 1995
). In the absence of hypertonic stress, vacuoles from wild-type or the
nhx1 mutant had a similar appearance (Fig. 3af
). Upon exposure to hypertonic medium, vacuoles of wild-type cells shrank within 6 min, with the surface rapidly changing from smooth to crenellated (Fig. 3gi
). Longer-term exposure to sorbitol (Fig. 3mo
) or growth for several days in 1 M sorbitol (data not shown) largely reversed these changes, with persistent staining of perivacuolar vesicles that are likely to represent the prevacuolar compartment as observed previously (Nass & Rao, 1998
). In contrast, the majority of
nhx1 cells showed significantly smaller changes in vacuolar volume and appearance in the short-term response to hyperosmotic challenge (Fig. 3jl
). Furthermore, the changes in vacuolar structure persisted for longer times in the mutant (Fig. 3pr
), suggesting that the response to osmotic stress was delayed or compromised in the absence of Nhx1. These observations suggest that Na+/H+ exchange by Nhx1 may be involved in regulating vacuolar volume in response to hypertonic stress.
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DISCUSSION |
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In this study we examined the role of the endosomal Na+/H+ exchanger Nhx1 under osmotically stressful conditions. Following inoculation of wild-type and nhx1 cells from a postdiauxic-phase culture into sorbitol-supplemented medium, the mutant showed a reduction in osmotolerant growth, indicating that Nhx1 does play a role in the response to hyperosmotic stress. In the absence of osmotic stress, the wild-type and
nhx1 mutant grew to similar cell densities; this is similar to the effect of null mutants of the MAP kinase cascade required for glycerol accumulation in acquired osmotolerance (HOG1 or PBS2; Brewster et al., 1993
), and a null mutant required for vacuole biogenesis and the acute response to hyperosmotic shock (ssv1-2; Latterich & Watson, 1993
). Interestingly, the reduction in growth of the
nhx1 mutant in sorbitol-containing medium was observed within 3040 h post-inoculation, but upon further growth to saturation, similar cell densities were observed for both mutant and wild-type. These results show that the
nhx1 mutant is compromised in its ability to initially respond to the osmotic stress, but following prolonged exposure can apparently acclimatize and begin growing to levels similar to wild-type.
The activity and induction of the glycerol-producing enzyme GPDH is highest when cells use glucose as the carbon source (Rios et al., 1997 ; Serrano, 1996
). Thus, carbon catabolite repression conditions, in which cells are grown in glucose, produce the greatest amounts of glycerol (Serrano, 1996
). We examined the contribution of Nhx1 to osmotolerance using the partially derepressing carbon source galactose (Fraenkel, 1982
) to minimize the contribution of high intracellular glycerol concentrations. Consistent with previous conclusions (Rios et al., 1997
), galactose-grown cultures of both wild-type and the
nhx1 mutant were significantly more sensitive to osmotic stress, and similar to glucose-grown cultures,
nhx1 cells were more osmotically sensitive relative to the isogenic control.
Yeast cells pass through distinct physiological phases during growth in glucose media (Blomberg et al., 1988 ; Werner-Washburne et al., 1993
). It has long been observed that postdiauxic-phase and stationary-phase cells are more tolerant to many environmental stresses, including osmotic stress (Schenberg-Frascino & Moustacchi, 1972
; Mackenzie et al., 1986
; Blomberg et al., 1988
; Elliott & Futcher, 1993
; Werner-Washburne et al., 1993
). We show here that the physiological state of the cells affects Nhx1-mediated osmotolerance. Exponential-phase cells have greater sensitivity to sorbitol, as observed by the lower IC50 of 900 mM in wild-type (Fig. 2a
). Furthermore, Nhx1 appears to contribute to osmotolerance only when the cells are in the postdiauxic/stationary phase upon exposure to sorbitol. It is possible that induction of Nhx1 activity or expression may occur at, or following, the diauxic shift, or that there are other mechanisms that contribute to osmotolerance in exponential-phase cells which compensate for the absence of Nhx1.
The yeast vacuole plays an important role in pH and ion homeostasis and contributes to protein trafficking, sorting and degradation (Banta et al., 1988 ; Klionsky et al., 1990
; Bryant & Stevens, 1998
). Osmotolerance has been directly linked to normal vacuole morphology and function (Banta et al., 1988
; Mager & Varela, 1993
; Latterich & Watson, 1993
). We show here that the recently identified late endosomal/prevacuolar Na+/H+ exchanger Nhx1 contributes to osmotolerance (Nass et al., 1997
; Nass & Rao, 1998
). Under normal culture conditions, it is likely that Nhx1-mediated sequestration of Na+ in the late endosome/vacuole provides an osmoticum that serves to retain water in the vacuoles (Morris et al., 1986
; Serrano, 1996
). The maintenance of turgor pressure in the late endosome and vacuole may be important for the proper trafficking of essential proteins required for osmotic tolerance or normal cellular function.
Yeast cells respond to an immediate hyperosmotic stress by decreasing cell size, possibly through activation of plasma membrane mechanosensitive ion channels (Gustin et al., 1988 ) and water loss. It is possible that cell-shrinkage-mediated activation of the Na+/H+ exchange activity of Nhx1 may allow exit of Na+ ions, and consequently water, from the vacuoles, contributing to the observed changes in vacuolar morphology (Fig. 4
). The flow of water from the vacuole to the cytoplasm may be an important initial response to counter hyperosmotic cell shrinkage. In the long-term, transport of Na+ into intracellular organelles by Nhx1 would protect sodium-sensitive proteins in the cytoplasm and contribute to the accumulation of osmotic equivalents in the vacuole; this may explain the delayed cell growth and short exponential phase of the
nhx1 mutant. Future studies will need to address the molecular mechanisms of osmosensing and signal transduction involved in the Nhx1-mediated response to hyperosmotic challenge.
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ACKNOWLEDGEMENTS |
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Received 22 April 1999;
revised 29 June 1999;
accepted 9 July 1999.