Report |
Address correspondence to Dr. Haruo Saito, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115. Tel.: (617) 632-3814. Fax: (617) 632-5951. E-mail: h-saito{at}ims.u-tokyo.ac.jp
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Abstract |
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Key Words: signal transduction; high osmolarity stress; histidine kinase; two-component system; HOG MAPK pathway
* Abbreviations used in this paper: Cre1, cytokinin response 1; HOG, high osmolarity glycerol; TM, transmembrane.
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Introduction |
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Results and discussion |
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The subcellular distributions of Sln1 and Sho1 are consistent with such functional specialization. Sho1 is predominantly associated with the cytoplasmic membrane at the places of polarized growth, and does not change its localization on osmotic shock (Fig. 1 C). In contrast, Sln1 is distributed uniformly throughout the cytoplasmic membrane, except perhaps the regions where Sho1 is localized (Fig. 1, C and D). Furthermore, the localization of Sln1 changes in response to hyperosmotic stress by rapidly clustering into dotlike structures (Fig. 1 D). This relocalization of Sln1 is transient and is independent of Hog1 kinase activation (Fig. 1 D, right panels).
Activation of the HOG pathway in response to high osmolarity stress is accompanied by a dynamic reorganization of the actin cytoskeleton (Brewster and Gustin, 1994). Our data (Fig. 2 A) show that the levels of Hog1 activity are affected neither by actin-destabilizing drug latrunculin A (Ayscough et al., 1997), nor by actin-stabilizing drug jasplakinolide (Ayscough, 2000). Thus, the dynamic actin structures are not involved in regulation of the HOG pathway.
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Reduction in turgor, induced by high osmolarity, leads concomitantly to shrinkage of cytosolic volume and an increase in the distance between plasma membrane and cell wall. Thus, monitoring of turgor pressure by the Sln1 osmosensor might be affected through the contact between plasma membrane and cell wall. To simulate the conditions of membrane detached from cell wall, we enzymatically removed the yeast cell wall using zymolyase. To keep the spheroplasts from rupturing, they are formed in media containing 1 M sorbitol. Although this concentration of sorbitol induces a transient activation of Hog1, the Hog1 activity returns to its basal level during the 2-h preincubation in sorbitol media before addition of zymolyase (in such adapted cells, Hog1 can be reactivated with additional osmotic stress). Removal of the cell wall gave rise to Hog1 activation in spheroplasts (Fig. 2 F), and this activation was through the SLN1 pathway because it is abrogated in ssk2 ssk22 double mutant, but not in ste11 mutant. Our preparation of spheroplasts could respond to high osmolarity stress by further activation of Hog1 (Fig. 2 F), likely due to remaining patches of intact cell wall in the spheroplasts. The activation of the SLN1 branch is not indirectly induced by degradation of the membrane Sln1 protein by proteolytic activity in zymolyase preparations (Fig. S1, B and C). Together, these observations indicate that there is a causal link between the pressure of the plasma membrane against the cell wall and the stimulation of Hog1 through the SLN1 branch. Perhaps consistent with this model, nystatin treatment did not activate the mammalian osmo-stress responsive p38 MAPK, an orthologue of Hog1, in wall-less HeLa cells (Fig. 3 C).
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How do Sln1 and Cre1 histidine kinases monitor changes in turgor pressure? It is possible that Sln1 and Cre1 mediate a physical contact between cell wall and plasma membrane, perhaps through a specific binding site in their periplasmic ectodomains. However, the results of systematic deletion analysis in the Sln1 ectodomain argues, rather, against this possibility. We identified only a sequence of 13 amino acids (aa 138150; Sln19) in the Sln1 ectodomain that was essential for its function (Fig. 4 A and Fig. S4 A). However, because Sln1
9 had altered cellular distribution as well as the total protein level (Fig. 4, C and D), we believe that these defects are responsible for the loss-of-function phenotype. The fact that the phenotype of sln1
9 could be suppressed by overexpression of Vps10, a protein involved in protein sorting, supports this explanation (Fig. 4, D and E). Although contact of Sln1 with the cell wall could be, in principle, mediated by N-linked oligosaccharides, this is also unlikely because the mutations of the two putative N-glycosylation sites in the essential part of ectodomain did not affect the function of Sln1 (Fig. 4 B and Fig. S4 B).
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In summary, we have presented evidence that turgor pressure is a key factor that regulates the activity of yeast osmosensor Sln1. Loss of turgor inactivates the Sln1 histidine kinase activity, leading to subsequent phosphorylation and activation of the HOG MAPK pathway. Also, we have shown that the plant cytokinin receptor Cre1, when activated by cytokinin, can substitute the Sln1 osmosensing function, and that its kinase activity is similarly regulated by turgor pressure. The compatibility of Cre1 with yeast hyperosmotic stress signaling pathway suggests that a mechanistically analogous osmosensing system could exist in plants.
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Materials and methods |
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Microscopy
Microscopic analyses were performed as described previously (Reiser et al., 1999). Fluorescent and differential interference contrast images were acquired using a microscope (Eclipse TE300; Nikon) equipped with a GFP filter set and a differential interference contrast objective. Images were captured using the MetaMorph® imaging software (Universal Imaging Corp.).
Immunoblot analysis
Hog1 phosphorylation was determined by immunoblot analysis of cell lysates using an anti-phospho-p38 antibody (Cell Signaling). HA-tagged Sln1 variants were analyzed by an anti-HA antibody conjugated to HRP (Roche).
Zymolyase treatment and spheroplast preparation
Logarithmic cell cultures were spun down and resuspended in YPD media containing 1.0 M sorbitol, buffered with 20 mM sodium phosphate (pH 7.5), and were shaken gently (60 rpm) for 2 h. Zymolyase 100T (Seikagaku Co.) at final concentration 50 mU µl-1 and 10 mM 2-mercaptoethanol was added to cell suspension and incubated for 30 min. Formation of spheroplasts was scored by light microscopy as a relative number of cells sensitive to lysis by 2% SDS. Hog1 phosphorylation was analyzed in samples with >90% of cells converted to spheroplasts.
Cell volume measurement
Cells were fixed directly in media by addition of 3.7% formaldehyde for 15 min, washed several times with distilled water, sonicated to remove cell aggregates, and cell volume was measured using a particle counter (Coulter Counter® Z1 series; Coulter International Corporation). Each measurement was repeated three times.
Assay of Hog1 phosphorylation and cell growth in the sln1 strains
The sln1 mutant is nonviable due to a hyperactivation of Hog1 kinase (Fig. S2; Maeda et al., 1994). The lethality of the sln1
deletion is rescued in the strain TM182 (sln1
GAL1-PTP2), or its derivative BVRY179 (sln1
ste11
GAL1-PTP2), by overexpression of the Ptp2 protein tyrosine phosphatase, induced by galactose, but repressed by glucose (Fig. S2). For determination of Hog1 activity in the sln1
strains carrying various SLN1, CRE1, or SLN1-CRE1 hybrid DNA constructs, cells were grown overnight in galactose media (PTP2 expression on) and then shifted for 6 h to glucose media supplemented (or not in the control experiments) with 10 µM zeatin. At this time point, Ptp2 is diluted sufficiently to observe activation of Hog1, but cells are still viable (Fig. S2). For complementation analysis of the sln1
mutant growth phenotype on solid media, cell cultures were grown overnight in galactose media and then dropped directly onto tested plates containing galactose or glucose and grown for 3 d at 30°C.
Online supplemental material
Online supplemental material demonstrates that Hog1 activation is not induced by ergosterol depletion or proteolytic degradation (Fig. S1). The growth curve of the sln1 mutant is shown in Fig. S2. Fig. S3 illustrates regulation of Sln1 and Cre1 by high omolarity stress. Fig. S4 shows the details of Sln1 ectodomain deletion analysis. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200301099/DC1.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (GM56699); the Ministry of Education, Culture, Sports, Science, and Technology of Japan; and the Asahi Glass Foundation (to H. Saito). V. Reiser is a recipient of Human Frontiers Scientific Program Long-term Fellowship (LT0312/2000 M).
Submitted: 24 January 2003
Revised: 2 May 2003
Accepted: 2 May 2003
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