Hypo-osmotic Stress Activates Plc1p-dependent Phosphatidylinositol 4,5-Bisphosphate Hydrolysis and Inositol Hexakisphosphate Accumulation in Yeast*

Polyphosphoinositide-specific phospholipases (PICs) of the δ-subfamily are ubiquitous in eukaryotes, but an inability to control these enzymes physiologically has been a major obstacle to understanding their cellular function(s). Plc1p is similar to metazoan δ-PICs and is the only PIC in Saccharomyces cerevisiae. Genetic studies have implicated Plc1p in several cell functions, both nuclear and cytoplasmic. Here we show that a brief hypo-osmotic episode provokes rapid Plc1p-catalyzed hydrolysis of PtdIns(4,5)P2 in intact yeast by a mechanism independent of extracellular Ca2+. Much of this PtdIns(4,5)P2 hydrolysis occurs at the plasma membrane. The hydrolyzed PtdIns(4,5)P2 is mainly derived from PtdIns4P made by the PtdIns 4-kinase Stt4p. PtdIns(4,5)P2 hydrolysis occurs normally in mutants lacking Arg82p or Ipk1p, but they accumulate no InsP6, showing that these enzymes normally convert the liberated Ins(1,4,5)P3 rapidly and quantitatively to InsP6. We conclude that hypo-osmotic stress activates Plc1p-catalyzed PtdIns(4,5)P2 at the yeast plasma membrane and the liberated Ins(1,4,5)P3 is speedily converted to InsP6. This ability routinely to activate Plc1p-catalyed PtdIns(4,5)P2 hydrolysis in vivo opens up new opportunities for molecular and genetic scrutiny of the regulation and functions of phosphoinositidases C of the δ-subfamily.

Phosphoinositide-based regulatory systems are ubiquitous in eukaryotes and contribute to many processes, including signaling from cell surface receptors, assembly/disassembly of the actin cytoskeleton, and vesicle trafficking. Receptor signaling through activation of PtdIns(4,5)P 2 1 hydrolysis by phosphoinositide-specific phospholipases C (phosphoinositidases C; PICs) is the prototype of such regulatory systems.
Of the five known PIC families, PIC␦s are ubiquitous in eukaryotes and receptor-controlled PICs of the ␤, ␥, and ⑀ subfamilies (1-3) are only found in metazoans. PIC has been detected only in sperm (4). A prokaryotic PIC that integrated into an emerging proto-eukaryote was probably the common ancestor of all eukaryote PICs (5), and it seems likely that this was more similar to modern PIC␦s than the later-evolved signaling PICs (1)(2)(3). PIC␦s might even retain some of the original functions of this ancestral PIC, so it is unfortunate that we understand so little about their regulation and functions. Improved understanding of PIC␦s is likely to come most readily from organisms that express only one, PIC␦-like, PIC and that lack the PtdIns(4,5)P 2 -consuming Type I phosphoinositide 3-kinases. One such is Saccharomyces cerevisiae, with Plc1p (encoded by PLC1) its sole PIC.
The activity of Plc1p thus influences many cell activities. Some, such as chromatin maintenance, transcription, and mRNA export, are nuclear, whereas others, including vacuole homeostasis and proteasome activity, reside in the cytosol compartment. It therefore seems that the pleiotropic phenotypes of ⌬plc1 cells are consequences of multiple flaws in several fundamental processes, in at least two cell compartments.
Despite this substantial body of genetic evidence, there is scant information on what controls Plc1p activity in vivo. It was suggested that glucose re-admission to glucose-deprived yeast might activate Plc1p (23), but this response was later attributed mainly to polyphosphoinositide deacylation (24). It was suggested that glucose re-admission might provoke phosphoinositide turnover and activate a plasma membrane H ϩ pump, with Plc1p needed for both responses (25), but again deacylation may have caused much of the observed phosphoinositide loss. Nitrogen re-addition to nitrogen-starved yeast provokes rapid Ins(1,4,5)P 3 formation (26), but this seems not to need Plc1p (27). Hypo-osmotic shock evokes a [Ca 2ϩ ] i rise in yeast (28) and in some animal and plant cells, and hypo-osmotic shock may sometimes activate PIC (29,30). However, the underlying phosphoinositide changes in these responses, and how these are linked to other cellular events, remain uncertain.
In this study, we present evidence that hypo-osmotic stress speedily activates Plc1p-catalyzed PtdIns(4,5)P 2 hydrolysis at the plasma membrane in S. cerevisiae, and we define the source of the hydrolyzed PtdIns(4,5)P 2 and the metabolic fate of the liberated Ins(1,4,5)P 3 . Table I lists the yeast strains used.

Growth of [ 3 H]Inositol-labeled Yeast and Procedure for Hypo-osmotic
Shock-Cells were grown exponentially with [ 3 H]inositol (5 Ci per ml), and maintained in the presence of this label throughout all manipulations until they were killed. They were acclimatized to hypertonic saline medium during 2-h incubations, in media supplemented first with 0.5 M NaCl and then with 0.9 M NaCl. The acclimatized cells (3 ϫ 10 6 ml Ϫ1 , 5 ml) were sedimented (3000 ϫ g, 5 min, 23°C) and resuspended in 0.5 ml of medium supplemented with 0.9 M NaCl at 23°C. 15 min later they were diluted 4-fold with medium containing 0.9 M NaCl (no osmotic shock) or lacking added NaCl (hypo-osmotic stress). ⌬stt4 cells, which are osmotically fragile, were grown throughout in medium supplemented with 0.9 M NaCl.
The upper phase plus interface, containing water-soluble inositol polyphosphates, were incubated at 30°C for 30 min, centrifuged (10 5 ϫ g, 30 min, 30°C) and the interfacial pellet discarded. The volume was made 2 ml with H 2 O, and acid was neutralized with an appropriate volume of 1 M NaOH containing 2.5 mM EDTA, 2.5 mM EGTA, and 37.5 mM HEPES, and samples were stored at Ϫ20°C. They were analyzed by HPLC on a 250 ϫ 4.6 mm Partisphere 5-SAX column, eluted at 1 ml/min with a complex gradient: Solution A was H 2 O, and Solution B was 1.25 M (NH 4 ) 2 HPO 4 , pH 3.8. The gradient was: 0 -5 min, 0% B; 5-10 min, ramp to 7% B; 10 -40 min, ramp to 9% B; 40 -110 min, ramp to 100% B; then isocratic to 120 min. Radioactivity was measured with a flow detector (see above). A different gradient, using the same column and Solutions A and B, was used to confirm that the major inositol polyphosphate that accumulated during hypo-osmotic shock was InsP 6 ( Fig. 1): 0 -5 min, 0% B; 5-10 min, ramp to 7% B; 10 -25 min, ramp to 70% B; 25-95 min, ramp to 78% B; 95-110 min, ramp to 100% B; then isocratic to 120 min. Fractions were collected for static scintillation counting.
Construction of a ⌬stt4 Strain in BY4742 Background-Genomic DNA retrieved from diploid stt4::kanMX4/STT4 cells by standard techniques was transformed into haploid wild-type BY4742 cells, with 1.0 M sorbitol present during transformation and selection. Kanamycin-resistant colonies were selected on geneticin-agar plates, and replacement of STT4 with the kan r marker was confirmed when PCR amplification of genomic DNA from the kanamycin-resistant colonies yielded an ϳ3.0-kb fragment (not shown).
Inositol Polyphosphate Kinase Assays-The kinase activities of GST-Arg82p and GST-Ipk1p, singly and in combination, were assayed by incubating 5 g of each protein for 0 -10 min at 28°C with 25,000 cpm of Measurements of GFP Fluorescence-The dimeric GFP-PLD␦-PH domain construct pTL336 was a gift from T. Levine (36). Changes in plasma membrane and cytosolic GFP intensity in hypo-osmoticallyshocked cells were visualized on a Nikon Eclipse E600 Microscope with an XF100 -3 filter cube (Omega Optical). Images were acquired with an ORCA digital camera (Hamamatsu, Japan) and changes measured using an Intensity Threshold program in Simple PCI (Compix Imaging Systems.

RESULTS
Our experiments used S. cerevisiae that were in exponential growth until shortly before environmental perturbations were imposed. They were cultured for 5-6 generations with medium containing [2-3 H]inositol, to label all inositol-containing cell constituents close to isotopic equilibrium with the intracellular precursor mixture of [ 3 H]inositol and inositol made de novo by the resident inositol 3-phosphate synthase. Under these conditions, rapid changes in labeling of cellular inositol phospholipids and phosphates will reflect approximately equivalent changes in the relative quantities of the molecules present.
Hypo-osmotic Shock Stimulates PtdIns(4,5)P 2 Hydrolysis-We compared the effects of hypo-osmotic stress on the PtdIns (4,5)P 2 complements of wild-type and ⌬plc1 yeast. Suspensions of equilibrium-labeled cells were adapted to high osmolarity, abruptly diluted 4-fold, and after various periods lipids were extracted, deacylated, and the resulting glycerophosphoesters separated by anion-exchange HPLC (see "Materials and Methods"). Fig. 1A shows typical chromatograms from wild-type and ⌬plc1 cells that were hypo-osmotically stressed for 2 min, and Fig. 1B records the amounts of phosphoinositides in the control and unstressed cells.
The PtdIns(4,5)P 2 complement of hypo-osmotically stressed wild-type cells started to decrease after ϳ20 s (Fig. 1, C and D), declined by about a half within ϳ2 min (Fig. 1, A-D) and started to return toward the starting value over the following 5-10 min (Fig. 1, C and D). This response was relatively straininvariant (Fig. 1D).
Expression of Plc1p from a multicopy plasmid in a ⌬plc1 strain restored [ 3 H]PtdIns(4,5)P 2 breakdown in response to hypo-osmotic stress (not shown). When multicopy overexpression of Plc1p was achieved in wild-type cells, it caused no substantial modification either of basal or stimulated [ 3 H]PtdIns(4,5)P 2 metabolism (see, for example, Fig 2C). Fig. 1 employed an extraction procedure that achieves close to quantitative [ 3 H]PtdIns(4,5)P 2 recovery (35). In most subsequent experiments, we also needed to recover water-soluble phosphates in a state suitable for anion-exchange HPLC analysis. For this, we used a method employing less aggressive acid chloroformmethanol extraction followed by phase partition (see "Materials and Methods"): this method retrieved inositol polyphosphates efficiently but failed to extract about one-third of [ 3 H]PtdIns(4,5)P 2 (not shown). We have not corrected for this under-extraction, so from Fig. 2 onwards the total [ 3 H]PtdIns(4,5)P 2 complements of the extracted cells would have been about 50% greater than the figures reported.

InsP 6 Accumulation Accompanies Stress-induced PtdIns(4,5)-P 2 Depletion-The experiments shown in
When aqueous phases from unstressed [ 3 H]inositol-labeled yeast were analyzed, there were substantial labeled peaks coincident with InsP 6 (the largest peak, Fig. 2A) and with multiple InsP and InsP 2 isomers (not show), and a small peak of PP-InsP 5 eluted after InsP 6 (at ϳ113 min: Fig. 2A). InsP and InsP 2 changed little during stress and were not studied in detail. Very little radioactivity eluted in parts of the chromatogram where the various isomers of InsP 3 , InsP 4 , and InsP 5 emerge ( Fig. 2A). This region of the chromatogram was devoid of clear-cut peaks in most experiments, but small but distinct peaks were seen in others, whether because of better HPLC or slight variations in metabolism we do not know.
To confirm the identity of the putative InsP 6 , an aqueous extract from [ 14 C]inositol-labeled and stressed cells was cochromatographed with authentic [ 3 H]InsP 6 in a second HPLC system (see "Materials and Methods"). The constancy of the 14 C: 3 H ratio across the InsP 6 peak confirmed its identity (Fig. 2B).
When wild-type cells were hypo-osmotically stressed for 2 min or more, the most striking change in inositol polyphosphate complement was an approximate doubling of the already substantial InsP 6 concentration (Fig. 2, A and C). ⌬plc1 cells, unstressed or following hypo-osmotic stress, behaved differently: they were devoid of inositol polyphosphates with three or more phosphate groups (Fig. 2A).
The information in Fig. 2, D and E, reinforces these deductions. First, the concurrent time courses of PtdIns(4,5)P 2 depletion and InsP 6 accumulation (Fig. 2D) suggest a direct relationship between PtdIns(4,5)P 2 loss and InsP 6 synthesis.
We determined which inositol polyphosphates accumulate during hypo-osmotic stress in mutants lacking Arg82p, Ipk1p or Kcs1p, with the results in Fig. 3 and Table II. Hypo-osmotic stress provoked an essentially normal loss of PtdIns(4,5)P 2 in all of these mutants (Table II).
⌬arg82 cells contained a small peak of Ins(1,4,5)P 3 (Fig. 3B, inset; note the expanded scale) but the main feature of the inositol phosphate complement in these cells was the presence FIG. 2. InsP 6 accumulates rapidly in hypo-osmotically shocked S. cerevisiae. A, the inositol polyphosphate complements of unstressed [ 3 H]inositol-labeled wild-type (BY4742) and ⌬plc1 (BY4742 plc1::kanMX4) are compared with cells that were hypo-osmotically shocked for 2 min. B, authentic 3 H-labeled InsP 6 (solid line) precisely co-migrated during anion-exchange HPLC with the 14 C-labeled InsP 6 formed in [ 14 C]inositollabeled yeast during 2 min of hypo-osmotic shock (for details, see "Materials and Methods"). C, inositol polyphosphates other than InsP 6 changed little during a 2-min hypo-osmotic shock. D, reciprocal decrease in PtdIns(4,5)P 2 and increase in InsP 6 when an hypo-osmotic shock was applied to cells that overexpressed Plc1p (BY4742 plc1::kanMX4 ϩ pUG36 -PLC1): wild-type cells behaved similarly. E, the phosphoinositidase C inhibitor U73122 inhibited both PtdIns(4,5)P 2 depletion and InsP 6 accumulation during hypo-osmotic stress. The bar at the left represents unstressed cells and that at the right cells that were hypo-osmotically stressed (2 min) in the absence of U73122. For each panel, the results are representative of 2-4 experiments that yielded similar results.

Comparison of the inositol polyphosphate complements of control and hypo-osmotically stressed wild-type yeast with the inositol phosphate complements of similarly treated cells lacking the various inositol polyphosphate kinases
For each strain, the information is representative of that gathered from 2-4 experiments. Where no figure is recorded, the quantity of 3 H detected in the relevant compound was near or below the detection limit for the experiment.  of greatly increased amounts of multiple isomers of InsP 2 , notably Ins(1,4)P 2 , which is likely to be a direct dephosphorylation product of Ins(1,4,5)P 3 (44,45) (Fig. 3B). The ⌬arg82 cells were devoid of other inositol phosphates with 3 or more phosphate groups (Fig. 3B and Table II). The Ins(1,4,5)P 3 peak became larger during hypo-osmotic stress, but none of the Plc1p-generated Ins(1,4,5)P 3 was converted to more highly phosphorylated products. Instead, much of the PtdIns(4,5)P 2derived radioactivity accumulated as the Ins(1,4,5)P 3 metabolite Ins(1,4)P 2 (Table II and Fig. 3B).
⌬ipk1 cells also accumulated no InsP 6 , but they did show major accumulations of InsP 5 and its pyrophosphorylated derivative PP-InsP 4 , together with small amounts of InsP 4 isomers. All of these were more abundant after hypo-osmotic shock ( Fig. 3C and Table II).
In most respects, ⌬kcs1 cells behaved like wild-type cells (Table II). However, they had higher basal and stimulated InsP 6 complements and, as expected, they made no PP-InsP 5 . They accumulated more InsP 5 and PP-InsP 4 than wild-type cells, both with and without hypo-osmotic stress ( Table II).
Stimulated PtdIns(4,5)P 2 Hydrolysis Occurs at the Plasma Membrane-To determine where PtdIns(4,5)P 2 hydrolysis occurs in hypo-osmotically stressed cells, we expressed a dimeric GFP-PH domain construct based on the PtdIns(4,5)P 2 -selective PH domain of PIC␦1 in the cells (36). Much of this construct localized around the cell periphery, and there were no obvious concentrations of fluorescence on any intracellular organelles (Fig. 6, inset). This suggests that much of the PtdIns(4,5)P 2 in S. cerevisiae is at the inner face of the plasma membrane.
We then analyzed, in parallel, the time-courses of three hypo-osmotically induced events in the cells expressing GFP-PIC␦1: changes in plasma membrane fluorescence, PtdIns(4,5)P 2 depletion and InsP 6 accumulation (Fig. 4). [PtdIns(4,5)P 2 ] declined to a nadir of ϳ60% of the starting value at 2 min, before gradually rising again. As before, the relationship between PtdIns(4,5)P 2 depletion and InsP 6 accumulation was approximately reciprocal (Fig. 4). In the experiment shown, [PtdIns(4,5)P 2 ] rose briefly after imposition of the hypo-osmotic stress, maybe as a secondary effect of the early increase in its precursor PtdIns4P (see above).
Plasma membrane GFP fluorescence tracked the PtdIns(4,5)P 2 changes remarkably closely (Fig. 4), indicating that much of the Plc1p-accessible PtdIns(4,5)P 2 was at the plasma membrane. There was no detectable labeling with GFP-PIC␦1-PH of other cellular organelles whose membranes will contain an unknown proportion of cell PtdIns(4,5)P 2 (e.g. nucleus, Golgi), so we have no indication whether Plc1p-catalyzed PtdIns(4,5)P 2 occurred at any of those sites.
Ca 2ϩ Entry Is Not the Immediate Activator of Plc1p-There is evidence from animal cells that hypo-osmotic shock sometimes triggers Ca 2ϩ entry, and that in such situations the consequent rise in [Ca 2ϩ ] i may sometimes activate PIC␦ (for references, see the Introduction). We therefore determined whether changing the availability of extracellular Ca 2ϩ would influence the S. cerevisiae response to hypo-osmotic stress.
Salt-acclimatized cells were held briefly in a medium that had been depleted of Ca 2ϩ and to which EGTA and BAPTA were added (see "Materials and Methods"). Extracellular Ca 2ϩ was then reintroduced, or not, and simultaneously the cells were hypo-osmotically stressed.
Hypo-osmotic shock caused rapid PtdIns(4,5)P 2 hydrolysis and InsP 6 synthesis both in cells with a normal extracellular Ca 2ϩ supply and in Ca 2ϩ -deprived cells (Fig. 5), indicating that the underlying Plc1p activation cannot require any substantial rise in cytosolic [Ca 2ϩ ] that arises from Ca 2ϩ influx. All phosphoinositidases C rely on submicromolar concentrations of Ca 2ϩ for activity (1,3), so it must be assumed that yeast maintain intracellular [Ca 2ϩ ] at a sufficient level to support this stimulated activity even when they are Ca 2ϩ -deprived.
This experiment also gave an intriguing, and unexplained, result. Readmission of Ca 2ϩ to Ca 2ϩ -deprived cells provoked a larger accumulation of an Ins(1,4,5)P 3 -like molecule than we saw under any other condition, and hypo-osmotic stress appeared to drive the conversion of this Ins(1,4,5)P 3 to InsP 6 (Fig. 5).
pik1-63 cells at raised temperature and ⌬lsb6 cells both maintained substantial concentrations of PtdIns(4,5)P 2 (Table  III). By contrast, the PtdIns(4,5)P 2 complement of unstressed ⌬stt4 cells was one-fifth to one-tenth that of wild-type cells. Moreover, there was no discernable fluorescence at the plasma membrane when the GFP-PLC␦1-PH construct was used to localize PtdIns(4,5)P 2 in living ⌬stt4 cells (not shown). Hypo-osmotic stress provoked normal PtdIns(4,5)P 2 hydrolysis and InsP 6 accumulation in the ⌬lsb6 cells and in the temperature-sensitive pik1-63 cells at their non-permissive temperature (Table III). However, it provoked no change in the already low PtdIns(4,5)P 2 complement of ⌬stt4 cells, and no additional InsP 6 accumulated in those cells during iso-osmotic stress (Table III).
These results suggest that Stt4p makes most of the PtdIns(4,5)P 2 in S. cerevisiae, and that knocking out STT4 eliminates the synthesis of PtdIns(4,5)P 2 at the plasma membrane. It also seems likely that that Stt4p makes all of the PtdIns(4,5)P 2 that is susceptible to hydrolysis by hypo-osmotically activated Plc1p. DISCUSSION There is still little understanding of the biological function(s) of PIC␦s or of how they are controlled in vivo (2,3). However, mouse sperm need PIC␦4 to initiate the acrosome reaction and achieve efficient fertilization (56), and spore germination is aberrant in Dictyostelium that lack this organism's only PIC␦ (57). Moreover, PLC␦1 accumulates abnormally in the neurofibrillary tangles of Alzheimer's disease (58,59) and in brain subjected to hyperoxic stress (60) or aluminum toxicity (61), and Alzheimer's patients and spontaneously hypertensive rats harbor unusual PLC␦1 alleles (62)(63)(64).
There has been a common hope that PIC␦s might transduce  receptor signals, and studies of mammalian PIC␦s have suggested some possible activating G-proteins (2). Two G proteins regulate Dictyostelium PIC␦ (65). In particular, a receptor for Conditioned Medium Factor liberates G ␤ ␥ from association with G ␣1 and stimulates PIC (66). However, an alternative model has implicated elevated intracellular [Ca 2ϩ ] as a possible PIC␦ activator, maybe as a result of Ca 2ϩ entry through capacitative channels (67). Plc1p, yeast's only PIC, is PIC␦-like and has been genetically implicated in numerous cell functions (see the Introduction). The traditional view of PICs is that their primary function is to make the second messengers sn-1,2-diacylglycerol and Ins(1,4,5)P 3 . However, recent genetic and biochemical studies have suggested that Ins(1,4,5)P 3 formed by Plc1p-catalyzed PtdIns(4,5)P 2 hydrolysis in yeast is converted to InsP 6 and related inositol polyphosphates, and some of these have previously unsuspected functions in the nucleus (Refs. 17-20, 22, 68, and see the Introduction). Our results confirm the earlier observation that S. cerevisiae must contain Plc1p if they are to make InsP 6 (68).
Previous work has not provided clear information on how Plc1p is supplied with PtdIns(4,5)P 2 or how its activity is regulated. The experiments reported in this paper newly establish or reinforce several important features of the Plc1p pathway. First, Stt4p and Mss4p convert PtdIns to the Plc1psensitive PtdIns(4,5)P 2 . Second, hypo-osmotic stress rapidly activates Plc1p-catalyzed PtdIns(4,5)P 2 hydrolysis. Third, much of the stress-induced PtdIns(4,5)P 2 hydrolysis occurs at the plasma membrane. Fourth, the burst of Ins(1,4,5)P 3 that is liberated following Plc1p activation is immediately converted to InsP 6 by Arg82p and Ipk1p. Finally, Kcs1p pyrophosphorylates some of this InsP 6 to PP-InsP 5 . Not only do these results confirm the previously reported roles of Arg-82, Ipk1p, and Kcs1p in converting Plc1p-derived Ins(1,4,5)P 3 to InsP 6 and its pyrophosphorylated derivatives, but they also demonstrate that the normal cell complement of these enzymes is capable of keeping pace with the explosive production of Ins(1,4,5)P 3 that is triggered by hypo-osmotic challenge.
S. cerevisiae has three PtdIns 4-kinases: Pik1p (49, 51), Stt4p (50, 52, 69 -71), and Lsb6p (54,72). Our results suggest that Stt4p makes most of a yeast cell's PtdIns4P and, in particular, that this includes all of the PtdIns4P that is precursor to the plasma membrane PtdIns(4,5)P 2 hydrolyzed by stress-activated Plc1p (Table III). This tallies with a recent demonstration that much of cellular Stt4p is at the plasma membrane (71). Earlier genetic studies ascribed Stt4p a function upstream of Mss4p (50) and offered evidence that Stt4p, Mss4p, and Plc1p all lie on a single pathway (73). Our study vindicates these genetic deductions.
Our conclusion that Stt4p makes the bulk of yeast PtdIns4P is in apparent conflict with a previous study that assigned approximately equal roles in PtdIns4P (and PtdIns(4,5)P 2 ) synthesis to Stt4p and Pik1p (52). How are these studies to be reconciled? Readily, since the previous study used a brief period of pulse-chase [ 3 H]inositol labeling to label yeast phospholipids. Although this is a convenient technique for labeling cells it does not label lipid pools to close to equilibrium with added inositol, which means that it cannot validly be used to determine the relative rates of synthesis of one product by multiple enzymes in vivo.
It has long been apparent that much of the PtdIns(4,5)P 2 in animal cells is at the plasma membrane (74): the best available estimate puts that proportion at 60 -70% (75). There is no equivalent information for yeast, but our evidence that most of its Plc1p-sensitive PtdIns(4,5)P 2 is at the plasma membrane tallies with indications from other work. The first pointer was that Mss4p-generated PtdIns(4,5)P 2 is needed for the integrity of the subplasmalemmal cytoskeleton (46). While this manuscript was under consideration, it also became apparent that Mss4p only makes PtdIns(4,5)P 2 efficiently when it is at the plasma membrane (76): Mss4p in the nucleus makes little PtdIns(4,5)P 2 .
Growing and unstressed cells contain about half as much InsP 6 as cells that have been acutely hypo-osmotically stressed, so cells that are receiving no overt stimulus must tonically support a slow but continuous rate of PIC-catalyzed PtdIns(4,5)P 2 hydrolysis. How this slow and sustained Plc1p activity is regulated and where in the cell this basal PtdIns(4,5)P 2 hydrolysis occurs remain to be determined.
The GFP-PIC␦1-PH construct reported that the plasma membrane PtdIns(4,5)P 2 complement never decreased by more than about one-half during hypo-osmotic stress, even when the stressed cells were overexpressing Plc1p. This suggests that cells maintain close control of this pathway even when they contain Plc1p in abundance. It also suggested that some form of feedback control must restrain further stress-stimulated PtdIns(4,5)P 2 hydrolysis after a couple of minutes, at a time when the PtdIns(4,5)P 2 level reaches its nadir and [InsP 6 ] stabilizes at a new, and roughly doubled, plateau concentration.
How does hypo-osmotic stress activate Plc1p sufficiently for about half of the PtdIns(4,5)P 2 in a cell to be hydrolyzed within a couple of minutes? One view is that PIC␦ activation is a simple response to elevation of cytosolic [Ca 2ϩ ] (67). MDCK cells seem to provide the only precedent for translocation of PIC␦ to the plasma membrane and activation in response to hypo-osmotic shock, but this apparently occurs without a need for Ca 2ϩ entry (29), and our results suggest that a rise in cytosolic [Ca 2ϩ ] does not trigger hypo-osmotic Plc1p activation in yeast (see "Results"). How Plc1p is activated remains to be determined. One possibility is that the primary sensor is a still-to-be-identified membrane stretch receptor protein, in which case the key question would be how its activation signal is transmitted onwards to Plc1p. Intriguingly, Plc1p activation seems not to be reversed immediately if the stress is removed. When isotonicity was quickly restored midway through the most rapid phase of hypo-osmotically driven PtdIns(4,5)P 2 hydrolysis, ongoing PtdIns(4,5)P 2 hydrolysis continued normally for at least the next minute or so. 2 Given the remarkable speed of PtdIns(4,5)P 2 hydrolysis and InsP 6 synthesis in the stressed yeast, without any substantial accumulation of intermediates, we wondered where in the cells the responsible enzymes and the inositol polyphosphate products were located. Similar events seem to occur in S. pombe, though in this case in response to hyper-osmotic challenge (41). Direct information on how inositol phosphates are distributed within eukaryotic cells is scant. The clearest data, from HL60 promyeloid cells, place most of the inositol polyphosphates, including InsP 6 and Ins(1,3,4,5,6)P 5 , either in the cytosol or in a pool that is in free and rapid exchange with that compartment (77). Moreover, the PP-InsP 5 that is made from InsP 6 seems to influence vacuolar morphology in the yeast cytoplasm (21). By contrast, much of the recent work on InsP 6 and its close metabolic relatives in yeast has pointed to important actions in the nucleus (17)(18)(19)(20)68).
We attempted to explore this further by comparing the intracellular distributions of biologically functional GFP-Plc1p, GFP-Arg82p, and GFP-Ipk1p constructs with the distribution of an over-expressed nuclear-targeted construct (the nuclear localization signal of SV40 large T antigen coupled to DsRed (78)). The DsRed construct was only in the nucleus, but the over-expressed GFP-Plc1p, GFP-Arg82p, and GFP-Ipk1p were present in both cytoplasm and nucleus, in each case at a higher concentration in the latter (not shown). This leaves the situation unresolved, and an important question for the future will be to determine whether Plc1p and the various inositol phosphate kinases really do carry out multiple functions in more than one cell compartment.
Here we have only discussed a relatively straightforward series of events that have at their center Plc1p activation in cells subjected to osmotic perturbation. We have not addressed how a lack of Plc1p and its products, the inositol polyphosphates discussed here and sn-1,2-diacylglycerol, cause dysregulation of multiple cell functions and thus the many ⌬plc1 phenotypes. Our observations make a method for physiologically controlling the catalytic activity of Plc1p available for the first time, and this should facilitate detailed examination of these other questions. We also have evidence, to be reported elsewhere, that activation of Plc1p-catalyzed PtdIns(4,5)P 2 hydrolysis participates in the responses of S. cerevisiae to high temperature, glucose readmission and nitrogen readmission, in surprisingly complex ways.