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Published as doi: 10.1096/fj.06-7898com.
 (The FASEB Journal. 2007;21:1813-1820.)
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Lipid perturbations sensitize osmotic down-shock activated Ca2+ influx, a yeast "deletome" analysis

Stephen H. Loukin*,1, Ching Kung*,{dagger} and Yoshiro Saimi*

* Laboratory of Molecular Biology, and

{dagger} Department of Genetics, University of Wisconsin–Madison, Madison, Wisconsin, USA

1Correspondence: Laboratory of Molecular Biology, University of Wisconsin, Madison, WI 53706, USA. E-mail: shloukin{at}wisc.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Osmotic down shock causes an immediate influx of Ca2+ in yeast, likely through a membrane stretch-sensitive channel. To see how this channel is constituted and regulated, we screened the collection of 4,906 yeast gene deletants for major changes in this response by luminomtery. We discovered deletants that responded very strongly to much milder down shocks than wild-type required, but show little changes in up-shock response. Of all the possibilities (general metabolism, ion distribution, cytoskeleton, cell wall, membrane receptors, etc.), most of the over-responders turned out to be deleted of proteins functioning in the biogenesis of phospholipids, sphingolipids, or ergosterol. Other over-responders are annotated to have vesicular transport defects, traceable to lipid defects in some cases. The deletant lacking the de novo synthesis of phosphatidylcholine, opi3{Delta}, is by far the strongest over-responder. opi3 deletion does not cause non-specific leakage but greatly sensitizes the force-sensing Ca2+-influx mechanism. Choline supplementation normalizes the opi3{Delta} response. Thus, the osmotic-pressure induced stretch force apparently controls channel activities through lipids. This unbiased examination of the yeast genome supports the view that forces intrinsic to the bilayer are determined by the geometry of the lipids and these forces, in turn, govern the activities of proteins embedded therein.—Loukin, S. H, Kung, C., Saimi, Y. Lipid perturbations sensitize osmotic down-shock activated Ca2+ influx, a yeast "deletome" analysis.


Key Words: calcium flux • lipids • membrane mechanics • osmotic force • mechanosensitive channels • yeast deletants


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OSMOTIC STRESS, THE HYDROSTATIC PRESSURE exerted on enclosed biological membranes by water fluxing passively across them in response to solute-induced disequilibrium, is a fundamental challenge to which all life has to face. In the niches of free-living cells such as yeast, this hydrostatic pressure can be both extremely positive, as when cells are rapidly diluted in rainwater, or extremely negative, as when cells are desiccated in the sun or slowly frozen. Besides the obvious existence of its cell wall, yeast has evolved dynamic membrane-physiological responses to hyper- and hypotonic stress (1) . Despite intensive and extensive investigations, however, the physical and molecular principles on how the hydrostatic pressure initiates these responses are still under current debate. Stretching the lipid bilayer opens purified bacterial mechanosensitive channels embedded therein (2) . Key to this mechanism are the biophysical properties of the bilayer, especially the magnitude, distribution, and symmetry of the forces within the bilayer, in which membrane proteins are embedded (3) . Fatty-acid chain length and asymmetric amphipaths govern the activities of bacterial mechanosensitive channels (4) .

The budding yeast Saccharomyces cerevisiae has several documented responses to hypotonic stress, including the rise in outward permeability of the Fps1 glycerol transporter (5) and the activation of the Pkc1 "cell integrity" pathway, in minutes (6) . Osmotic down-shock also causes a transient rise in cytoplasmic Ca2+ by an influx, within seconds (7) . The conduit for this immediate Ca2+ influx is unknown, but it likely detects the changes in the osmotic force directly. An attractive candidate is the 36-picoSiemens plasma-membrane ion channel, which can readily be opened by membrane stretches directly observed under patch clamp (8) but of yet unknown molecular composition.

To find the components and regulators of this channel, we surveyed the down-shock induced Ca2+ response of yeast gene-deletion strains. A collection of 4906 yeast strains, each deleted of a single nonessential gene (9) , is available. This "deletome" has typically been used to study long-term growth under various conditions in mixed cultures (9 , 10) , but is used here to examine an acute response of each deletants individually instead. The 4906 genes, the bulk of the yeast’s ~6000 genes, function in all aspects of the biology. A priori, some of these aspects, such as ion permeations (through channels, pumps, exchangers, and their regulators), cytoskeletal proteins, cell-wall integrity, etc. might be speculated to affect this Ca2+ response to down shock. Unexpectedly, most of the deletants that gave extremely large responses are those defective in lipid metabolism. The findings are surprisingly consistent with the notion that stretch-activated channels, and possibly other membrane proteins, are governed by their surrounding lipids (11) .


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast strains and media
A commercial copy of the yeast deletion library of parental strain BY4742 (MAT{alpha}, his3{Delta}1, leu2{Delta}0, lys2{Delta}0, ura3{Delta}0) (9) was purchased from Open Biosystems (Huntsville, AL, USA). Standard CMD or CMD leu– synthetic media (12) was used for normal growth. For luminometry assays, a sulfate-depleted variant of CMD described in (13) , referred to as "DCD" here, was used. For the experiment shown in Fig. 3C , 1 mM choline chloride was added to the DCD medium to culture opi3{Delta} cells.


Figure 3
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  		Figure 3. Response of wild-type and opi3{Delta} cells to different osmotic down shocks. A) Wild-type (left) or opi3{Delta} (right) cells were pregrown in DCD supplemented with 1.5 M sorbitol to produce stronger down shocks to the test solutions listed in the inset. Note that the wild-type response to the strongest down shock (from DCD+1.5M sorbitol to 10% DCD) is comparable to the saturated response of opi3{Delta} to a much milder shock (from DCD+1.5M sorbitol to DCD+0.2M sorbitol). B) Peak responses from (A), standardized to the peak response resulting from dilution into 10% DCD plotted against the magnitude of the downshock calculated from the difference between DCD + 1.5 M sorbitol and the final dilutions. C) The responses to the standard 10-fold dilution from DCD to buffer of opi3{Delta} cells cultured with or without the supplement of 1 mM choline.

Transformation of deletion library with pEVP11/AEQ (7)
The basic transformation protocol was similar to that by Gietz et al. (14) . Briefly, deletion library of the yeast in microplates was grown in 40 µl of YEPD for 2 d and then subcultured in additional 200 µl medium for 3–4 h. Cells were centrifuged (2000 rpm for 5 min on Beckman-Coulter Avanti J-E), liquid aspirated with Vaccu-Pette/96 (Scienceware, ISC BioExpress101), and cells were resuspended in 150 µl of 100 mM lithium acetate in TE (pH ~8.0). After incubation at 30°C for 30 min, the cells were spun down as above and liquid aspirated. Plasmids (~600 µg per plate) in the transformation liquid were added to cells and incubated at 42°C for ~60 min. Cells were centrifuged (2000 rpm or higher for 5 min) and resuspended in 150 µl of selective medium (DCD –leu+200 µg/ml G4–18) and incubate at 30°C. After one or two days, 5 to 10 µl of cells from the original transformation microplates were transferred into fresh selective medium (~100 µl) for further growth and then frozen away by adding an equal volume of 30% glycerol in the medium.

Detection of aequorin luminescence
For the high-throughput screening of the deletion library, replicants of the 96-well plates containing the pEVP11/AEQ transformed deletion library were grown overnight to early "stationary" phase in DCD-leu. From each well, 5 µl was transferred to 50 µl of DCD-leu containing 2µM coelenterazine (Invitrogen, Carlsbad, CA, USA) and grown for 24 h at 20°C in humidified chambers in the dark. Hypotonic shock was elicited and aequorin luminescence was detected using a Mirthras LB940 microplate luminometer (Berthold, Bad Wildbad, Germany) by injection of 200 µl of 10 mM Na 2-[N morpholino] ethanesulfonic acid (MES), pH 6.6, into 20 µl of cell culture. Luminescence data were acquired and analyzed using MikroWin2000 (Berthold). Those giving small or no response were retested similarly. For individual tests, cells were grown as above except in larger volumes in individual culture tubes in DCD-leu unless otherwise stated. Luminescence was measured using a Sirius luminometer (Berthold) and hypotonic responses were elicited as above unless otherwise stated. 1.5 M sorbitol was added to the coelenterazine cultures for the experiments in Fig. 3A, B . CaCl2, MgCl2, GdCl3, or EGTA were added to the Na MES injected to final concentrations reported in relevant experiments. Up-shock responses were elicited by the injection of 200 µl DCD supplemented with 1.75M sorbitol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The yeast "deletome" contains deletants hypersensitive to osmotic down shock
We used the fluorescence of transgenic aequorin to monitor the internal Ca2+ in each of the 4906 viable clones of the deletion library (9) . Deletants were each transformed with the aequorin-expressing plasmid (7) and individually screened for their Ca2+ responses to osmotic down shock. We found several deletants that responded to large down shocks weakly or with altered kinetics. They include bem2{Delta} (lacking a Rho GTPase activation protein), yke2{Delta} (lacking a part of the Gim complex), and YCR095C (lacking an unknown cytosolic protein). However, none of the deletants were completely devoid of this Ca2+ response, suggesting that the transmembrane gene products responsible are either essential or redundant. In contrast, we encountered several deletion strains that had surprisingly large increases in the transient hypotonic response to a 10-fold dilution of the culture medium with buffer (Fig. 1 A, individual responses of the entire "deletome" are in the Supplemental Material, Table 2). Whereas the collection’s median peak response to hypotonic shock by a 10-fold dilution was 560 RLU (relative luminescence units/sec), 22 deletants gave responses that are each larger than 37,000 RLU. As can be seen in Fig. 1B , the over-responders do not reflect chance deviations from the bulk of the "deletome". The bottom-responding 90% of the "deletome" population has a near-normal distribution of 626 ± 444 RLU (mean±SD) with a mean not far from the median. The over-responding outliers are clearly distinct from that population, having responses greater than 10 SD from the mean of the bottom 90%.


Figure 1
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  		Figure 1. Peak Ca2+ Responses to osmotic down shock of the "Yeast Deletome." A) Peak aequorin responses in RLU/sec of the 4906 deletants in response to a 10-fold culture-to-buffer dilution presented in alphabetical order of the names of the deleted open reading frames. Top 25 responders listed in Table 1 are highlighted with circles. The strongest responder is opi3{Delta}. B) Histogram of all but the top 2% of responders on 100-fold expanded RLU axis as compared to A to illustrate near-normal distribution of great majority of the deletants.

Over-responders to osmotic down shock do not over-respond to up shock
The strong responders from the initial high-throughput screen were retested individually and were found to retain their over-responsive phenotype (Fig. 2 A). The increased Ca2+ response of these mutants is not simply due to an increase in the concentration or efficacy of aequorin. Yeast also responds to osmotic up shock with a Ca2+ pulse, but by a mechanism distinct from down-shock response. Hypertonic challenge causes a Ca2+ release from the vacuole through TrpY1 (Yvc1), the vacuolar mechanosensitive cation channel of the TRP superfamily, (13 , 15 , 16) . Whereas the down-shock responses of the stronger mutants were hundreds of times that of the wild-type, their up-shock responses have the usual kinetics and are similar or nominally smaller in magnitude than the wild-type (Fig. 2B ). Interestingly, the smallest up-shock response comes from vps16{Delta}, a vacuolar mutant with highly compromised vacuolar structure.


Figure 2
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  		Figure 2. Osmotic responses of strong-responding deletants. A) Down-shock responses. Aequorin luminescence of 5 of the top10 over-responding deletants and the parental strain in response to a 10-fold dilution of the DCD medium. B) Up-shock responses. Luminescence of same cultures in response to 10-fold dilution into DCD supplemented with 1.75 M sorbitol. The differences between wild-type and the deletants in the up-shock response (B) are insignificant, compared to the dramatic differences in down-shock responses (A). Note RLU scale difference.

Most top over-responders have defects in lipid metabolism and/or membrane trafficking
The deleted functions of the majority of the strongest responders, as annotated in the databases, are not random. They fall into two categories (Table 1 ). Among the top 22 over-responders, 12 are deleted of genes related to membrane lipid metabolism and 9 to vesicular trafficking. This latter cell-biological phenotype can be traced to defects in lipid biochemistry in 4 cases and to defects in membrane dynamics of yet unclear physico-chemical causes in 5. Five of the top 22 over-responders, as annotated, do not belong to either the lipid or the trafficking category.


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  		Table 1. Peak luminometric responses of strongest hypotonic responders. Peak response to 10-fold dilution of culture medium with buffer is given in RLUs. Wild-type peak RLU is given for comparison. Annotations are from the Saccharomyces Genome Database (http://www.yeastgenome.org/)

The force-to-flux mechanism is sensitized in the top over-responder
The deletants’ strong response can come from a novel pathway, including non-specific leakage or from an existing pathway, albeit sensitized. To distinguish between these two possibilities, we compared the wild-type parent and the phosphatidylcholine (PC)-lacking opi3{Delta} (see Discussion). opi3{Delta}’s response, though variable, is by far the strongest. However, although opi3{Delta}’s response to the screening test (the 10-fold dilution from culture medium) is more than a 1000 times of the wild-type (Table 1 , Fig. 2A ), wild-type cells also give this large response (Fig. 3A , left) if challenged with larger osmotic down shocks achieved by prior growth in an osmotically supplemented medium. Both the wild-type and the opi3{Delta} responses are saturable. The difference between opi3{Delta} (Fig. 3A , right) and wild-type (left) is that opi3{Delta} reacts to milder osmotic shocks with large response and reaches saturation at milder shocks. The over-responders stand out simply because the wild-type response to the screening test is so small relative to the maximum capable. The parallel left shift of the responses to a range of down shocks in the opi3{Delta} (Fig. 3B ) indicates that the opi3 mutation sensitizes an existing machinery rather than creating a new pathway. That machinery, possibly a stretch-activated channel, seems to have a large dynamic range, sensitive to both small and large osmotic changes. Further, that sensitivity seems to be tuned by the surrounding lipids. This seems clear, at least in the case of opi3{Delta}, since cultural addition of choline, which restores PC through the Kennedy salvage pathway while retaining the mutation, resets opi3{Delta}’s response to the wild-type level (Fig. 3C ).

Over-responses are not due to cell lysis or leakage
That time courses of the saturable Ca2+ response are similar (Fig. 3A ), and the responses to the degree of down shocks are parallel in wild-type and opi3{Delta} (Fig. 3B ) indicate that the deletions do not create new pathways. Three additional independent lines of evidence show the increased Ca2+ response in the deletants is not merely due to hypotonically induced leakage or cell lysis, allowing the release of aequorin to combine with external Ca2+. First, we deliberately retested known osmo-labile or osmo-sensitive deletants, even though they were not found to be over-responders in our "deletome" analysis. bck1{Delta} and slg1{Delta}, known to be in the "cell-integrity" pathway, as well as fps1{Delta}, lacking the hypotonically-stimulated glycerol transporter, all had muted responses similar to wild-type in the down-shock (Fig. 4 A, left) as well as the up-shock test (Fig. 4A , right). Second, we note that the initial rate of luminescence increase of opi3{Delta}, observed in seconds, quickened by moderate increase in Ca2+ concentration (Fig. 4B , left, inset). This observation is consistent with the increased external [Ca2+] driving faster entry of Ca2+ into the cytoplasm and is inconsistent with aequorin release, since aequorin should react with Ca2+ within a few milliseconds and the contaminating Ca2+ in the solution will already be much higher than the dissociation constant of aeroquin-Ca2+ (17) . Furthermore, there is no reason to believe that the low 1 mM Ca2+ should encourage leakage or lysis. Note that the rate increase is Ca2+-specific; Mg2+ does not speed up signal (Fig. 4B , left, inset). Chelating Ca2+ had the opposite effect, as expected (Fig. 4B , right). (The chelator’s effects on kinetics differ partially from those in a previous report (7) , likely due to differences in culture stage and handling.) Finally, millimolar Gd3+, a blocker of mechanosensitive channels, blocks the wild-type response (7) , as well as the opi3{Delta} hyper-response (Fig. 4C ). This is inconsistent with lytic release of aequorin, since Gd3+ is known to elicit aequorin luminescence (18) .


Figure 4
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  		Figure 4. Evidence showing the down-shock induced luminescence is due to Ca2+ influx and not aequorin release from leaking or lysing mutants. A) Known osmotically labile deletants do not show large responses. Down-shock (left) and up-shock (right) tests were conducted on bck1{Delta}, slg1{Delta}, and fps1{Delta} as in Fig. 3A . Their responses are indistinguishable from that of the wild-type. B) The effect of Ca2+ on the opi3{Delta} down-shock response. (left) opi3{Delta} cells were down-shocked by a 10-fold dilution from DCD to buffer with or without 1 mM CaCl2 or MgCl2. The peak difference with or without MgCl2 is within experimental error. Inset expands the time axis at point of shock exposure, showing that added CaCl2, but not MgCl2, increases the rate of opi3{Delta} response. (right) Addition of the Ca2+ chelator EDTA reduced and slowed the response. C) Addition of GdCl3 blocks the opi3{Delta} response at 3 but not 1 mM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present examination of the yeast "deletome" was carried out to identify gene products that constitute or regulate the pathway from an acute osmotic down shock to the Ca2+ influx. We examined each of the 4,906 yeast gene deletants without any prior selection. Note also that the major results are from deletants that gave stronger rather than weaker responses, ensuring that the pathway from the stimulus (force) to the response (Ca2+ flux) is intact. A priori, various perturbations can affect the outcome of the response of live cells to an acute stress. Changes in the general energetic state due to metabolic mutations, in ion distribution due to mutations in channels, pumps, or exchangers, in the physical strength due to mutations in cytoskeleton or cell wall, etc. could all in theory affect the Ca2+ signal induced by down shock. A posteriori, the outcome presented here clearly supports the notion that the surrounding lipids govern the force-to-flux mechanism, presumably a stretch-sensitive Ca2+-passing channel. Two theories are current on how stretch forces reach the channel proteins: through the lipid bilayer or through other proteins (matrix or cytoskeletal proteins) (11 , 19 ) (Fig. 5 A). With the caveat of not being able to examine lethal deletants and those with redundant functions, our findings provide support for the former but not the latter in this case, since very few of the over-responders are found to be related to matrix or cytoskeletal proteins.


Figure 5
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  		Figure 5. Speculations on how the osmotic-force-to-Ca2+-influx pathway may detect osmotic force and be affected by lipid composition. The pathway is assumed to be a stretch-sensitive Ca2+-passing channel (double squares). A) The stretch force that opens the channel may be from the lipid bilayer (left) or from other proteins such as the cytoskeleton (right) (19) . That over-responders to the down-shock osmotic force were repeatedly found to have defects in lipids but rarely in cytoskeletal defects (Table 1) favors the former possibility and disfavors the latter one. B) Wild-type membrane composition dictates a set of forces internal to lipid bilayer. In the case of opi3{Delta}, the strongest over-responder, the loss of PC and the compensatory increase of PE with a smaller head (black dots) increase the monolayer curvature stress, and therefore the distribution of the forces within the bilayer (3) , creating an environment that favors channel opening (left). PE tends to be concentrated in the inner leaflet of the bilayer (29) . Unlike the hypothetical even distribution, the asymmetric distribution of phospholipids would also be expected to cause an asymmetry in the internal force profile as well as local bilayer curvature, which may favor channel opening (right).

The biochemical consequences of the deletants are telling. OPI3 encodes a phosphatidyl-N-methylethanolamine N-methyltransferase, which catalyzes the last two steps of de novo synthesis from phosphatidylethanolamine (PE) to phosphatidylcholine (PC), the most abundant membrane lipid. opi3{Delta} produces membranes virtually devoid of PC (20) and has the strongest effect on the down-shock response among all deletants (Table 1) . Cho2 catalyzes the first step of that synthesis. cho2{Delta} also over-responds (67th strongest of the 4906 tested. See Supplemental, Table 2), but to a lesser extent than opi3{Delta}. That cho2{Delta} responds less acutely is consistent with Opi3’s ability to weakly substitute for Cho2 in the first methylation step (21) . Deletions of the other enzymatic steps in the committed synthesis of the other major phospholipids could not be screened in our assay because they are either essential or redundantly encoded. Ino2, missing in the 6th-strongest responder, encodes a transcription factor which, together with Ino4 (the 52nd strongest. See Supplemental, Table 2.), derepresses of the major phospholipid synthetic genes, including OPI3, on depletion of the terminal substrates inositol and choline (22) . Drs2, missing in the 3rd strongest, encodes a flippase (aminophospholipid-translocating ATPase), which mediates the asymmetric distribution of PE, PC, and PS (phosphatidylserine) between the two monolayers (23) . The product missing in the 15th strongest deletant, Cdc50 physically interacts with Drs2 and is thought to regulate its flippase function during polarized cell growth (24) . The 9th (sac1{Delta}) and the 21st strongest (vps34{Delta}) deletant lack the phosphainositide (PI) phosphatase and PI 3-kinase, respectively (25) (26) . Vps15{Delta} (14th strongest) lacks a kinase that is a regulatory subunit, which acts together with Vps34 catalytic subunit of PI 3-kinase (26) .

The discovery that certain glycerol phospholipid perturbations sensitize the machinery that connects osmolarity to Ca2+ influx begs the question of how this can happen. PC, PE, PS, and PI are the major phospholipids of the yeast membrane (20) . It has been proposed that the PE/PC ratio as well as acyl-chain length is homeostatically regulated to maintain optimal membrane curvature (27) . According to the shape-structure concept of lipid polymorphism (28) , PC favors planer bilaminar structure, whereas PE induces negative membrane curvature because the cross-sectional diameter of its headgroup is smaller than that of its acyl chains. Depletion of PC and the compensatory increase of PE in opi3{Delta} (27) would be predicted in such a model to have influence on the forces internal to the bilayer due to monolayer curvature stress (3) (Fig. 5B , left). In addition, the asymmetric distribution of PE to the inner leaflet of the bilayer (29 , 30 ) can create asymmetry in the force profile in the bilayer that favors channel opening (Fig. 5B , right), explaining why mutations in lipid flippase (drs2{Delta}, cdc50{Delta}) can be as effective as mutations in synthetases. The 36-pS mechanosensitive conductance observed under patch clamp, an attractive candidate for the source of the Ca2+ flux, is known to be activated by membrane tension (8) , possibly from the lipid bilayer. MscL, MscS, and other mechnosensitive channels of prokaryotes have been thoroughly investigated by biophysical, biochemical, and crystallographic means (4) . Purified MscL reassembled into lipid bilayers retains mechanosensitivity (2) , so it is now incontrovertible that these are mechanical devises that gauge the internal forces and the lipid bilayer. Though there are clear indications (11 , 31) , how far the insights gained from the prokaryotic channels can be generalized to those of eukaryotes is still under current debate. The results of the present survey seem to have inadvertently supported the application of the force-from-lipid notion to a yeast channel. This notion can be further tested in the yeast system. Preliminary patch-clamp recording in the whole-spheroplast mode showed that the open probability of the wild-type mechanosensitive channels rises with applied pressure in a manner that can be fitted with a Boltzmann distribution. The spheorplast membrane of opi3{Delta}, however, ruptured at very low pressure so that channel activity could not reach the inflection point of a Boltzmann curve. Further experimentation is required to determine whether this apparent membrane fragility is due to the lack of PC.

In light of the notion that channels are controlled by surrounding lipids, it is not surprising that perturbations on lipids besides phosphoglyceral lipids can also be effective. Two of the strongest responders (ERG2, 3) are deleted for enzymes in the synthesis of ergosterol, the yeast analog of mammalian cholesterol that promotes membrane fluidity. PDX3 deletion (4th strongest) results in an 85% decrease in ergosterol content and a 50% increase in the ratio of saturated fatty acids (32) . All of the viable nonredundant mutants in ergosterol-synthesis pathway had down-shock responses within the top 2% of the deletome’s response profile: erg2{Delta}, erg3{Delta}, listed in Table 1 as well as erg5{Delta} (29th strongest responder), erg6{Delta} (34th), erg24{Delta} (42nd), and erg4{Delta} (61st among the 4906, in Table 2 in Supplemental Information).

Also among the strongest responders were deletants of genes encoding Tsc3, which stimulates sphingolipid biosynthesis (33) , as well as Fen2, the membrane transporter of pantothenate (34) , a precursor of acetyl-coenzyme A (CoA), the aliphatic carrier for many lipid and sterol biosynthetic steps. Sphingolipids constitute ~30% of content of the yeast plasma membrane and thus are a bulk membrane lipid. They have been implicated in signaling pathways as well (35) . The two enzymatic steps in the committed synthesis of dihydrosphingosine, carried out by Lcb1,2 and Tsc10, are essential and therefore their deletions could not be included in our screen. Tsc3, however, stimulates the activity of the Lcb1,2 (33) and its deletion causes a marked increase in the down-shock response (20th strongest). Fen1 elongates the acyl chains of sphingolipids from 20 to 24 carbons (36) , and its deletion causes over response (38th). fen2{Delta} (11th) lacks a plasma membrane transporter (34) of pantothenate, the precursor of CoA, which carries acyl chains to all of the lipid and sterol biosynthetic reactions. fen2 deletion is tolerated due to yeast’s ability to synthesize pantothenate de novo, albeit less efficiently, from spermine (37) . Several of the known phenotypes of fen2 mutants can be explained by a decrease in the availability of CoA and its effect on membrane composition, including its Fenproimorph sensitivity, "FEN." In retrospect, it is reasonable that the fen2{Delta} deletant would over-respond simply based on the inhibition of the ergosterol and sphingolipid synthetic pathways alone.

Besides contributing to the bulk, some lipids can be considered signaling lipids. Among the top responders are vps15{Delta} and vps34{Delta}, deleted for the regulatory and catalytic subunits of PI-3 kinase (38) . Curiously, sac1{Delta}, which is deleted for a PI phosphatase capable of catalyzing the reverse reaction in vitro and contains elevated levels of PI-3,5P2 and PI-3P (25) , also is a strong over-responder. How these "signaling lipids" may affect the composition of the bulk lipids and whether they directly affect the embedded channels remain to be determined. Note that a sphingomeyolinase D has recently been found to open a mammalian voltage-gate channel (39) , raising the possibility of sphingolipids being messengers and the likelihood of lipid regulations on channels besides those gated by stretch force.

Members of the second group of strong responding deletants were annotated as being involved with post-Golgi steps in vesicle trafficking (Table 1) . Among them are drs2{Delta}, sac1{Delta}, vps15{Delta}, and vps34{Delta}, which have defined lipid defects listed above. Their gene names reflect the original cell-biological phenotypes of their discovery. Here, we added enhancement of down-shock Ca2+ response to the phenotypic pleiotropism expected of perturbations of major membrane lipids. Strong responders also include vps16{Delta}, pep3{Delta}, and pep5{Delta} lacking components belonging of the HOPS (homotypic-vacuole-fusion and vacuole-protein sorting) 65S complex, which regulates both vesicle-to-vacuole and vacuole-to-vacuole fusion (40 , 41) . vps54{Delta}, the 25th strongest responder, lacks a product, which, together with Vps51, 52, and 53, forms a complex required for retrograde transport from the vacuole to the Golgi. Vps45{Delta} (the 18th) lacks a protein required for the fusion of Golgi-derived vesicles with the prevacuolar compartment. How the HOPS complex, Vsp51-to-54 complex etc. affect and are affected by the compositions and dynamics of the lipid bilayers are unclear. Lipid effects may not be the only cause of over responses to down shock, since over-responders include a few deletants lacking elements outside the two categories we have identified (Table 1) . Whether their effects can be explained by indirect effects on lipid metabolisms remain to be tested.

The work presented here opens new avenues for exploration. Though current literature on signal transduction emphasizes signaling lipids, the roles of bulk lipids are obviously important. The deletants identified here should be further explored to gain insights on how lipids, and therefore the biophysical properties of the bilayer, govern ion channels, vesicle trafficking, and possibly other membrane events. This survey also raises intriguing questions. For example, why does the up-shock sensitive channel, i.e., TRPY1 (Yvc1) in the vacuolar membrane respond differently to the same lipid perturbations from the down-shock channel (Fig. 2B )? Even in model animals such as the mouse, the fly, and the worm, bulk lipids are difficult to manipulate in vivo. Yeast is unique among eukaryotic models that can tolerate large changes in bulk lipids, as evidenced by the viable deletants examined here, and should be further exploited toward a deeper understanding on the relationship between lipids and proteins. The yeast deletion collection is a powerful tool at the cutting edge of contemporary research. It has been used typically to study long-term growth in mixed culture under various conditions (9 , 10 ). Here, we have employed it to examine individual deletants for a physiological response to an acute stimulus. Such an extension of the use of this and other "deletomes" should also be fruitful in the future.


   ACKNOWLEDGMENTS
 
We thank Dr. B. T. Dye for tips on yeast transformation in microplates. This work was supported by the Vilas Trust, University of Wisconsin, NIH GM 47856 to C. K. and NIH GM54867 to Y. S.

Received for publication December 8, 2006. Accepted for publication January 11, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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