Induction of repressible acid phosphatase by unsaturated fatty acid in

Saccharomyces cerevisiae

SYUICHI DOI1'*, MASAYASU WATANABE2, KAZUYUKI TANABE3't, MICHIKO NAKASAKO1 and

MASAO YOSHIMURA1

'Department of Legal Medicine and zDepartment of Bacteriology, Kinki University School of Medicine, Osaka-Say ama, Osaka 5S9, Japan^Department of Medical Zoology, Osaka City University Medical School, Asahi-machi, Abeno-ku, Osaka 545, Japan

* Author for correspondence•(•Present address: Laboratory of Biology, Osaka Institute of Technology, Ohmiya 5-chome, Asahi-ku, Osaka 535, Japan

Summary

We studied the induction of acid phosphatase(APase) by fatty acids in Saccharomyces cerevisiae.S. cerevisiae has two types of APase: constitutiveand repressible enzymes. The synthesis of the latterAPase is normally derepressed by depletion ofinorganic phosphate (P;) in the incubation medium.Of the saturated and unsaturated fatty acids tested,linoleic, linolenic and arachidonic acids inducedthe synthesis of APase even in the presence of a highconcentration of Pi, whereas palmitic, stearic andoleic acids did not. De novo protein synthesis butnot stimulation of secretion of the enzyme wasrequired for the induction. Genetic analyses usingplasmids carrying the genes, PHO5 and PHO3, thatcode for repressible APase and constitutive APase,respectively, showed that linolenic acid induced theformation of repressible APase. Linolenic acid in-

hibited the uptake of exogenous 32P; and simul-taneously lowered the intracellular level of P;.These circumstances indicate that linolenic acid-induced derepression of respressible APase is pri-marily caused by a fall in the intracellular level ofP;. However, cells that had been preincubated in thepresence of a high concentration of P; producedAPase shortly after the addition of linolenic acid. Itis, therefore, suggested that, as well as a normalregulatory mechanism for derepression of repres-sible APase, a mechanism independent of the exter-nal level of P; participates in the induction ofrepressible APase by linolenic acid.

Key words: acid phosphatase, fatty acid, yeast.

Introduction

Fatty acid has an important role in determining theproperties of cellular membranes, such as fluidity. Fur-thermore, there is growing evidence that fatty acidactively participates in regulation of cellular functions:transport systems (Overath et al. 1970; Wilson et al.1970; Linden et al. 1973) and biosynthesis, processingand assembly of membrane proteins and periplasmicproteins (Kimura and Izui, 1976; Ito et al. 1977; Pageset al. 1978; DiRienzo and Inouye, 1979), in E. coliactivation of protein kinase (Mcphail et al. 1981; Nishi-zuka, 1984; Murakami and Rottenberg, 1985) and Ca2+

ionophore-like activity in liposomes (Utsumie/ al. 1985).During the course of our study on the effect of fatty

acid on mating in Saccharomyces cerevisiae, we haveobserved that synthesis of agglutinin responsible forsexual agglutination during mating is induced by someunsaturated fatty acids (Doi, Watanabe, Tanabe, Naka-sako and Yoshimura, unpublished data). This finding has

Journal of Cell Science 94, 511-516 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

led us to try and determine whether fatty acid affects notonly the induction of the agglutinin but other secretoryproteins. We have examined the effect of fatty acids onsynthesis and/or secretion of acid phosphatase (APase,EC.3.1.3.2). The APase is assembled on intracellularmembranes and transported to the cell surface by mem-brane-bounded vesicles by exocytosis (Schekman andNovick, 1982; Schekman, 1985). In addition, the APaseis one of the genetically well-defined enzyme proteins ofS. cerevisiae (Oshima, 1982).

Cells of S. cerevisiae have two species of acid phospha-tase: one is synthesized constitutively in the presence ofphosphate (constitutive APase) and the other is syn-thesized only in the absence of exogenous phosphate(repressible APase) (Oshima, 1982). Constitutive APaseis encoded by the gene, PH03. Repressible APase isencoded by at least three different unlinked genes,PH05, PHOW and PHOll (Bostian et al. 1980; Kramerand Andersen, 1980; Rogers et al. 1982), of which PH05induces a major fraction of the activity (Oshima, 1982).

511

Derepression of repressible APase formation primarilyresults from de novo synthesis of polypeptides, due to theappearance of the corresponding translatable APasemRNAs (Rogers et al. 1982), depending on the concen-tration of exogenous inorganic phosphate (P,). Tait-Kamradt et al. (1986) demonstrated the reciprocal regu-lation of the genes PH05 and PH03. PH03 is expressedonly when PH05 is turned off. Conversely, PH05 isexpressed when transcription of PH03 is blocked. In thepresent study, we examine physiologically and geneticallythe effects of fatty acids on the induction of APase andpresent evidence that some unsaturated fatty acids inducerepressible APase even in the presence of a high concen-tration of exogenous P,.

Materials and methods

StrainsWild-type haploid strains of Saccliammvces cerevisiae, K21-1A(MATa, ATCC42332) and K21-1C (MATa, ATCC42333) werefrom our stocks. Diploid strain, 1A/1C (MATa/MATa), wasconstructed by crossing K21-1A with K21-1C. Haploid strain,GG100-14D (MATahis3 uraJ-52 trpl-1 pho3 pho5 can 1-106)and two strains derived from GG100-14D by transformationwith each of the two plasmids, SB1S-1 (PH03) and pSB24-2(PH05), in each of which a PHO gene was inserted in thevector, YEp24 (Tait-Kamradt et al. 1986), were generous giftsfrom Dr A. G. Tait-Kamradt.

Media and culture conditionYPD medium containing 2% glucose, 1 % peptone and 0.5%yeast extract was used for yeast culture and stocks. Yeast cellswere grown at 25°C with shaking (59-60strokesmin"1). YKmedium was YPD supplemented with KH2PO4 at a finalconcentration of 0.5%, unless otherwise mentioned. Prior toaddition of fatty acids to culture, fatty acid solution in waterdiluted from a stock solution (100 mM in ethanol) was brieflysonicated.

Assay for APaseAPase activity was assayed according to the method describedby Torriani (1960). The reaction mixture (2 ml) for the assayconsisted of 7.0mgml~ />-nitrophenylphosphate and enzymein 0.05 M-acetate buffer (pH4.0). Intact cells, after washingwith the above buffer, were used as a source of enzyme. Afterincubation at 35°C for lOmin, the reaction was stopped byadding the same volume of NaCO3-saturated solution. Afterremoval of cells by centrifugation, the absorbance of thesupernatant at 420 nm (>!42o) w a s determined. To yield thespecific activity, the absorbance of the cell suspension wasmeasured at 530 nm (̂ 530) before starting the incubation.Specific activity (SA) for APase was expressed as the ratioobtained by the equation, SA=/l42o/^s3o.

Uptake of32PO4

A 0.1ml sample of cell suspension (.453o=lO) was inoculatedinto 4.9 ml of YPD medium containing 100,i(M-linolenic acid,and then 50 /.A of stock solution (20 ,uCi) of 32PO4 was added atzero time. The cultures were shaken reciprocally at 50 strokesmin"1 at 25°C. At several intervals, samples were withdrawn,filtered through glass fiber filter (Whatman, GF/C) and thenthe cells were washed with 15 ml of ice-cold water. The filterswere immersed in water in vials and the radioactivity wascounted with a scintillation counter, Packard 460C.

Determination of intracelhdar inorganic phosphateAfter washing, cells were resuspended in 2 ml of 60 % methanolto extract intracellular inorganic phosphate (5 min at roomtemperature) (Huber-Welchli and Wiemken, 1979) and washedwith 1 ml of water. The supernatants were combined todetermine inorganic phosphate, which was determined using a'Phosphor C-Test Wako' kit (Wako Pure Chemicals Co. Ltd,Osaka, Japan), a modified version of the method of Fiske andSubbarow (1925).

ChemicalsFatty acids were obtained from Sigma (St Louis, MO, USA)and 32PO4 from New England Nuclear (Boston, MA, USA).

Sudan Black stainingCells incubated with 200f<M-linolenic acid in YPD medium, asdescribed in the legend for Fig. 1, were harvested, washed twicewith water and fixed with 1.85% formalin for 15 min at roomtemperature. The fixed cells were washed with water and 50%ethanol, successively. Then the cells were stained with SudanBlack saturated in 70% ethanol for 5 min at room temperature.After washing with 70% ethanol, the stained cells wereembedded in 0.5 % agar for microscopic observation.

Results

APase fonnation by fatty acidsProduction of repressible APase is repressible by anaddition of 0.5 % KH2PO4 to YPD medium (Toh-e et al.1973), which results in the cessation of APase synthesis(Bostian et al. 1980). The effects of exogenously addedfatty acids on APase formation under repressed conditionwere examined. Cells of strain 1A/1C from the logarith-mic phase in YPD were incubated in YK medium(YPD + 0.5 % KH2PO4) containing various fatty acids at25°C. At 4h after incubation, APase activity wasmeasured (Fig. 1). In the absence of fatty acid, a lowlevel of APase activity was detectable. This activity maybe attributable to constitutive APase. In the presence ofpalmitic and stearic acids (saturated fatty acids) and ofoleic acid (an unsaturated fatty acid) the enzyme activityremained at a low level at various concentrations of thefatty acids tested. On the other hand, the APase activityappreciably increased by addition of the unsaturated fattyacids, linoleic, linolenic and arachidonic acids. The levelsof APase activity were in order: linolenic, linoleic andarachidonic acid, at any concentration of the fatty acids.APase activity of cells in 200 /iM-linolenic acid was aboutfour times higher than the control level. We thereforeused linolenic acid for subsequent experiments. APaseproduction was significantly enhanced by linolenic acideven when cells were preincubated for 2h in YPDmedium containing 1 % of KH2PO4 to repress com-pletely the synthesis of APase (Fig. 2). Fig. 3 shows thetime course of APase formation by linolenic acid. In theabsence of fatty acid, APase activity was at the constitut-ive level throughout incubation. Addition of 100j-iu-linolenic acid induced APase formation as early as at 1 hand the maximum level of enzyme activity was reached at3h.

In order to measure the uptake of linolenic acid by the

512 S.Doietal.

cells, we examined staining of cells with a dye, SudanBlack, that stains lipids and fatty acid. Cells treated withlinolenic acid (Fig. 4B) were clearly stained with SudanBlack in contrast with the control cells (Fig. 4A). Itappears that the vacuoles are stained. Electron micro-scopic observation shows that vacuoles of cells treated

100

Fatty acid (JIM)

200

Fig. 1. APase formation in the presence of various fattyacids. Cells of strain 1A/1C at logarithmic phase in YPDwere harvested, washed and resuspended in fresh YKmedium (YPD + 0.5% KH2PO4) containing fatty acid, at anabsorbance at 530 nm of 0.2. After 4h of incubation at 25 °Cwith shaking (60 strokes min~ ), cells were harvested, washedand resuspended in 50 mM-acetate buffer (pH4.0) to assayAPase activity. (O) linoleic acid; ( • ) linolenic acid;(A) oleic acid; (A) arachidonic acid; ( • ) stearic acid; and( • ) palmitic acid.

- 2 0 2

Incubation time (h)

Fig. 2. APase formation by linolenic acid after repression byPj. Cells of 1A/1C prepared as in the legend to Fig. 1 wereresuspended in fresh YPD supplemented with 1 % KH2PO4

at/\53o=O.2 and incubated with shaking at 25 °C for 2h. Atzero time, the culture was split, and 200,UM-linolenic acid wasadded to one culture and an equivalent quantity of ethanolwas added to the other as a control. Then the two cultureswere incubated further. At intervals, samples were removedand APase activity was assayed. (O) no linolenic acid;( • ) 100^(M-linolenic acid.

D.

< " 2

Time (h)

Fig. 3. Time course of APase formation in the presence oflinolenic acid. Cells prepared as described in the legend forFig. 1 were incubated in fresh YPD (AS30=0.2) containinglOOjUM-linolenic acid at 25 °C. At several intervals, cells wereremoved and APase activity was measured. (O) No linolenicacid; ( • ) 100jUM-linolenic acid.

with linolenic acid are stained black by OsC>4 (Doi,unpublished data). This result shows that vacuolesaccumulate the fatty acid and/or its derivatives taken upfrom medium. Further, we examined localization of thesecretion of APase after treatment with linolenic acid byenzyme-specific staining (Field and Schekman, 1980)(data not shown). Without linolenic acid cells werefaintly stained and the faint staining was ascribed toconstitutive APase. In contrast, strong staining wasobserved in the bud portion, indicating that the APaseinduced by linolenic acid was mainly secreted in the budportion.

Effect of varying concentration of P, on linolenic acid-induced APase formationThe effect of linolenic acid on APase production at highconcentrations of P, was examined (Fig. 5). The APaselevel was slightly diminished by increasing the concen-tration of P, in both the presence and absence of linolenicacid. Nevertheless, the APase activity was considerablyhigher at a wide range of P, values in the presence oflinolenic acid than that in the absence of the fatty acid,thus indicating that linolenic acid stimulates the forma-tion of APase even at high concentrations of exogenousP-

Requirement of protein synthesis for linolenic acid-induced APase formationSecretion of APase does not require protein synthesis(Novick and Schekman, 1979). Experiments with cyclo-heximide were then carried out to see whether linolenicacid stimulates de novo synthesis of APase or secretion ofthe enzyme already synthesized and stored inside cells(Fig. 6). While cycloheximide did not significantly affectAPase levels during incubation in the absence of linolenicacid, it inhibited formation of APase in the presence oflinolenic acid. Hence, it seems that linolenic acid-inducedAPase formation requires de novo synthesis of the en-zyme protein.

APase formation by fatty acid in yeast 513

•• f

4A B

Fig. 4. Linolenic acid-treated cells stained with Sudan Black. Cells of 1A/1C prepared as described in the legend to Fig. 1 wereresuspended in YPD medium with (B) or without (A) 200,UM-linolenic acid. After 2h incubation at 25°C, cells were fixed andstained with Sudan Black as described in Materials and methods.

6 -

Fig. 5. APase formation in the presence of highconcentrations of Pj. Cells of 1A/1C were prepared asdescribed in the legend to Fig. 1 and resuspended at/l53O=0.2 in fresh YPD containing KH2PO4, with ( • ) orwithout (O) lOOjUM-linolenic acid. The culture was incubatedat 25 °C, with shaking. After 3 h, samples were removed andAPase activity was measured.

Relationship between APase formation and intracellularPi contentSince a high concentration of exogenous P, inhibits thesynthesis of repressible APase but does not affect thesynthesis of constitutive APase, the effect of linolenic acidon the uptake of 32PC>4 by cells was examined. As shownin Fig. 7, linolenic acid clearly suppressed the uptake ofthe label from the medium. Determination of concen-trations of intracellular P, revealed that cells havingincreased APase activity after treatment with linolenicacid had decreased intracellular levels of P, (Fig. 8).

Time (h)

Fig. 6. Inhibition of APase formation by cycloheximide.Cells of 1A/1C were prepared as described in the legend toFig. 1 and resuspended in fresh YPD (/\53o=0.2) containinglinolenic acid with (closed symbols) or without (opensymbols) cycloheximide (20(.ignil"1), and then incubated at25 °C with shaking. At intervals, samples were withdrawn andAPase activity was measured. ( A , A ) 100;/M-linolenic acid;(O,#) no linolenic acid.

These data suggest that linolenic acid inhibits the uptakeof exogenous P,, resulting in a decrease in the intracellu-lar level of P,, and consequently induces the formation ofrepressible APase.

The above suggestion was analysed genetically usingAPase-deficient cells carrying plasmids containing thegenes, PH03 and/or PH05. Constitutive and repressibleAPases are coded by PH03 and PH05, respectively. InGG100-14D cells, which are deficient in APase formation(Tait-Kamradt et al. 1986), APase activity was hardlydetectable, irrespective of the presence of linolenic acid(Table 1). APase activity in cells carrying the plasmid

514 S.Doietal.

gX

Ic1

o'•3

Table 1. APase fonuatioii by linolenic acid intransfonnants with plasmids inserted ivith PHO gene

0 1

Time (h)

Fig. 7. Uptake of 32PO4 in the presence of linolenic acid.Cells were preepared as in the legend to Fig. 1 and incubatedin fresh YPD (AS30=0-2) with ( • ) or without (O) 100 /m-linolenic acid at 25 °C. At intervals, samples were withdrawnand the radioactivity was counted.

0 100 200Linolenic acid (/JM)

Fig. 8. Relationship between intracellular P, and APaseformation. Cells were prepared as described in the legend toFig. 1 and incubated in fresh YPD (A530=0.2) containingvarious concentrations of linolenic acid at 25°C. After 3 h,cells were sampled and subjected to determination ofintracellular contents of P, and APase activity as described inMaterials and methods. (O) APase activity; ( • ) intracellularP, content.

inserted with intact PH05 synthesized APase in thepresence of linolenic acid. These results clearly show thatlinolenic acid induces the gene, PH05, to express repres-sible APase.

Discussion

Induction of repressible APase by linolenic acidThe present study demonstrates that of the saturated andunsaturated fatty acids tested, linoleic, linolenic and

Strain

GG100-14DGG100-14DGG100-14D

(SB15-1)*(pSB24-2)*

Genotype

phoJ pho5PH03PH05

APase

-

0.030.100.27

activity!"

+0.050.132.78

* These strains were transformed with the plasmids inserted withthe PHO gene shown in parenthesis.

f APase activity was measured in the absence ( —) or presence ( + )of 100|<M-linolenic acid and expressed as specific activity (seeMaterials and methods).

arachidonic acids, stimulate the synthesis of APase.APase was induced in the presence of high concentrationof external P[ by those fatty acids that normally repressrepressible APase. Experiments with cycloheximideshowed that enhanced activity of APase by linolenic acidwas due to de novo synthesis of APase, but not to thestimulation of APase secretion. Gordon et al. (1972)reported that an unsaturated fatty acid (such as Tween80) stimulated the synthesis of a mitochondrial enzymeand of cellular proteins in Saccharomyces cerevisiae.The linolenic acid-induced APase synthesis, however,did not result from general stimulation of cellular proteinsynthesis, since the specific activity of APase increasedafter induction by the fatty acid.

Constitutive APase and repressible APase are tran-scribed from the structural genes, PH03 and PIIO5,respectively (Oshima, 1982). Since cells of GG100-14Dcarrying the plasmid inserted with PH05 gene producedAPase in the presence of linolenic acid (Table 1), weconclude that linolenic acid-induced APase formationresults from transcription of the PH05 gene, indicatingthat induced APase is the repressible type.

Mechanism for the induction of repressible APaseLinolenic acid induced repressible APase even at a highconcentration of external P|. Measurement of the uptakeof 32PO4 and determination of the intracellular P, levelafter treatment with linolenic acid suggested that lino-lenic acid inhibited the uptake of P,, resulting in adecrease in the intracellular P,, and consequently inducedrepressible APase. In Saccharomyces cerevisiae there aretwo transport system for P,: one having a high affinity forexternal P, and the other having a low affinity (Tamaiet al. 1985). The activity of the high-affinity P, transportsystem is coordinately regulated with the PH05 gene byexternal P, and requires the function of the PH02 gene(Tamai et al. 1985). The P, binding protein of Saccham-myces cerevisiae is localized at the cell wall (Jeanjeanet al. 1986). The high-affinity P, transport system may beinduced by linolenic acid, because this system is coordi-nately regulated with expression of the PH05 gene.Nevertheless, linolenic acid inhibited the uptake ofexogenous P,. Therefore, it is likely that the inhibition ofP, uptake may result from either interaction betweenlinolenic acid and the P, transport system or inactivationof the transport system by a change in membrane fluidity.

APase formation by fatty acid in yeast 515

Fully repressed cells, which were preincubated for 2h inthe presence of a high concentration of P,, synthesizedAPase shortly after addition of linolenic acid in thecontinuous presence of P,. This finding suggests anothermechanism for the induction of APase under repressedconditions, which is independent of the intracellular levelof Pj. In this context is our unpublished observation thatlinolenic acid induced the synthesis of agglutinin, a cellsurface glycoprotein responsible for sexual agglutinationin S. cerevisiae, even in the absence of a matingpheromone of opposite mating type is relevant (Doi,Watanabe, Tanabe, Nakasako and Yoshimura, unpub-lished data). These circumstances thus suggest the pres-ence of another unknown regulatory mechanism for theproduction of repressible APase in S. cerevisiae, inaddition to that regulated by the external level of P,. Theregulation of production of repressible APase by linolenicacid is being undertaken.

We are grateful to Dr A. G. Tait-Kamradt for providingstrains (GG100-14D) carrying plasmid inserted with PHOgene.

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(Received 20 March 1989 -Accepted, in revised fonn, 12 July 1989)

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