Vol. 173, No. 19JOURNAL OF BACTERIOLOGY, OCt. 1991, p. 6124-61310021-9193/91/196124-08$02.00/0Copyright C 1991, American Society for Microbiology

Regulation of Phosphatidylglycerolphosphate Synthase inSaccharomyces cerevisiae by Factors Affecting

Mitochondrial DevelopmentPAULETTE M. GAYNOR, SUSAN HUBBELL, ANDREW J. SCHMIDT, R. ANDREA LINA,

STACEY A. MINSKOFF, AND MIRIAM L. GREENBERG*

Department of Biological Chemistry, University of Michigan Medical School, 1301 Catherine Road,Ann Arbor, Michigan 48109-0606

Received 18 March 1991/Accepted 16 July 1991

Phosphatidylglycerolphosphate synthase (PGPS; CDP-diacylglycerol glycerol 3-phosphate 3-phosphatidyl-transferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found inthe mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinatelyregulated with general phospholipid synthesis and is repressed when cells are grown in the presence of thephospholipid precursor inositol (M. L. Greenberg, S. Hubbell, and C. Lam, Mol. Cell. Biol. 8:47734779,1988). In this study, we examined the regulation of PGPS in growth conditions affecting mitochondrialdevelopment (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affectingmitochondrial function. PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbonsource (glycerol-ethanol), and this derepression was independent of the presence of inositol. PGPS derepressedtwo- to fourfold as cells entered the stationary phase of growth. Stationary-phase derepression occurred in bothglucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol andcholine. PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen. Inmutants lacking mitochondrial DNA ([rhoo] mutants), PGPS activity was 30 to 70% less than in isogenic [rho']strains. PGPS activity in [rhoo] strains was subject to inositol-mediated repression. PGPS activity in [rhoo] cellextracts was derepressed twofold as the [rhoo] cells entered the stationary phase of growth. No growth phasederepression was observed in mitochondrial extracts of the [rhoo] cells. Relative cardiolipin content increasedin glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of thephospholipid precursors inositol and choline. These results demonstrate that (i) PGPS expression is regulatedby factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent ofcross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.

Cardiolipin (CL) is found only in the mitochondrial innermembrane (7, 9, 20) and is necessary for several aspects ofmitochondrial function. In the yeast Saccharomyces cerevi-siae, CL is required for cytochrome oxidase (CO) activity(41, 42) and may be involved in import of proteins into themitochondrion (10, 11). In higher eucaryotes, CL is aneffector of the cytochrome P-450-dependent cholesterol side-chain cleavage enzyme (31) and is required for activities ofCO (34) and the mitochondrial phosphate carrier protein(21). An understanding of the regulation of CL biosynthesiswould therefore provide insight into mitochondrial mem-brane biogenesis as well as the role played by this phospho-lipid in mitochondrial function.The synthesis of CL involves three sequential reactions (7,

28, 37). The enzyme phosphatidylglycerolphosphate synthase(PGPS) catalyzes the committed step in CL synthesis, involv-ing the conversion of the liponucleotide CDP-diglyceride(CDP-DG) and glycerol 3-phosphate to phosphatidylglycerol-phosphate (PGP). PGP phosphatase (PGPase) dephosphory-lates PGP to phosphatidylglycerol, which subsequently isconverted to CL by CL synthase (CLS). In procaryotes, CLis synthesized from two molecules of phosphatidylglycerol,while in higher eucaryotes, the CLS reaction involves thecondensation of phosphatidylglycerol and CDP-DG. Tamaiand Greenberg (39) have recently shown that S. cerevisiae,

* Corresponding author.

like higher eucaryotes, utilizes CDP-DG as a substrate in thesynthesis of CL.We initially postulated that mitochondrial phospholipid

synthesis may be affected by at least two sets of factors: (i)those that affect general phospholipid synthesis and (ii) thosethat affect mitochondrial development. In a previous study(14), our laboratory showed that expression of PGPS in S.cerevisiae is indeed regulated by the water-soluble phospho-lipid precursors inositol and choline. These precursors alsorepress the enzymes of the phosphatidylinositol (PI) andphosphatidylcholine (PC) branches of phospholipid synthe-sis (6). However, inositol repression of PGPS is not medi-ated by the same genetic regulatory circuit that controls thePI and PC branches, since the IN02-IN04-OPIJ regulatorygenes which control synthesis of PI and PC branch enzymesdo not regulate PGPS expression (14).

In this study, we focused on the regulation of PGPSexpression by factors affecting mitochondrial development.An early study by Jakovcic et al. (20) indicated that therelative CL content in yeast mitochondrial membranes de-pends on carbon source, growth stage, and oxygen availabil-ity. Since PGPS catalyzes the committed step in CL synthe-sis, we examined its activity under these conditions. Wedemonstrated that PGPS activity is subject to control byfactors affecting mitochondrial development. In addition, weshowed that unlike mitochondrial respiratory enzymes as-sayed previously (32), PGPS activity is never fully re-pressed, even during anaerobic growth. Our results indicate

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TABLE 1. Strains used in this workStrain Genotype

S. cerevisiaeD273-1OB............ MATat met6 [rho+]D273-1OB [rhoo] ........... MATat met6 [rhoo]Ade5............ MATa adeS [rho+]S288C........... MATa gal2 [rho']

S. carlsbergensisCB1l........... MATa adel [rho+]CB11 [rhoo]........... MATa adel [rhoo]

that PGPS serves as a mitochondrial marker which is inde-pendent of respiratory function and thus correlates withmitochondrion-specific membrane biogenesis.

MATERIALS AND METHODS

Strains. The S. cerevisiae and Saccharomyces carlsber-gensis strains used in this study are described in Table 1.Growth media. Strains were maintained in 15% glycerol at

-80°C for long-term storage and on YEPD (1% yeast ex-tract, 2% peptone, 2% glucose) slants at 4°C for short-termstorage. Synthetic medium consisted of salts (23), vitamins(8), and glucose (2%) or glycerol (3%) plus ethanol (0.95%).This medium is essentially vitamin-free yeast base as de-scribed in the Difco Manual, omitting glucose, histidine,methionine, and tryptophan. Adenine (0.15 mM) and methi-onine (0.002%) were added as required to supplement aux-otrophies. Where indicated, inositol and choline were addedto 75 ,uM and 1 mM, respectively.

In the anaerobic growth experiments, media were supple-mented with Tergitol (5 g/liter), Tween 80 (2.5 ml/liter),ergosterol (20 mg/liter), and antifoam A (0.5 mllliter).

Materials. All chemicals were reagent grade. Yeast ex-tract, peptone, and Bacto agar were obtained from Difco.Horse heart cytochrome c (type VI) and buffer and enzymeassay components were purchased from Sigma ChemicalCo. CDP-DG was obtained from Life Science Resources.32Pi (carrier-free) and [3H]glycerol (40 Ci/mmol) were ob-tained from Dupont, NEN Research Products.

Synthesis of [3H]glycerol 3-phosphate. [3H]glycerol 3-phos-phate was synthesized with [3H]glycerol and ATP by usingglycerol kinase as previously described (15, 16). The reac-tion was 98% complete as determined by chromatography onWhatman no. 1 paper in isopropanol-water-30% ammoniumhydroxide (7:2:1) (33).Growth conditions. Liquid cultures were inoculated from

YEPD slants or plates and incubated overnight. Experimen-tal cultures were inoculated from these overnight culturesand were grown to the indicated growth stage. Overnightcultures were always grown in the same medium as theexperimental cultures. Cultures were incubated at 30°C in arotary shaker at 200 rpm. Cells were harvested by centrifu-gation at 4°C and washed once with buffer 1 (50 mMTris-hydrochloride buffer [pH 7.5], 1 mM EDTA, 300 mMsucrose, 10 mM P-mercaptoethanol) and stored at -80°C.For anaerobic growth conditions, cells were grown under

a continuous stream of deoxygenated argon and then chilledin ice water immediately before harvesting. Purified argonwas deoxygenated by passage through alkaline dithionite (10mg/ml in 0.1 M phosphate buffer, pH 8.0).

Preparation of cell extracts. Cells were suspended in buffer1 at a concentration of 1 g/ml (wet weight) and were broken

open by vortexing with glass beads for five 1-min intervals,with cooling of the cells on ice between intervals. Extractswere centrifuged at 3,000 x g for 5 min, and supernatantswere transferred to 15-ml Corex tubes. The 3,000 x gcentrifugation was repeated two additional times, each timetransferring the supernatants and discarding the pellets.After the third centrifugation, aliquots of the supernatantwere stored at -80°C.

Preparation of mitochondrial extracts. Mitochondria wereisolated by differential centrifugation as previously de-scribed (14). Briefly, cell extracts were prepared as de-scribed above. The supernatants of the third centrifugationwere transferred to small Oak Ridge tubes and centrifuged at27,000 x g for 10 min to obtain the mitochondrial pellet. Themitochondrial pellet was washed twice in buffer 1, sus-pended in buffer 2 (50 mM Tris-hydrochloride buffer [pH7.5], 20% glycerol, 10 mM P-mercaptoethanol) to a concen-tration of 2.5 mg/,u (wet weight) and stored at -80°C.

Assays for protein and enzyme activity. Mitochondrial andcell extracts were assayed for protein concentration by themethod of Bradford (4) with a protein assay kit (Bio-RadLaboratories), using bovine serum albumin as a standard.PGPS activity was assayed at 30°C as previously de-

scribed (14) by the method of Carman and Belunis (5).Briefly, the incorporation of 0.5 mM [3H]glycerol 3-phos-phate (4,000 dpm/nmol for mitochondrial extracts or 40,000dpmlnmol for cell extracts) into chloroform-soluble materialwas measured for 20 min in the presence of 50 mM morpho-lineethanesulfonic acid HCl (pH 7.0), 0.1 mM MnCl2, 0.2mM CDP-DG, 1 mM Triton X-100, and mitochondrial or cellextract (containing 50 or 150 ,ug of protein, respectively) in atotal volume of 0.1 ml. The specific activity of PGPS isdefined as units per milligram of protein, where 1 U is theamount of enzyme that catalyzes the formation of 1 nmol ofproduct per min under the assay conditions described.Cytochrome c oxidase (CO) was assayed spectrophoto-

metrically at 23°C by established procedures (35, 36).Briefly, 0.93 ml of 50 mM potassium phosphate buffer (pH7.1) and 0.07 ml of reduced cytochrome c were added to a1-ml cuvette. Cytochrome c was prepared as a 1% (wt/vol)solution in 50 mM Tris-chloride (pH 8.0) and reduced withsodium dithionite. The reaction was initiated by adding cellor mitochondrial extract (80 ,ug of protein) to the cuvette,and the decrease in A550 was observed against a blank in aBeckman DU-64 spectrophotometer. The blank contained0.93 ml of 50 mM potassium phosphate buffer (pH 7.1) and0.07 ml of potassium ferricyanide-oxidized cytochrome c ina 1-ml cuvette. The concentration of cytochrome c wasdetermined spectrophotometrically by using the extinctioncoefficients of 2.99 x 104, 0.89 x 104, and 2.1 x 104cm2/mmol for reduced, oxidized, and reduced minus oxi-dized cytochrome c, respectively, as determined by Massey(27). The first-order rate constant -kobs was calculated fromthe slope of the curve by using the formula ln (A2- A,/Al-AoI)/t2 - tl. Reaction velocities were calculated from theproduct of the rate constant and the initial concentration ofcytochrome c as described by Smith (36). A unit of COactivity is defined as the amount of enzyme that oxidizes 1,umol of cytochrome c per min. Specific activity is defined asunits per milligram of protein.

Phospholipid composition analysis. Cultures (25 ml) weregrown at 30°C in the indicated medium in the presence of 32p-(50 ,uCi) to steady-state labeling as previously described (1).Cells were harvested by centrifugation at the indicatedgrowth stage and washed with buffer 1. Cells were resus-pended in 1 ml of buffer 1, glass beads were added (ca. 0.5 g),

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TABLE 2. Effect of carbon source on PGPS expression in cell extracts

Growth conditionsa Sp act (U/mg, mean ± SEb)

Carbon Inositol S288C D273-1OB Ade5source (75 ,uM) PGPS CO PGPS CO PGPS CO

Glucose - 0.008 ± 0.001 7 ± 1 0.010 ± 0.003 26 ± 7 0.016 ± 0.003 10 ± 2Glycerol-ethanol - 0.020 ± 0.005 133 ± 53 0.031 ± 0.01 96 ± 27 0.027 ± 0.005 113 ± 14Glucose + 0.005 ± 0.001 6 ± 3 0.010 ± 0.002 35 ± 6 0.008 ± 0.002 7 ± 2Glycerol-ethanol + 0.014 ± 0.002 155 ± 65 0.027 ± 0.008 104 ± 55 0.019 ± 0.003 130 ± 29

a Cells were grown in synthetic medium with glucose or glycerol-ethanol as the sole carbon source in the presence or absence of inositol. Cells were harvestedat the midexponential phase (A550 = 0.4 to 0.5).bn = 3.

and the cell suspension was vortexed for five 1-min intervals.Cell extract was separated from the glass beads by centrif-ugation. An additional 0.5 ml of buffer 1 was added to theglass beads, and the cell extract was again separated andcombined with the first extract. The combined cell extractwas subjected to low-speed centrifugation, and the resultingsupernatant was centrifuged in an Eppendorf centrifuge for20 min to obtain the mitochondrial fraction. The mitochon-drial fraction was resuspended in 0.8 ml of water, andchloroform (1 ml) and methanol (2 ml) were added. Sampleswere vortexed intermittently for 1 h. Phospholipids wereextracted by the method of Bligh and Dyer (3). The chloro-form phase was dried under nitrogen and resuspended in 25,ul of chloroform-methanol (1:1). A portion was removed andused to determine the total chloroform-extractable lipid. Theremaining lipids were spotted on boric acid-ethanol-treatedWhatman SG-81 silica-impregnated paper (13) and separatedby ascending chromatography first in chloroform-methanol-acetic acid (65:25:8) and then in chloroform-methanol-water-ammonium hydroxide (120:75:6:2). Phospholipids were iden-tified by comparison of their migration with that of standardphospholipids, as well as by chromatographic analysis ofproducts of mild alkaline hydrolysis (39). Chromatogramswere autoradiographed, and radioactive spots were cut outand counted by liquid scintillation.

RESULTS

Derepression of PGPS expression during growth in a non-fermentable carbon source. When S. cerevisiae cells aregrown aerobically in medium containing a high concentra-tion of glucose (i.e., >0.1%), catabolite repression reducesthe expression of respiratory enzymes, and the glucose isfermented (12). Perlman and Mahler (32) have shown thatsome respiratory enzymes (constitutive) derepress less thanfivefold, while others (derepressible) derepress more thansixfold during growth on nonfermentable carbon sources.For constitutive enzymes, therefore, derepression is ob-served in cell extracts while the amount of enzyme per unitof mitochondrial mass is relatively constant. For derepress-ible enzymes, the increase would be apparent both in cellextracts and in amount per unit of mitochondrial mass. Todetermine the effect of carbon source on PGPS expression,we assayed PGPS activity in cell extracts and mitochondrialextracts of cells grown in the presence of glucose or glycerol-ethanol as the sole carbon source. We also examined CO asa marker enzyme for respiratory function. We studied threecommonly used wild-type strains. Strain D273-10B is usedextensively in mitochondrial function studies (43); strainAde5 is used in studies of phospholipid biosynthesis (6); andstrain S288C is commonly used in many other more general

studies. Since PGPS expression is repressed during growthin the presence of phospholipid precursors (inositol alone orinositol and choline) (14), we studied the effect of carbonsource on PGPS in both the presence and absence ofinositol.

Interestingly, while the extent of derepression of therespiratory enzyme CO exhibited strain dependence, theextent of PGPS derepression varied little among the strainstested. In cell extracts of all wild-type strains examined,PGPS derepressed 2- to 3-fold during growth in glycerol-ethanol, while CO derepressed 10- to 20-fold in S288C andAde5 but only 3- to 4-fold in D273-1OB (Table 2). The extentof PGPS derepression was independent of the presence ofinositol in the growth medium.The level of derepression of PGPS in cell extracts of

respiring cells suggests that PGPS is a constitutive enzyme,i.e., one that increases in amount per cell but not in amountper unit of mitochondrial mass (32). Consistent with thishypothesis, when we examined PGPS in mitochondrial ex-tracts of respiring cells, we observed no derepression inAde5 and less than twofold derepression in S288C andD273-1OB (data not shown).

Derepression of PGPS during the stationary phase ofgrowth. Mitochondrial development is more prominent inthe stationary phase of growth than in the exponential phase(38). To determine whether PGPS expression is regulated bygrowth stage, we examined the activities of PGPS and CO inmitochondrial extracts as the cells progressed from theexponential to the stationary phase of growth (Fig. 1 and 2).When D273-1OB cells were grown in glucose medium in theabsence of inositol and choline, PGPS derepressed twofoldas cells entered the stationary phase of growth (Fig. 1B). Inthe presence of inositol and choline, the extent of derepres-sion in the stationary phase was threefold, although theoverall activity of PGPS decreased (Fig. 1D). CO alsoderepressed in the presence of glucose as the cells enteredthe stationary phase of growth (Fig. 1B and D) as previouslyshown (32). When D273-1OB cells were grown in glycerol-ethanol medium, PGPS derepressed twofold in the absenceand fourfold in the presence of inositol and choline as thecells entered the stationary phase of growth (Fig. 2B and D).CO remained at its maximally expressed level throughoutthe growth phase in glycerol-ethanol.

Derepression of PGPS in strain Ade5 as the cells enteredthe stationary phase of growth was similar to derepressionobserved in D273-1OB (data not shown).PGPS is not repressed in mitochondria during anaerobic

growth. Mitochondria are present in anaerobically growncells, although the inner mitochondrial membrane is lessdeveloped than in aerobically grown cells (38). To determinethe effect of oxygen on PGPS activity, we grew cells

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FIG. 1. PGPS expression during growth in glucose. S. cerevisiae D273-1OB was grown in glucose synthetic medium in the absence (A andB) or presence (C and D) of inositol (75 p.M) plus choline (1 mM). Cells were harvested at the indicated times, and mitochondrial extracts wereprepared. PGPS (-) and CO (O) activities were assayed as described in the text. Viable cell number (A and C) was determined by serialdilution and plating on YEPD.

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FIG. 2. PGPS expression during growth in glycerol-ethanol. S. cerevisiae D273-10B was grown in glycerol-ethanol medium in the absence(A and B) or presence (C and D) of inositol (75 ,uM) plus choline (1 mM). Cells were harvested at the indicated times, and mitochondrialextracts were prepared. PGPS (-) and CO (O) activities were assayed as described in the text. Viable cell number (A and C) was determinedby serial dilution and plating on YEPD.

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FIG. 3. PGPS activity in [rho'] mutants. The parent [rho']strains (D273-10B and CB11) (-) and isonuclear [rho°] mutants (L)were grown in glucose synthetic medium in the absence (I-) orpresence (1+) of inositol (75 F.M) as indicated. Cells were harvestedat the mid-log phase (A550 = 0.5); mitochondrial extracts wereprepared and assayed as described in the text. Specific activities arebased on the mean + standard error (n = 5), normalized to thespecific activity of PGPS in the parent [rho'] strain grown inmedium without inositol.

aerobically and anaerobically in synthetic medium contain-ing glucose and supplements required for anaerobic growth(sterol and unsaturated fatty acids). CO, repressed in theabsence of oxygen (32), was measured as a control foranaerobic conditions. In D273-1OB cells grown aerobically inglucose, CO activity was measurably greater than that in theother strains tested. Undetectable CO activity thereforeindicated that stringent anaerobic conditions were present.In striking contrast to CO, PGPS expression in mitochon-drial extracts was nearly identical in aerobic and anaerobicconditions. Observed PGPS specific activities were 0.198and 0.190 U/mg for aerobic and anaerobic extracts, respec-tively (standard error was ±0.005, with n = 3). The specificactivity of CO was 140 + 71 U/mg in aerobic extracts. NoCO activity was detected in anaerobic extracts.

Decreased PGPS expression in strains with mutations af-fecting mitochondrial function. Nuclear mutations in genesaffecting mitochondrial function (PET genes) do not lead toabnormal mitochondrial morphology (38). However, in[rhoo] strains, which lack detectable mitochondrial DNA,mitochondrial morphology is aberrant (38). While double-membrane structures are present in [rhoo] mutants, nointernal organization is visible, and these promitochondiahave no respiratory capacity (38). We measured PGPSactivity in mitochondrial extracts from several pet mutantsand found no significant difference in activity between themutants and their isogenic parent strains (data not shown).In contrast, PGPS expression in [rhoo] mutants was less thanthat observed in respective isonuclear [rho'] strains. Figure3 shows PGPS activity in mitochondrial extracts of [rhoo]and [rho+] cells of strain D273-1OB as well as strain CB11 (acommonly used tester strain for complementation of [rhoo]mutations). Results indicate that PGPS in [rhoo] extracts wasonly about 70% of [rho'] PGPS levels.We sought to determine whether the decrease in PGPS

expression in [rhoo] mutants was independent of regulationby cross-pathway control and by growth phase. As seen inFig. 3, PGPS expression was decreased in the [rhoo] mutantsgrown in the presence or absence of inositol. Therefore, the[rho0] effect and the inositol effect are independent. We alsoexamined the effects of [rhoo] mutation on PGPS expressionas a function of growth phase. Figure 4B shows that in cell

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FIG. 4. PGPS activity in [rho'] mutant during growth in glucose.S. cerevisiae strains D273-10B [rho'] (-) and D273-1OB [rho0] (0)were grown in glucose synthetic medium. Cells were harvested atthe indicated times, and cell (B) and mitochondrial (C) extracts wereprepared and assayed as described in the text. Viable cell number(A) was determined by serial dilution and plating on YEPD.

extracts of both D273-10B [rho'] and D273-1OB [rhoo]strains, PGPS activity is derepressed as cells enter thestationary phase, although activity in the [rho°] mutant isless than that observed in the [rho'] parent. Growth phasederepression of PGPS was not observed in mitochondrialextracts of the [rhoo] mutant (Fig. 4C).

Effects of carbon source, growth stage, and inositol andcholine on relative CL content. To determine whether factorswhich influence PGPS expression affect CL content inmitochondrial membranes, we measured the relative CLcontent in D273-10B and Ade5 strains as a function of carbonsource, growth stage, and the presence of inositol andcholine in the growth medium. These results are summarizedin Tables 3 and 4.

Relative CL content was 1.5-fold (D273-10B) to 2.5-fold(Ade5) greater in glycerol-ethanol-grown rells than in glu-cose-grown cells in the exponential phase of growth. In thestationary phase of growth, this difference in relative CLcontent as a function of carbon source was less pronouncedfor both strains. The relative CL content was not increasedin the stationary phase of growth for either strain comparedto the corresponding exponential-growth-phase condition.

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TABLE 3. Effect of carbon source, growth stage, andphospholipid precursors on relative CL content of

Ade5 mitochondrial extracts

Growth conditionsa Relative phospholipid composition'Carbon source and Inositol- PC PE PI PS CL Otherc

growth phase choline

GlucoseExponential 52 19 4 3 4 18Stationary 50 21 9 4 6 10

Glycerol-ethanolExponential 49 20 5 3 10 13Stationary 50 17 8 4 10 11

Glucose +Exponential 47 20 13 3 4 13Stationary 44 27 10 4 6 9

Glycerol-ethanol +Exponential 48 22 6 3 9 12Stationary 48 22 7 3 9 11a Cells were grown in synthetic medium with glucose or glycerol-ethanol as

the sole carbon source in the absence or presence of inositol (75 ,uM) pluscholine (1 mM). Cells were harvested in the exponential (A550 = 0.4 to 0.5) andstationary (24 h after exponential) phases of growth. The data are repre-sentative of a minimum of four independent experiments.

b The relative phospholipid composition is expressed as the 32p; incorpo-rated into a specific phospholipid divided by the 32p; incorporated into totalphospholipid, multiplied by 100. Abbreviations: PE, phosphatidylethanol-amine; PS, phosphatidylserine.

c Pooled percentages of minor phospholipid species.

The presence of inositot and choline in the growth mediumhad no measurable effect on relative CL content.

DISCUSSION

The biosynthetic enzyme PGPS catalyzes the synthesis ofa mitochondrial phospholipid. Therefore, we expected thatthis enzyme might be regulated by factors affecting generalphospholipid synthesis as well as by factors controllingmitochondrial development. In an earlier study, we showed

TABLE 4. Effect of carbon source, growth stage, andphospholipid precursors on relative CL content of

D273-1OB mitochondrial extracts

Growth conditions' Relative phospholipid compositionb

Carbon source and Inositol- PC PE PI PS CL Othercgrowth phase choline

GlucoseExponential 46 25 4 3 7 15Stationary 45 26 9 5 7 8

Glycerol-ethanolExponential 46 24 4 4 11 11Stationary 51 20 8 3 9 9

Glucose +Exponential 44 23 9 3 7 14Stationary 40 28 14 3 7 8

Glycerol-ethanol +Exponential 46 23 10 4 10 7Stationary 47 22 11 3 10 8

a.b,c See Table 3, footnotes a, b, and c.

that PGPS is subject to cross-pathway control by the phos-pholipid precursor inositol, although not via the same regu-latory genes that mediate general phospholipid synthesis(14). In this study, we demonstrated that PGPS is regulatedby factors controlling mitochondrial development. We con-clude the following: (i) carbon source, growth stage, andmutations in the mitochondrial genome affect PGPS expres-sion; (ii) regulation by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fullyrepressed, even during growth in the absence of oxygen.The extent of PGPS derepression observed was two- to

threefold in glycerol-ethanol- versus glucose-grown cells(Table 2) and two- to fourfold in stationary- versus exponen-tial-phase cultures (Fig. 1, 2, and 4). Perlman and Mahler(32) showed that mitochondrial enzymes fall into two classesdistinguishable by the extent of their derepression. Enzymesof the constitutive class increase in amount per cell (up tofivefold) but not in amount per mitochondrial mass. Dere-pressible enzymes increase in amount per unit of mitochon-drial mass (generally by more than sixfold). By these crite-ria, PGPS appears to fall into the first class of enzymes, inwhich increases in amount per cell probably coincide withthe increase in mitochondrial volume (38). While Perlmanand Mahler (32) found that CO derepressed less than sixfold,our experiments indicated that the extent of derepression ofthis enzyme is strain dependent, as seen in Table 2.A previous study by Homann et al. (18) showed that

phospholipid biosynthetic enzymes involved in the de novosynthesis of phospholipids, including CDP-DG synthase,phosphatidylserine synthase, and the phospholipid N-meth-yltransferases are repressed 2.5- to 5-fold as cells enter thestationary phase. In contrast, the enzymes PI kinase andphosphatidate phosphatase derepress as cells enter the sta-tionary phase (17, 19, 29). PGPS appears to be regulated likePI kinase and phosphatidate phosphatase with regard toderepression in the stationary phase, as shown in Fig. 1 and2.

Regulation of PGPS by carbon source, growth stage, andmitochondrial genome is independent of regulation by cross-pathway control. Thus, while PGPS expression in mitochon-drial extracts was reduced during growth in the presence ofinositol, cells were nevertheless able to derepress PGPS asthey entered the stationary phase of growth in both glucose-and glycerol-ethanol-grown cells (Fig. 1 and 2). Similarly,repression of PGPS in [rho'] mutants occurred to the sameextent (30%) in the presence or absence of inositol, althoughPGPS expression in both [rho'] and [rho'] cells was less inmedium containing inositol than in medium lacking inositol(Fig. 3). Therefore, it is likely that different regulatorymechanisms bring about regulation by inositol and regulationby factors affecting mitochondrial development.Why is PGPS activity decreased in [rho'] mutants? Parikh

et al. (3d) hypothesized the existence of a retrograde path ofcommunication from mitochondria to nucleus in yeast cells.This hypothesis is supported by several reports demonstrat-ing altered expression of nuclear genes in [rho'] mutants.Regulation of expression of CITJ and CIT2, genes encodingmitochondrial and peroxisomal forms of yeast citrate syn-thase, is altered in [rho'] cells. CITI expression is 1.6- to13.8-fold lower in [rho'] than in wild-type cells, while CIT2expression is 8- to 11-fold greater in [rho'] mutants (25).Kaisho et al. (22) showed that [rhoo] strains exhibited10-fold-increased transcription of the human lysozyme geneon an expression plasmid under control of the GAL1Opromoter. Similarly, transcription from mitochonrdrial pro-moter plasmids is three- to fourfold more abundant in [rhoo]

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strains than in [rho'] cells (26). These observations suggestthat the mitochondrial genome can influence expression ofnuclear genes, possibly including the gene encoding PGPS.The enzyme PGPS is similar to constitutive respiratory

enzymes in that the amount of enzyme per mitochondrialmass is relatively constant. However, it is strikingly differentfrom these enzymes in that PGPS is never fully repressed,even in mitochondria from anaerobically grown cells. It ispossible that PGPS, unlike respiratory enzymes, is essentialfor cell viability. Since mitochondria are essential to theeucaryotic cell even during anaerobic metabolism (2), it islikely that cells require enzymes for synthesis of mitochon-drial membrane phospholipids.How does regulation of PGPS expression relate to regu-

lation of the second and third CL pathway enzymes, PGPaseand CLS, respectively? While PGPS expression in mito-chondrial extracts is repressed by inositol (14), PGPase andCLS do not appear to be similarly regulated. Neither PGPase(23) nor CLS (39) expression is repressed in mitochondrialextracts during growth in the presence of inositol. In fact,PGPase activity in mitochondrial membranes is actuallyslightly higher in the presence of inositol. However, thespecific activity of PGPase in mitochondria is at least 50-foldhigher than the specific activity of PGPS (23). Thus, cross-pathway control by inositol is exerted primarily at the levelof PGPS expression. With respect to regulation by carbonsource, PGPase and CLS expression do not vary in mito-chondrial extracts of glucose- versus glycerol-ethanol-growncells (23, 40). In cell extracts, specific activities of these twoenzymes could not be definitively ascertained for severalreasons. PGPase activity in crude cell extracts did notappear to vary with carbon source (24). However, it ispossible that phosphatases other than PGPase in the cellextract are capable of dephosphorylating PGP, obscuringaccurate determination of specific activity. CLS specificactivity in cell extracts was too low to detect (40).How is regulation ofPGPS expression reflected in relative

CL content? Because the regulation of phospholipid biosyn-thetic pathways is complex, the control of a particularenzyme is not always reflected in relative composition of thephospholipid product. Homann and coworkers (18) showedthat relative PC and PE composition did not decrease duringthe stationary phase, even though expression of PC pathwayenzymes was reduced. Regulation of the CL pathway issimilarly complex. We showed that the enzyme PGPS isregulated by cross-pathway control (14) as well as by carbonsource (Table 2), growth stage (Fig. 1 and 2), and mitochon-drial genome (Fig. 3 and 4). The degree of regulation ofPGPS expression by these factors is three- to fourfold, whilethe extent of regulation of relative CL composition is two-fold or less (Tables 3 and 4). CL composition in the presentstudy is in agreement with data from an earlier study byJakovcic and coworkers (20) in which relative CL composi-tion varied by no more than twofold as a function of carbonsource and growth stage. While we did not see the sametwofold increase in relative CL composition in the stationaryphase as did Jakovcic and coworkers (20), several factorsmay account for this difference. The Jakovcic study wasdone with aneuploid strains grown in complex media, incontrast to the present study in which we employed haploidstrains grown in synthetic minimal medium. Furthermore, itis difficult to compare the time in the growth cycle in whichstationary-phase cells were harvested for phospholipid anal-ysis in the two studies.The mechanisms by which cross-pathway control, carbon

source, growth stage, and the mitochondrial genome control

PGPS expression and CL composition remain to be eluci-dated. Since PGPS expression correlates well with mito-chondrion-specific development independent of respiratoryfunction, this enzyme is an excellent indicator of mitochon-drial membrane biogenesis. The gene(s) encoding this en-zyme will therefore serve as a useful molecular tool for theanalysis of mitochondrial development. Furthermore, iden-tification of the gene(s) encoding PGPS will permit geneticmanipulation of CL content and in vivo analysis of the roleof CL in membrane function. Thus, the characterization ofPGPS expression and CL synthesis described in this andrelated studies (14, 20, 23, 24, 39, 40) lays the groundworkfor molecular characterization of CL function and mitochon-drial development.

ACKNOWLEDGMENTS

We are grateful to Beth Kelly for critical review of the manu-script, Didi Robins for helpful discussions, John Granger for experttechnical assistance, and Ruby Hogue for preparation of the manu-script.This work was supported by Public Health Service grant GM

37723 from the National Institutes of Health. Paulette M. Gaynorwas supported in part by a Thurnau postdoctoral fellowship. StaceyA. Minskoff was supported in part by Public Health Service traininggrant T32GM07544 from the National Institutes of Health.

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