JOURNAL

OF BACTERIOLOGY, OCt. 1991, p. 6124-6131 0021-9193/91/196124-08$02.00/0 Copyright C 1991, American Society for Microbiology

Vol. 173, No. 19

Regulation of Phosphatidylglycerolphosphate Synthase in Saccharomyces cerevisiae by Factors Affecting Mitochondrial Development PAULETTE 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-phosphatidyltransferase; EC 2.7.8.5) catalyzes the first step in the synthesis of cardiolipin, an acidic phospholipid found in the mitochondrial inner membrane. In the yeast Saccharomyces cerevisiae, PGPS expression is coordinately regulated with general phospholipid synthesis and is repressed when cells are grown in the presence of the phospholipid 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 mitochondrial development (carbon source, growth stage, and oxygen availability) and in strains with genetic lesions affecting mitochondrial function. PGPS derepressed two- to threefold when cells were grown in a nonfermentable carbon source (glycerol-ethanol), and this derepression was independent of the presence of inositol. PGPS derepressed two- to fourfold as cells entered the stationary phase of growth. Stationary-phase derepression occurred in both glucose- and glycerol-ethanol-grown cells and was slightly greater in cells grown in the presence of inositol and choline. PGPS expression in mitochondria was not affected when cells were grown in the absence of oxygen. In mutants 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] cell extracts was derepressed twofold as the [rhoo] cells entered the stationary phase of growth. No growth phase derepression was observed in mitochondrial extracts of the [rhoo] cells. Relative cardiolipin content increased in glycerol-ethanol-grown cells but was not affected by growth stage or by growth in the presence of the phospholipid precursors inositol and choline. These results demonstrate that (i) PGPS expression is regulated by factors affecting mitochondrial development; (ii) regulation of PGPS by these factors is independent of cross-pathway control; and (iii) PGPS expression is never fully repressed, even during anaerobic growth.

Cardiolipin (CL) is found only in the mitochondrial inner membrane (7, 9, 20) and is necessary for several aspects of mitochondrial function. In the yeast Saccharomyces cerevisiae, CL is required for cytochrome oxidase (CO) activity (41, 42) and may be involved in import of proteins into the mitochondrion (10, 11). In higher eucaryotes, CL is an effector of the cytochrome P-450-dependent cholesterol sidechain cleavage enzyme (31) and is required for activities of CO (34) and the mitochondrial phosphate carrier protein (21). An understanding of the regulation of CL biosynthesis would therefore provide insight into mitochondrial membrane biogenesis as well as the role played by this phospholipid 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, involving the conversion of the liponucleotide CDP-diglyceride (CDP-DG) and glycerol 3-phosphate to phosphatidylglycerolphosphate (PGP). PGP phosphatase (PGPase) dephosphorylates PGP to phosphatidylglycerol, which subsequently is converted to CL by CL synthase (CLS). In procaryotes, CL is synthesized from two molecules of phosphatidylglycerol, while in higher eucaryotes, the CLS reaction involves the condensation of phosphatidylglycerol and CDP-DG. Tamai and Greenberg (39) have recently shown that S. cerevisiae, *

Corresponding author. 6124

like higher eucaryotes, utilizes CDP-DG as a substrate in the synthesis 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) those that 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 phospholipid precursors inositol and choline. These precursors also repress the enzymes of the phosphatidylinositol (PI) and phosphatidylcholine (PC) branches of phospholipid synthesis (6). However, inositol repression of PGPS is not mediated by the same genetic regulatory circuit that controls the PI and PC branches, since the IN02-IN04-OPIJ regulatory genes which control synthesis of PI and PC branch enzymes do not regulate PGPS expression (14). In this study, we focused on the regulation of PGPS expression by factors affecting mitochondrial development. An early study by Jakovcic et al. (20) indicated that the relative CL content in yeast mitochondrial membranes depends on carbon source, growth stage, and oxygen availability. Since PGPS catalyzes the committed step in CL synthesis, we examined its activity under these conditions. We demonstrated that PGPS activity is subject to control by factors affecting mitochondrial development. In addition, we showed that unlike mitochondrial respiratory enzymes assayed previously (32), PGPS activity is never fully repressed, even during anaerobic growth. Our results indicate

PGPS IN S. CEREVISIAE

VOL. 173, 1991

TABLE 1. Strains used in this work Strain

Genotype

S. cerevisiae D273-1OB ............ D273-1OB [rhoo] ........... Ade5 ............ S288C ...........

MATat met6 [rho+] MATat met6 [rhoo]

MATa adeS [rho+] MATa gal2 [rho']

S. carlsbergensis

CB1l ........... CB11 [rhoo] ...........

MATa adel [rho+] MATa adel [rhoo]

that PGPS serves as a mitochondrial marker which is independent of respiratory function and thus correlates with mitochondrion-specific membrane biogenesis. MATERIALS AND METHODS

Strains. The S. cerevisiae and Saccharomyces carlsbergensis 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 extract, 2% peptone, 2% glucose) slants at 4°C for short-term storage. 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 described in the Difco Manual, omitting glucose, histidine, methionine, and tryptophan. Adenine (0.15 mM) and methionine (0.002%) were added as required to supplement auxotrophies. Where indicated, inositol and choline were added to 75 ,uM and 1 mM, respectively. In the anaerobic growth experiments, media were supplemented 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 extract, peptone, and Bacto agar were obtained from Difco. Horse heart cytochrome c (type VI) and buffer and enzyme assay components were purchased from Sigma Chemical Co. CDP-DG was obtained from Life Science Resources. 32Pi (carrier-free) and [3H]glycerol (40 Ci/mmol) were obtained from Dupont, NEN Research Products. Synthesis of [3H]glycerol 3-phosphate. [3H]glycerol 3-phosphate was synthesized with [3H]glycerol and ATP by using glycerol kinase as previously described (15, 16). The reaction was 98% complete as determined by chromatography on Whatman no. 1 paper in isopropanol-water-30% ammonium hydroxide (7:2:1) (33). Growth conditions. Liquid cultures were inoculated from YEPD slants or plates and incubated overnight. Experimental cultures were inoculated from these overnight cultures and were grown to the indicated growth stage. Overnight cultures were always grown in the same medium as the experimental cultures. Cultures were incubated at 30°C in a rotary shaker at 200 rpm. Cells were harvested by centrifugation at 4°C and washed once with buffer 1 (50 mM Tris-hydrochloride buffer [pH 7.5], 1 mM EDTA, 300 mM sucrose, 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 chilled in ice water immediately before harvesting. Purified argon was deoxygenated by passage through alkaline dithionite (10 mg/ml in 0.1 M phosphate buffer, pH 8.0). Preparation of cell extracts. Cells were suspended in buffer 1 at a concentration of 1 g/ml (wet weight) and were broken

6125

open by vortexing with glass beads for five 1-min intervals, with cooling of the cells on ice between intervals. Extracts were centrifuged at 3,000 x g for 5 min, and supernatants were transferred to 15-ml Corex tubes. The 3,000 x g centrifugation was repeated two additional times, each time transferring the supernatants and discarding the pellets. After the third centrifugation, aliquots of the supernatant were stored at -80°C. Preparation of mitochondrial extracts. Mitochondria were isolated by differential centrifugation as previously described (14). Briefly, cell extracts were prepared as described above. The supernatants of the third centrifugation were transferred to small Oak Ridge tubes and centrifuged at 27,000 x g for 10 min to obtain the mitochondrial pellet. The mitochondrial pellet was washed twice in buffer 1, suspended in buffer 2 (50 mM Tris-hydrochloride buffer [pH 7.5], 20% glycerol, 10 mM P-mercaptoethanol) to a concentration of 2.5 mg/,u (wet weight) and stored at -80°C. Assays for protein and enzyme activity. Mitochondrial and cell extracts were assayed for protein concentration by the method of Bradford (4) with a protein assay kit (Bio-Rad Laboratories), using bovine serum albumin as a standard. PGPS activity was assayed at 30°C as previously described (14) by the method of Carman and Belunis (5). Briefly, the incorporation of 0.5 mM [3H]glycerol 3-phosphate (4,000 dpm/nmol for mitochondrial extracts or 40,000 dpmlnmol for cell extracts) into chloroform-soluble material was measured for 20 min in the presence of 50 mM morpholineethanesulfonic acid HCl (pH 7.0), 0.1 mM MnCl2, 0.2 mM CDP-DG, 1 mM Triton X-100, and mitochondrial or cell extract (containing 50 or 150 ,ug of protein, respectively) in a total volume of 0.1 ml. The specific activity of PGPS is defined as units per milligram of protein, where 1 U is the amount of enzyme that catalyzes the formation of 1 nmol of product per min under the assay conditions described. Cytochrome c oxidase (CO) was assayed spectrophotometrically at 23°C by established procedures (35, 36). Briefly, 0.93 ml of 50 mM potassium phosphate buffer (pH 7.1) and 0.07 ml of reduced cytochrome c were added to a 1-ml cuvette. Cytochrome c was prepared as a 1% (wt/vol) solution in 50 mM Tris-chloride (pH 8.0) and reduced with sodium dithionite. The reaction was initiated by adding cell or mitochondrial extract (80 ,ug of protein) to the cuvette, and the decrease in A550 was observed against a blank in a Beckman DU-64 spectrophotometer. The blank contained 0.93 ml of 50 mM potassium phosphate buffer (pH 7.1) and 0.07 ml of potassium ferricyanide-oxidized cytochrome c in a 1-ml cuvette. The concentration of cytochrome c was determined spectrophotometrically by using the extinction coefficients of 2.99 x 104, 0.89 x 104, and 2.1 x 104 cm2/mmol for reduced, oxidized, and reduced minus oxidized cytochrome c, respectively, as determined by Massey (27). The first-order rate constant -kobs was calculated from the slope of the curve by using the formula ln (A2- A,/Al AoI)/t2 - tl. Reaction velocities were calculated from the product of the rate constant and the initial concentration of cytochrome c as described by Smith (36). A unit of CO activity is defined as the amount of enzyme that oxidizes 1 ,umol of cytochrome c per min. Specific activity is defined as units per milligram of protein. Phospholipid composition analysis. Cultures (25 ml) were grown 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 indicated growth stage and washed with buffer 1. Cells were resuspended in 1 ml of buffer 1, glass beads were added (ca. 0.5 g),

GAYNOR ET AL.

6126

J. BACTERIOL. TABLE 2. Effect of carbon source on PGPS expression in cell extracts Sp act (U/mg, mean ± SEb)

Growth conditionsa Carbon source

Glucose

Glycerol-ethanol Glucose Glycerol-ethanol

S288C

Inositol (75 ,uM)

+ +

PGPS

0.008 0.020 0.005 0.014

± ± ± ±

0.001 0.005 0.001 0.002

Ade5

D273-1OB CO

7 133 6 155

± ± ± ±

PGPS

1 53 3 65

0.010 0.031 0.010 0.027

± ± ± ±

0.003 0.01 0.002 0.008

PGPS

CO

26 96 35 104

± ± ± ±

7 27 6 55

0.016 0.027 0.008 0.019

± ± ± ±

0.003 0.005 0.002 0.003

CO

10 113 7 130

± ± ± ±

2 14 2 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 harvested at 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 centrifugation. An additional 0.5 ml of buffer 1 was added to the glass beads, and the cell extract was again separated and combined with the first extract. The combined cell extract was subjected to low-speed centrifugation, and the resulting supernatant was centrifuged in an Eppendorf centrifuge for 20 min to obtain the mitochondrial fraction. The mitochondrial fraction was resuspended in 0.8 ml of water, and chloroform (1 ml) and methanol (2 ml) were added. Samples were vortexed intermittently for 1 h. Phospholipids were extracted by the method of Bligh and Dyer (3). The chloroform phase was dried under nitrogen and resuspended in 25 ,ul of chloroform-methanol (1:1). A portion was removed and used to determine the total chloroform-extractable lipid. The remaining lipids were spotted on boric acid-ethanol-treated Whatman SG-81 silica-impregnated paper (13) and separated by ascending chromatography first in chloroform-methanolacetic acid (65:25:8) and then in chloroform-methanol-waterammonium hydroxide (120:75:6:2). Phospholipids were identified by comparison of their migration with that of standard phospholipids, as well as by chromatographic analysis of products of mild alkaline hydrolysis (39). Chromatograms were autoradiographed, and radioactive spots were cut out and counted by liquid scintillation. RESULTS

Derepression of PGPS expression during growth in a nonfermentable carbon source. When S. cerevisiae cells are grown aerobically in medium containing a high concentration of glucose (i.e., >0.1%), catabolite repression reduces the expression of respiratory enzymes, and the glucose is fermented (12). Perlman and Mahler (32) have shown that some respiratory enzymes (constitutive) derepress less than fivefold, while others (derepressible) derepress more than sixfold during growth on nonfermentable carbon sources. For constitutive enzymes, therefore, derepression is observed in cell extracts while the amount of enzyme per unit of mitochondrial mass is relatively constant. For derepressible enzymes, the increase would be apparent both in cell extracts and in amount per unit of mitochondrial mass. To determine the effect of carbon source on PGPS expression, we assayed PGPS activity in cell extracts and mitochondrial extracts of cells grown in the presence of glucose or glycerolethanol as the sole carbon source. We also examined CO as a marker enzyme for respiratory function. We studied three commonly used wild-type strains. Strain D273-10B is used extensively in mitochondrial function studies (43); strain Ade5 is used in studies of phospholipid biosynthesis (6); and strain S288C is commonly used in many other more general

studies. Since PGPS expression is repressed during growth in the presence of phospholipid precursors (inositol alone or inositol and choline) (14), we studied the effect of carbon source on PGPS in both the presence and absence of inositol. Interestingly, while the extent of derepression of the respiratory enzyme CO exhibited strain dependence, the extent of PGPS derepression varied little among the strains tested. In cell extracts of all wild-type strains examined, PGPS derepressed 2- to 3-fold during growth in glycerolethanol, while CO derepressed 10- to 20-fold in S288C and Ade5 but only 3- to 4-fold in D273-1OB (Table 2). The extent of PGPS derepression was independent of the presence of inositol 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 amount per unit of mitochondrial mass (32). Consistent with this hypothesis, when we examined PGPS in mitochondrial extracts of respiring cells, we observed no derepression in Ade5 and less than twofold derepression in S288C and D273-1OB (data not shown). Derepression of PGPS during the stationary phase of growth. Mitochondrial development is more prominent in the stationary phase of growth than in the exponential phase (38). To determine whether PGPS expression is regulated by growth stage, we examined the activities of PGPS and CO in mitochondrial extracts as the cells progressed from the exponential to the stationary phase of growth (Fig. 1 and 2). When D273-1OB cells were grown in glucose medium in the absence of inositol and choline, PGPS derepressed twofold as cells entered the stationary phase of growth (Fig. 1B). In the presence of inositol and choline, the extent of derepression in the stationary phase was threefold, although the overall activity of PGPS decreased (Fig. 1D). CO also derepressed in the presence of glucose as the cells entered the stationary phase of growth (Fig. 1B and D) as previously shown (32). When D273-1OB cells were grown in glycerolethanol medium, PGPS derepressed twofold in the absence and fourfold in the presence of inositol and choline as the cells entered the stationary phase of growth (Fig. 2B and D). CO remained at its maximally expressed level throughout the growth phase in glycerol-ethanol. Derepression of PGPS in strain Ade5 as the cells entered the stationary phase of growth was similar to derepression observed in D273-1OB (data not shown). PGPS is not repressed in mitochondria during anaerobic growth. Mitochondria are present in anaerobically grown cells, although the inner mitochondrial membrane is less developed than in aerobically grown cells (38). To determine the effect of oxygen on PGPS activity, we grew cells

PGPS IN S. CEREVISIAE

VOL. 173, 1991 7.6

A

7.5

,

x

7.4

z

Mx

N

7.2

D

6127

7.1

7.0 B

0.4

400

D D

B

~~~~~~~~~~~~~~~~~E

4

300

0.3

0 j 2200*

E0

100

IL

a.0.1

0

0A. 15

20

25

30

35

40

45 15

25

20

Time, hr

30

35

40

45

Time, hr

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

37.5 Z

I9 CD

N

7.4

7.2N 7.1

4~~~~~~~~~~~~~~~~~~~ 7.0

0.6

BD50

0.5

o

40

0.4 1 S 5 0.3

30

35

40

45

so

55 25

30

35

40

4S

S0

5S300

j0

0.2

2 0.1

0.0. 2S

100 30

35

40 45 Time, hr

50

55 25

30

35

40 45 Time, hr

50

55

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 mitochondrial extracts were prepared. PGPS (-) and CO (O) activities were assayed as described in the text. Viable cell number (A and C) was determined by serial dilution and plating on YEPD.

6128

J. BACTERIOL.

GAYNOR ET AL. 1.0

7.6 r A

-

tD a

E E

E

0.8-

7.4

z

i en

c

0.

0.4-

cn

a.

0.20.0

4)

7.2 1

C.)

7.0

a

0.6-

cm

121,

11+ D273-lOB

0

CS J

6.8

6.6 1-

1+

0.015

CB11

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-) or presence (1+) of inositol (75 F.M) as indicated. Cells were harvested at the mid-log phase (A550 = 0.5); mitochondrial extracts were prepared and assayed as described in the text. Specific activities are based on the mean + standard error (n = 5), normalized to the specific activity of PGPS in the parent [rho'] strain grown in medium without inositol.

B

E

0.010 0.

cm n 0.

0.005

Innn-

nn

0.6

aerobically and anaerobically in synthetic medium containing glucose and supplements required for anaerobic growth (sterol and unsaturated fatty acids). CO, repressed in the absence of oxygen (32), was measured as a control for anaerobic conditions. In D273-1OB cells grown aerobically in glucose, CO activity was measurably greater than that in the other strains tested. Undetectable CO activity therefore indicated that stringent anaerobic conditions were present. In striking contrast to CO, PGPS expression in mitochondrial extracts was nearly identical in aerobic and anaerobic conditions. Observed PGPS specific activities were 0.198 and 0.190 U/mg for aerobic and anaerobic extracts, respectively (standard error was ±0.005, with n = 3). The specific activity of CO was 140 + 71 U/mg in aerobic extracts. No CO activity was detected in anaerobic extracts. Decreased PGPS expression in strains with mutations affecting mitochondrial function. Nuclear mutations in genes affecting mitochondrial function (PET genes) do not lead to abnormal mitochondrial morphology (38). However, in [rhoo] strains, which lack detectable mitochondrial DNA, mitochondrial morphology is aberrant (38). While doublemembrane structures are present in [rhoo] mutants, no internal organization is visible, and these promitochondia have no respiratory capacity (38). We measured PGPS activity in mitochondrial extracts from several pet mutants and found no significant difference in activity between the mutants and their isogenic parent strains (data not shown). In contrast, PGPS expression in [rhoo] mutants was less than that observed in respective isonuclear [rho'] strains. Figure 3 shows PGPS activity in mitochondrial extracts of [rhoo] and [rho+] cells of strain D273-1OB as well as strain CB11 (a commonly used tester strain for complementation of [rhoo] mutations). Results indicate that PGPS in [rhoo] extracts was only about 70% of [rho'] PGPS levels. We sought to determine whether the decrease in PGPS expression in [rhoo] mutants was independent of regulation by cross-pathway control and by growth phase. As seen in Fig. 3, PGPS expression was decreased in the [rhoo] mutants grown in the presence or absence of inositol. Therefore, the [rho0] effect and the inositol effect are independent. We also examined the effects of [rhoo] mutation on PGPS expression as a function of growth phase. Figure 4B shows that in cell

C

E° 0

0.5

0.4 "

0.3

en

0.2

0.3

"I-V

0.1

I0

20

30 Tlime, hr

40

50

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 at the indicated times, and cell (B) and mitochondrial (C) extracts were prepared 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 the stationary phase, although activity in the [rho°] mutant is less than that observed in the [rho'] parent. Growth phase derepression of PGPS was not observed in mitochondrial extracts of the [rhoo] mutant (Fig. 4C). Effects of carbon source, growth stage, and inositol and choline on relative CL content. To determine whether factors which influence PGPS expression affect CL content in mitochondrial membranes, we measured the relative CL content in D273-10B and Ade5 strains as a function of carbon source, growth stage, and the presence of inositol and choline in the growth medium. These results are summarized in 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 glucose-grown cells in the exponential phase of growth. In the stationary phase of growth, this difference in relative CL content as a function of carbon source was less pronounced for both strains. The relative CL content was not increased in the stationary phase of growth for either strain compared to the corresponding exponential-growth-phase condition.

VOL. 173, 1991

PGPS IN S. CEREVISIAE

TABLE 3. Effect of carbon source, growth stage, and phospholipid precursors on relative CL content of Ade5 mitochondrial extracts Growth conditionsa Relative phospholipid composition' Carbon source and Inositol- PC PE PI PS CL Otherc choline growth phase Glucose Exponential 4 52 19 3 4 18 50 21 Stationary 9 4 6 10

Glycerol-ethanol Exponential Stationary

Glucose Exponential Stationary

+

Glycerol-ethanol Exponential Stationary

+

49 50

20 17

5 8

3 4

10 10

13 11

47 44

20 27

13 10

3 4

4 6

13 9

48 48

22 22

6 7

3 3

9

12

9

11

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) plus choline (1 mM). Cells were harvested in the exponential (A550 = 0.4 to 0.5) and stationary (24 h after exponential) phases of growth. The data are representative of a minimum of four independent experiments. b The relative phospholipid composition is expressed as the 32p; incorporated into a specific phospholipid divided by the 32p; incorporated into total phospholipid, multiplied by 100. Abbreviations: PE, phosphatidylethanolamine; PS, phosphatidylserine. c Pooled percentages of minor phospholipid species. a

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

DISCUSSION

The biosynthetic enzyme PGPS catalyzes the synthesis of a mitochondrial phospholipid. Therefore, we expected that this enzyme might be regulated by factors affecting general phospholipid synthesis as well as by factors controlling mitochondrial development. In an earlier study, we showed TABLE 4. Effect of carbon source, growth stage, and phospholipid precursors on relative CL content of D273-1OB mitochondrial extracts Growth conditions'

Carbon source and

Inositol-

growth phase Glucose Exponential Stationary

choline

Glycerol-ethanol Exponential Stationary Glucose Exponential Stationary

+

Glycerol-ethanol Exponential

+

Relative phospholipid compositionb PE PI PS CL Otherc

PC

46 45

25 26

4 9

3 5

7 7

15 8

46

24 20

4 8

4 3

11

51

9

11 9

44 40

23 28

9 14

3 3

7 7

14 8

46

23

10 11

4 3

10 10

7 8

47 22 Stationary a.b,c See Table 3, footnotes a, b, and c.

6129

that PGPS is subject to cross-pathway control by the phospholipid precursor inositol, although not via the same regulatory genes that mediate general phospholipid synthesis (14). In this study, we demonstrated that PGPS is regulated by factors controlling mitochondrial development. We conclude the following: (i) carbon source, growth stage, and mutations in the mitochondrial genome affect PGPS expression; (ii) regulation by these factors is independent of crosspathway control; and (iii) PGPS expression is never fully repressed, 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 exponential-phase cultures (Fig. 1, 2, and 4). Perlman and Mahler (32) showed that mitochondrial enzymes fall into two classes distinguishable by the extent of their derepression. Enzymes of the constitutive class increase in amount per cell (up to fivefold) but not in amount per mitochondrial mass. Derepressible enzymes increase in amount per unit of mitochondrial mass (generally by more than sixfold). By these criteria, PGPS appears to fall into the first class of enzymes, in which increases in amount per cell probably coincide with the increase in mitochondrial volume (38). While Perlman and Mahler (32) found that CO derepressed less than sixfold, our experiments indicated that the extent of derepression of this 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 novo synthesis of phospholipids, including CDP-DG synthase, phosphatidylserine synthase, and the phospholipid N-methyltransferases are repressed 2.5- to 5-fold as cells enter the stationary phase. In contrast, the enzymes PI kinase and phosphatidate phosphatase derepress as cells enter the stationary phase (17, 19, 29). PGPS appears to be regulated like PI kinase and phosphatidate phosphatase with regard to derepression in the stationary phase, as shown in Fig. 1 and 2. Regulation of PGPS by carbon source, growth stage, and mitochondrial genome is independent of regulation by crosspathway control. Thus, while PGPS expression in mitochondrial extracts was reduced during growth in the presence of inositol, cells were nevertheless able to derepress PGPS as they entered the stationary phase of growth in both glucoseand glycerol-ethanol-grown cells (Fig. 1 and 2). Similarly, repression of PGPS in [rho'] mutants occurred to the same extent (30%) in the presence or absence of inositol, although PGPS expression in both [rho'] and [rho'] cells was less in medium containing inositol than in medium lacking inositol (Fig. 3). Therefore, it is likely that different regulatory mechanisms bring about regulation by inositol and regulation by factors affecting mitochondrial development. Why is PGPS activity decreased in [rho'] mutants? Parikh et al. (3d) hypothesized the existence of a retrograde path of communication from mitochondria to nucleus in yeast cells. This hypothesis is supported by several reports demonstrating altered expression of nuclear genes in [rho'] mutants. Regulation of expression of CITJ and CIT2, genes encoding mitochondrial and peroxisomal forms of yeast citrate synthase, is altered in [rho'] cells. CITI expression is 1.6- to 13.8-fold lower in [rho'] than in wild-type cells, while CIT2 expression is 8- to 11-fold greater in [rho'] mutants (25). Kaisho et al. (22) showed that [rhoo] strains exhibited 10-fold-increased transcription of the human lysozyme gene on an expression plasmid under control of the GAL1O promoter. Similarly, transcription from mitochonrdrial promoter plasmids is three- to fourfold more abundant in [rhoo]

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strains than in [rho'] cells (26). These observations suggest that the mitochondrial genome can influence expression of nuclear genes, possibly including the gene encoding PGPS. The enzyme PGPS is similar to constitutive respiratory enzymes in that the amount of enzyme per mitochondrial mass is relatively constant. However, it is strikingly different from these enzymes in that PGPS is never fully repressed, even in mitochondria from anaerobically grown cells. It is possible that PGPS, unlike respiratory enzymes, is essential for cell viability. Since mitochondria are essential to the eucaryotic cell even during anaerobic metabolism (2), it is likely that cells require enzymes for synthesis of mitochondrial membrane phospholipids. How does regulation of PGPS expression relate to regulation of the second and third CL pathway enzymes, PGPase and CLS, respectively? While PGPS expression in mitochondrial extracts is repressed by inositol (14), PGPase and CLS do not appear to be similarly regulated. Neither PGPase (23) nor CLS (39) expression is repressed in mitochondrial extracts during growth in the presence of inositol. In fact, PGPase activity in mitochondrial membranes is actually slightly higher in the presence of inositol. However, the specific activity of PGPase in mitochondria is at least 50-fold higher than the specific activity of PGPS (23). Thus, crosspathway control by inositol is exerted primarily at the level of PGPS expression. With respect to regulation by carbon source, PGPase and CLS expression do not vary in mitochondrial extracts of glucose- versus glycerol-ethanol-grown cells (23, 40). In cell extracts, specific activities of these two enzymes could not be definitively ascertained for several reasons. PGPase activity in crude cell extracts did not appear to vary with carbon source (24). However, it is possible that phosphatases other than PGPase in the cell extract are capable of dephosphorylating PGP, obscuring accurate determination of specific activity. CLS specific activity in cell extracts was too low to detect (40). How is regulation of PGPS expression reflected in relative CL content? Because the regulation of phospholipid biosynthetic pathways is complex, the control of a particular enzyme is not always reflected in relative composition of the phospholipid product. Homann and coworkers (18) showed that relative PC and PE composition did not decrease during the stationary phase, even though expression of PC pathway enzymes was reduced. Regulation of the CL pathway is similarly complex. We showed that the enzyme PGPS is regulated by cross-pathway control (14) as well as by carbon source (Table 2), growth stage (Fig. 1 and 2), and mitochondrial genome (Fig. 3 and 4). The degree of regulation of PGPS expression by these factors is three- to fourfold, while the extent of regulation of relative CL composition is twofold or less (Tables 3 and 4). CL composition in the present study is in agreement with data from an earlier study by Jakovcic and coworkers (20) in which relative CL composition varied by no more than twofold as a function of carbon source and growth stage. While we did not see the same twofold increase in relative CL composition in the stationary phase as did Jakovcic and coworkers (20), several factors may account for this difference. The Jakovcic study was done with aneuploid strains grown in complex media, in contrast to the present study in which we employed haploid strains grown in synthetic minimal medium. Furthermore, it is difficult to compare the time in the growth cycle in which stationary-phase cells were harvested for phospholipid analysis in the two studies. The mechanisms by which cross-pathway control, carbon source, growth stage, and the mitochondrial genome control

J. BACTERIOL.

PGPS expression and CL composition remain to be elucidated. Since PGPS expression correlates well with mitochondrion-specific development independent of respiratory function, this enzyme is an excellent indicator of mitochondrial membrane biogenesis. The gene(s) encoding this enzyme will therefore serve as a useful molecular tool for the analysis of mitochondrial development. Furthermore, identification of the gene(s) encoding PGPS will permit genetic manipulation of CL content and in vivo analysis of the role of CL in membrane function. Thus, the characterization of PGPS expression and CL synthesis described in this and related studies (14, 20, 23, 24, 39, 40) lays the groundwork for molecular characterization of CL function and mitochondrial development. ACKNOWLEDGMENTS

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

37723 from the National Institutes of Health. Paulette M. Gaynor

was supported in part by a Thurnau postdoctoral fellowship. Stacey A. Minskoff was supported in part by Public Health Service training

grant T32GM07544 from the National Institutes of Health.

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