ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
169,
577-590
Biogenesis The Effects
of Altered
MARZUKI,
Department
of Mitochondria
Steady-State
Mitochondrial-Energy SANGKOT
(197%
Membrane
Metabolism GARY
of Biochemistry,
l Lipid Composition
in Saccharomyces
S. COBON, J. M. HASLAM, LINNANE Monash Received
University, February
Clayton,
Victoria,
on
Cerevkiae AND
ANTHONY
W.
3168, Australia
6, 1975
A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cereuisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression. The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated. The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%. In cells which contained lipids with 22-28s unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the leveis of cytochromes aa, and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.
One approach our laboratory has adopted to study mitochondrial membrane structure-function relationships has involved the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae grown in glucose batch cultures. It has been reported that a decrease in the mitochondrial membrane unsaturated fatty acid content below the wild-type level of 75% resulted in a decrease in the efficiency of oxidative phosphorylation. Oxidative phosphorylation is completely uncoupled ’ This paper Mitochondria.”
is No. 38 in the series, “Biogenesis No. 37 is Molloy et al., 1975 (27).
in isolated mitochondria with unsaturated fatty acids representing 1528% of the total (1, 2). At this low level of unsaturated fatty acids, mitochondrial cytochrome levels and respiration appeared to be normal (3) although mitochondria isolated from such cells contained a greatly reduced ability to establish and maintain K+ and H+ gradients (4). Only cells in which unsaturated fatty acid content was depleted to less than 11% of the total fatty acid content by growth of the mutant to stationary phase in a glucose batch culture showed a 40% reduction in the whole cell respiratory activity and some decrease in the whole
of 577
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
578
MARZUKI
cell cytochrome content (3). Interpretation of these results was complicated by several factors. First, cells grown on glucose to a low unsaturated fatty acid level are catabolite repressed so that the observed reductions in respiratory activity and cytochrome levels may be a consequence of either low unsaturated fatty acid content or of catabolite repression (3). Second, the cells extensively depleted of unsaturated fatty acids were in a stationary phase of growth so that the observed decrease in respiratory activity may have been due to the culture conditions rather than a consequence of a change in the molecular organization of the mitochondrial membrane. Finally, in batch cultures, it is not possible to determine with any reasonable degree of precision the unsaturated fatty acid level at which oxidative phosphorylation becomes uncoupled and other functional changes occur. In the present communication, a technique of cell culture is described which overcomes these difficulties. Cells were grown in a chemostat using glucose-limiting media in such a way that the steadystate glucose level could be maintained below that which induces catabolite repression. Furthermore, cultures were maintained for a number of generations at chosen cellular unsaturated fatty acid levels so that the comparison of cells containing high and low cellular unsaturated fatty acid levels was facilitated. Differences observed were a direct result of the variation in the cellular unsaturated fatty acid content. We found that even small changes in unsaturated fatty acid content below wildtype cell levels resulted in a decrease in the molar growth yield, which is an in vivo determination of the efficiency of oxidative phosphorylation. At a cellular unsaturated fatty acid content of about 45%, the molar growth yield approached that of a respiratory-deficient mutant, indicating that, at this level of unsaturated fatty acid content, mitochondrial oxidative phosphorylation was about 90% uncoupled. The mitochondrial cytochrome levels and whole cell respiration were normal even down to 34% unsaturated fatty acid content but when the steady-state cellular unsaturated fatty
ET AL.
acid content was further decreased to 22-28%, the levels of mitochondrial cytochromes aa3, b, and cc, were also reduced to less than 10% of the normal levels. Cyanide-sensitive respiration approached zero. An investigation into the nature of the changes in the mitochondrial proteinsynthesizing system accompanying unsaturated fatty acid depletion is the topic of the accompanying paper (5) in which it is reported that unsaturated fatty acid-depleted cells do not form functional mitochondrial ribosomes. MATERIALS
AND
METHODS
Strain of Yeast Saccharomyces cereuisiae KD115 (a ole~ p+), an unsaturated fatty acid auxotroph, was obtained from Dr. Resnick (6). A spontaneous revertant, strain KD115-2, was isolated from a culture of KD115 grown in medium lacking unsaturated fatty acids. The petite mutant of KD115 (o( ale- p”) used in this study was selected from an unsaturated fatty acid-depleted culture of KD115 as described previously (7).
Reagents Tween-80 was obtained from H. B. Selby (Nottinghill, Victoria, Australia). The fatty acid composition of the batch of Tween-80 used was 81% oleic acid, 6% palmitoleic acid, 2% myristoleic acid, and 11% saturated fatty acids. Yeast extract was obtained from Difco Laboratories (Detroit MI), ATP, peroxidase (EC 1.11.1.7). glucose oxidase (EC 1.1.3.4), and hexokinase (EC 2.7.1.1) were purchased from the Sigma Chemical Company (St. Louis, MO), and alcohol dehydrogenase (EC 1.1.1.1) from Calbiochem Australia Pty. Ltd. (Sydney, N.S.W. Australia). [32P]Orthophosphate (carrier-free) was purchased from the Australian Atomic Energy Commission, Lucas Heights N.S.W. All other materials used were of reagent grade.
Culturing Procedure Yeast cells were grown in chemostat cultures (8) at 28°C by the following procedure, except when otherwise specified. Cells were grown in 7.5-liter capacity fermenters (New Brunswick) with a working volume of 4 liters. The medium contained a salts mixture as described previously (2) and yeast extract, 10 g/liter. Glucose was autoclaved separately as 75% stock solutions and added to the medium to give a final concentration of 10 g/liter. The medium was supplemented with various concentrations of Tween-80 as a source of unsdurated fatty acids. The inoculum for the 4-liter cultures was cells
YEAST
MITOCHONDRIAL
grown to exponential phase in batch cultures supplemented with an excess of Tween-80 (4000 &ml) and added to the continuous culture to give an initial cell concentration of approximately 0.1 mg cell dry weight/ml. The flow rate of fresh glucose medium into the culture was 400 ml/h (dilution rate D = 0.1 h-l). The cultures were grown under forced aeration (5-8 liters aidmin) and stirred at 380 rpm. Under these conditions, the steady-state cell density of the OLE revertant strain KD115-2 increased linearly with the glucose concentration in the input medium up to a glucose concentration of at least 10 g/liter, indicating that glucose was the growth-limiting factor.
Revertant and Petite Cells in Cultures The reversion rate of KD115 is relatively high and the revertant levels in the cultures were, therefore, routinely determined by plating samples of cultures on solid medium containing a salts mixture (2), yeast extract 2 g/liter, glucose 10 g/liter, and agar 20 g/liter, The frequency of OLE revertants in the cultures was minimized by ensuring a low level of revertants in the inoculum, which was achieved through frequent subcloning of the stock culture. Only those chemostat cultures containing a final level of less than 0.1% OLE revertants were used in the biochemical studies. The frequency of respiratory incompetent (petite) cells was determined as described previously (7).
Preparation of Mitochondria Mitochondria were prepared from glusulasedigested cells as described by Cobon et al. (9). Owing to the fragility of the mitochondria containing low levels of unsaturated fatty acid, these organelles were isolated in 0.9 M sorbitol medium rather than 0.6 M as described previously (9).
Analytical
Methods
The following published analytical procedures were used: cell-density determination by measurement of packed cell volume (3); polarographic measurement of whole-cell respiratory activity (3); enzymatic determination of glucose (10) and ethanol (11) concentrations; and P:O ratios in isolated mitochondria (12). The cytochrome spectra of the whole cells and isolated mitochondria were measured at room temperature or at the temperature of liquid nitrogen as described by Clarke-Walker and Linnane
(13).
Unsaturated Fatty Acid Analyses Whole-cell and mitochondrial samples were saponified in 7 M KOH for 3 h at 100°C and nonsaponifiable material was then extracted into ether. The aqueous fraction was acidified and the fatty acids were extracted into ether which was subsequently
ENERGY
579
METABOLISM
dried over anhydrous sodium removed by evaporation and were methylated in a solution by heating at 60°C for 1 h. extracted into hexane and chromatography in an F and 810 or a Pye Unicam Model
sulfate. The ether was the recovered fatty acids of 14% BF, in methanol The methyl esters were analyzed by gas-liquid M chromatograph Model R.
Phospholipid Analyses Chemostat cultures with 350-ml working volumes were maintained at the desired unsaturated fatty acid level for three to four generations after reaching steady state in the presence of 1.25 PCi [3ZP]orthophosphate/ml of input media. Cells were harvested, washed, and a whole-cell sample was disrupted by passage through a French pressure cell at 1.8 x lo5 k pascals. The remainder of the cells were used for the preparation of mitochondria. The lipid components were extracted from the whole cell and the mitochondrial samples and separated by two-dimensional tic on silica gel as previously described (9). Areas of gel containing lipids were scraped into scintillation vials and the radioactivity determined in a Philips liquid scintillation analyzer.
Calculation of Molar Growth Yields and Phosphorylation Efficiency in Vivo The molar growth yield for glucose cells formed per mole of substrate calculated as follows: Y,l”
= cws,
Yglu (g dry wt of consumed) was
- S,)
where x is the cell density in the steady state (mg cell dry wt/ml), S, is the molar glucose concentration in the inflowingmedium, and S, is the remaining molar glucose concentration in the culture medium. It was found that when the cells were grown in the presence of radioactively labeled glucose, the cells incorporated into the cell mass about 10% of the amount of glucose utilized (unpublished observation). Consequently, the Y,,, calculated was corrected for the proportion of glucose incorporated and thus not available for energy production. The Y,,” was also corrected for the energy used for the maintenance process as described by Pirt
(14). The molar growth yield for ATP, Y,.r, (g dry wt of cells formed per mole of ATP) was calculated as 0.5 x Y *,” of a respiratory incompetent mutant, assuming the following stoichiometry: Glucose
+ 2 ADP
+ 2 P, = 2 ethanol
+ 2 CO,
+ 2 ATP
The phosphorylation efficiency under other conditions was calculated using this determined value for YATP and the experimentally determined Y,,” (15).
580
MARZUKI
ET
AL.
functions. In agreement with these workers, a shift to a fermentative metabolism Mitochondrial samples were prepared for electron was observed as the dilution rate was microscopy as described by Watson et al. (16). increased above 0.2 h-’ (corresponding to RESULTS the maximum specific growth rate of the revertant in ethanol). This change is indiGrowth Characteristics of KDl15 Related cated by a decrease in the cell density to Strains in Glucose-Limited Chemostat about 1.5 mg cell dry wt/ml and the Cultures accumulation of ethanol in the culture To determine the growth conditions medium. The actual glucose concentration under which metabolism was purely fer- in the culture remained below 0.5 mM until mentative and those under which both the dilution rate was about 0.3 h-’ and fermentative and oxidative processes then increased sharply as the critical diluwould occur, the effect of dilution rate on tion rate of about 0.35 h-’ was approached. culture parameters of the OLE revertant For comparison, a respiratory incompewas investigated. Figure la shows the tent strain, which had no mitochondrial growth yield of the OLE revertant plotted DNA, KD115 Po, was used to establish the as a function of the specific growth rate p culture parameters when the metabolism which, in the steady state, equals the was purely fermentative (Fig. lb). This dilution rate D. With a 10 g/liter initial petite mutant grown in an excess of unsatglucose concentration (55.5 mM), the urated fatty acid at a dilution rate of 0.1 steady-state cell density was constant at h-’ attained a steady-state cell density of about 5.5 mg cell dry wt/ml when the about 1.2 mg cell dry wt/ml which was dilution rate was between 0.025 and 0.2 constant over the whole range of dilution h-‘. Over this range of dilution rate, the rate below the critical dilution rate of 0.3 metabolism of the cells was purely oxida- h-‘. tive as shown by the low level of ethanol accumulated in the culture medium. The Manipulation of Membrane Lipid slight decrease in the cell density as the Composition of Strain KD115 in dilution rate was lowered has been attribContinuous Culture uted by Fiechter and von Meyenburg (17) The steady-state cellular unsaturated to an increase in the proportion of the energy required for maintenance of cellular fatty acid content could be varied from the Electron Microscopy
GLUCOSE
CELL e-.-e
i D,U~T,ON RATE D = SPECIFIC
JO
DENSITY -*--
0.1 GROWTH RATE p
7 :
20
:,
10
5 I
r
:
0.2
0.3
t
(hr-‘)
1. The effect of dilution rate on continuous culture parameters of KD115related strains. Cultures of the OLE revertant KD115-2 (Fig. la) in the absence of unsaturated fatty acid supplement or of the respiratory-deficient KD115 p0 (Fig. lb) in the presence of 4000 pg Tween80/ml of input media were grown at different dilution rates in chemostat cultures. The growth-limiting substrate in the inflowing media was glucose (10 g/liter) in both cases. At each dilution rate, after a steady state had been established, samples were taken at intervals over a period of at least 46 h. Cell density, e-0, ethanol, A-A, and glucose O--O concentrations were determined on each sample. Each point represents the average of at least three determinations. FIG.
YEAST
MITOCHONDRIAL
ENERGY
wild-type level of 75% to as low as 15% by altering the level of Tween-80 in the input medium. For example, at input levels of 4000 and 1000 pg of Tween-80 per ml of medium, the steady-state cellular unsaturated fatty acid contents were 75% and 50% of the total fatty acids, respectively (Table I). As in batch cultures (2), an increase in the level of short-chain fatty acids was observed as the unsaturated fatty acid content was lowered (Table I). There was no significant difference between the mitochondrial and the total cellular fatty acid compositions (data not shown). However, there were differences in the phospholipid compositions of whole cells and mitochondria. These differences varied with unsaturated fatty acid composition (Table II). Cells with a low unsaturated fatty acid content were lower in diphosphatidyl glycerol and phosphatidyl serine and higher in phosphatidic acid than cells grown with a high unsaturated fatty acid content. These phospholipid differences were much more pronounced in the mitochondria from such cells where for mitochondria with high and low unsaturated fatty acid contents, respectively, the percentages of diphosphatidyl glycerol were 15.0 and 2.6, phosphatidyl ethanolamine 23.4 and 14.9, phosphatidyl inositol 10.8 and 16.4, and phosphatidic acid 2.0 and 9.8. In all cases, mitochondria were richer in diphosphatidyl glycerol and phosMANIPULATION
Tween-80
OF CELLULAR
4000 2000
1000 500 350 200
100 KD 115-2
phatidyl ethanolamine than the whole cells, a result similar to those previously reported for yeast (9, 18) and rat liver (19) mitochondria. The Nature of Substrate Limitation at Low Levels of Unsaturated Fatty Acid Supplementation At a dilution rate of 0.1 h-l KD115 chemostat cultures supplemented with 4000 pg of Tween-BO/ml were glucose limited; the steady-state concentration of glucose in the culture was about 0.3 mM (Table III). At such glucose levels the cells were not subject to catabolite repression, as the cells were capable of growth by oxidative metabolism as indicated by negligible accumulation of ethanol in the medium and the high cell yield (Fig. la). Since many mitochondrial functions are sensitive to catabolite repression, it was critical to demonstrate that when the level of unsaturated fatty acid supplement was lowered, the unsaturated fatty acid did not become growth limiting, with a consequential increase in the concentration of glucose in the culture and concomitant catabolite repression. Analysis of the medium showed that the remaining glucose concentration was less than 0.5 mM under all conditions even though the residual unsaturated fatty acid concentration in the medium decreased from about 1400 pg/ml to 38 pg/ml as the level of Tween-80 in the input
TABLE I UNSATURATED FATTY ACID LEVELS OF STRAIN KD115 CHEMOSTAT CULTUREP
(fig/ml)
Weight c,:,
581
METABOLISM
14:o
1.7 * 0.2 4.0 * 1.0 5.6 i 0.2 13.1 * 2.8 15.0 * 1.2 12.9 + 0.6 12.0 * 1.3 6.0 z+ 0.3
C 16 0 - LB 0 * 34.9 * 43.5 i 41.7 * 49.9 * 63.0 zt 79.1 * 20.1 * 22.3
1.2
1.0 2.3 2.2 3.0 2.1
1.9 1.2
% of total
fatty
acids Cl,:,
Cl,:, 7.9 * 0.5 5.9 * 0.1 4.0 * 0.2 5.1 * 0.2 3.6 * 0.3 1.6 i- 0.5 1.5 * 0.2 36.0 zt 2.0
IN GLUCOSE-LIMITED
* 1.7 53.1 * 0.2 45.5 * 2.1 38.3 + 2.8 30.0 * 2.1 20.3 ,z 1.8 10.5 * 0.1 36.9 ze 1.8
66.5
Total
UFA
75.2 * i* 44.2 i 34.1 zt 22.0 i 12.8 + 73.3 *
60.3 50.2
1.4 0.1 3.5 3.0 2.3 1.4
1.3 1.2
n KD115 was grown at a dilution rate of 0.1 h-’ to a steady state in chemostat cultures supplemented with the indicated levels of Tween-80 in the input media. Samples were taken at various times after the steady state was reached. The cells were harvested, water washed, and the unsaturated fatty acid composition was determined by gas-liquid chromatography as described in Methods. Results are expressed as molar R of total fatty acids * standard deviations. At least three independent samples were taken from each culture,
582
MARZUKI
PHOSPHOLIPID
Unjatu.ated fatty acid content (% total fatty cids) Whole cells Mitochondria Whole cells Mitochondria
75
15
COMPOSITION
Phosphatidyl inositol
ET AL.
TABLE II OF CELLS AND MITOCHONDRIA OF HIGH AND Low UNSATURATED Phospholipid (Mole % total lipid phosphorus) Phosphatidyl serine
Phosphatidic acid
Phosphatidyl ethanolamine
FATTY ACID CONTENT
T Diphosphatidyl glycerol
Phosphatidyl glycerol
Unidentified
4.6 + 0.6
0.8 * 0.1
5.9 i 1.8
* 0.4
1.0 f il.3
3.0 * 1.1
:
: !l.l
* 1.0
11.4 * 1.1
3.4 zt 1.5
10.6 zt 0.7
1.0.8 * 1.2
4.4 * 0.3
2.0 * 0.2
23.4 e 2.1
2!2.6 z+ 0.1
6.1 I 0.7
9.7 f 3.0
11.7 * 0.3
1.5 * 0.1
0.6 zr 0.1
3.7 * 0.3
16.4 i 0.0
6.8 rt 1.2
9.8 * 0.2
14.9 * 0.5
2.6 ze 0.2
0.3 * 0.1
3.9 * 0.2
I.5.0
1
L
o Cells were grown in chemostat cultures containing the appropriate unsaturated fatty acid supplement and 1.25 HCi [32P]orthophosphate/ml as described in Methods. Lipids were extracted from cells and mitochondria and separated by thin-layer chromatography. Radioactivity in each lipid species was determined and is expressed as mole % of total lipid phosphorus i- standard deviation of four determinations.
medium decreased from 4000 pg/ml to 100 pg/ml (Table III). It was concluded that glucose was the growth-limiting substrate at all levels of unsaturated fatty acid supplementation tested. The Effect of Unsaturated Fatty Acid Depletion on Glucose Utilization Two of the most useful parameters in studying the efficiency of energy metabolism in microorganisms are the molar growth yield Y (g dry wt of cells formed per mole of substrate consumed) and the molar growth yield on ATP, YATp (g dry wt of cells formed per mole of ATP). The molar growth yield on a given substrate is a constant for a given organism. If the metabolic pathway is known, YATp can be calculated from Y. On the other hand, since YATP is also a biological constant, if this value for an organism is known or can be calculated from one set of conditions, it may be used to estimate the ATP yield for metabolic processes carried out under another set of conditions (15).
The Yglu of the culture supplemented with excess Tween-80 (cellular unsaturated fatty acid 75%) was calculated from the steady-state cell density at a dilution rate of 0.1 h-’ as described in Methods. At this level of cellular unsaturated fatty acid, the ym was about 123 g/mole, similar to that of the revertant strain (Table IV). A 22% decrease in Y,,, was observed when the cellular unsaturated fatty acid content was decreased to 50%. AS no accumulation of ethanol in the culture was observed, oxidation of ethanol was less efficient in terms of energy yield. A further comparatively small decrease in the unsaturated fatty acid content to 44% resulted in a pronounced decrease in YgIU and substantial amounts of ethanol accumulated in the culture. In this case, not only was the efficiency of oxidative phosphorylation reduced but the ability of the cells to utilize ethanol was also impaired. Progressive decrease in the unsaturated fatty acid content to 15% resulted in a Y,,, of 21.8 and a large accumulation of ethanol. This Y,,, corresponded to the
YEAST TABLE
MITOCHONDRIAL
III
ANALYSIS OF FAW ACID AND GLUCOSE CONCENTRATIONS IN CULTURE SUPERNATANT OF STRAIN KD115 GROWN WITH VARIOUS CONCENTRATIONS OF TWEEN80 SUPPLEMENT IN THE INPUT MEDIAN Tween-80 (r&l)
input
Steady-state culture supernatant Fatty acid (~cg/ml)
4000 2000 1000 500 350 100
1419 213 109 79 57 38
Glucose (mM) 0.29 0.16 0.26 0.34 0.42 0.37
0 KD115 was grown as described in the legend to Table I. Samples were taken from the culture at various times after reaching the steady state, cells were removed by centrifugation, and the fatty acid and glucose concentrations in the supernatants were determined as described in Methods. The results are representative of at least two determinations.
basic fermentation value obtained for the respiratory deficient p” mutant of KD115 when grown under conditions of either high or low unsaturated fatty acid content (Table IV). We have previously reported that growth at low cellular unsaturated fatty acid levels leads to the induction of cytoplasmic petite mutants (7). However, it is clear that the increase in YBIU at low levels of unsaturated fatty acid content is not attributable to the accumulation of petites. At a cellular unsaturated fatty acid level of 34%, for example, the petite frequency of the culture when harvested was 11.5% of the total cell population; the Y,,, expected from such a culture on the basis of a simple mixture of petite and grande cells would be 111 g/mole whereas the observed Yg,,, was only 38.5 g/mole. Calculation of Oxidative Phosphorylation Efficiency in Vitro and in Vivo We have previously reported that the oxidative phosphorylation efficiency of mitochondria isolated from batch-grown yeast cells decreased with the unsaturated fatty acid content of the mitochondria (2). In the present study, mitochondria isolated
ENERGY
583
METABOLISM
from cells grown in the chemostat to various steady-state levels of unsaturated fatty acid were also examined for their oxidative phosphorylation efficiency, and the results are in a very close agreement to the previous observations (Table V). With 75% of the fatty acids unsaturated, the mitochondria had a P:O ratio of 1.15, which was comparable with the level in the revertant KD115-2 (Table V), and was in the normal range for Saccharomyces cerevisiae mitochondria. With the decreasing unsaturated fatty acid level in the mitochondria, there was a concomitant fall in the P:O ratios, which at the 50% unsaturated fatty acid level was about half that of normal mitochondria, and essentially no oxidative phosphorylation could be detected at about the 34% unsaturated fatty acids level (Table V). TABLE
IV
THE EFFECT OF UNSATURATED ON GLUCOSE METABOLISM Organism
KD115
KD 115-2 KD 115 p”
Cellular unsaturated fatty acid (% total fatty acid) 75 60 50 44 34 22 15 73 75 21
FATTY ACID DEPLETION BY STRAIN KD115”
YglU (g cell dry weight/ mole glucose) 122.6 113.2 95.7 43.2 38.5 22.4 21.8 121.9 22.1 19.8
lo Petite cell level of harvested culture
Ethanol (culture supernatant, mM)
0.8 1.2 2.2 2.2 11.5 32 34 1.0 100 100
2 8 4 44 50 71 84 9 103 99
a The fatty acid desaturase mutant KD115 was grown to steady state as described in the legend to Table I to give the indicated cellular unsaturated fatty acid content. The revertant strain KD115-2 was grown to steady state under identical conditions in the absence of Tween-80 supplement. The respiratorydeficient mutant derived from KD115, KDll5 pO, was grown under identical conditions in the presence of either 4000 pg/ml Tween-80 or 300 pg Tween-BO/ml to give the indicated cellular unsaturated fatty acid content. For each culture the Y,,” was calculated and the petite level and ethanol concentration in the culture supernatants were determined as described in Methods. Results are representative of at least three determinations,
584
MARZUKI TABLE
V
OXIDATNE PHOSPHORYLATION ACTIVITIES OF MITOCHONDRIA ISOLATED FROM KD115 GROWN TO VARIOUS STEADY-STATE CELLULAR UNSATURATED FAT-TV ACID LEVELS~ Cellular unsaturated fatty acid (% total fatty acids) 75 60 50 44 34 22 KD115-2
Substrates ETOH
1.15 0.92 0.56 0.38
OF BIOCHEMISTRY
AND
BIOPHYSICS
169,
577-590
Biogenesis The Effects
of Altered
MARZUKI,
Department
of Mitochondria
Steady-State
Mitochondrial-Energy SANGKOT
(197%
Membrane
Metabolism GARY
of Biochemistry,
l Lipid Composition
in Saccharomyces
S. COBON, J. M. HASLAM, LINNANE Monash Received
University, February
Clayton,
Victoria,
on
Cerevkiae AND
ANTHONY
W.
3168, Australia
6, 1975
A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cereuisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression. The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated. The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%. In cells which contained lipids with 22-28s unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the leveis of cytochromes aa, and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.
One approach our laboratory has adopted to study mitochondrial membrane structure-function relationships has involved the use of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae grown in glucose batch cultures. It has been reported that a decrease in the mitochondrial membrane unsaturated fatty acid content below the wild-type level of 75% resulted in a decrease in the efficiency of oxidative phosphorylation. Oxidative phosphorylation is completely uncoupled ’ This paper Mitochondria.”
is No. 38 in the series, “Biogenesis No. 37 is Molloy et al., 1975 (27).
in isolated mitochondria with unsaturated fatty acids representing 1528% of the total (1, 2). At this low level of unsaturated fatty acids, mitochondrial cytochrome levels and respiration appeared to be normal (3) although mitochondria isolated from such cells contained a greatly reduced ability to establish and maintain K+ and H+ gradients (4). Only cells in which unsaturated fatty acid content was depleted to less than 11% of the total fatty acid content by growth of the mutant to stationary phase in a glucose batch culture showed a 40% reduction in the whole cell respiratory activity and some decrease in the whole
of 577
Copyright @ 1975 by Academic Press, Inc. All rights of reproduction in any form reserved
578
MARZUKI
cell cytochrome content (3). Interpretation of these results was complicated by several factors. First, cells grown on glucose to a low unsaturated fatty acid level are catabolite repressed so that the observed reductions in respiratory activity and cytochrome levels may be a consequence of either low unsaturated fatty acid content or of catabolite repression (3). Second, the cells extensively depleted of unsaturated fatty acids were in a stationary phase of growth so that the observed decrease in respiratory activity may have been due to the culture conditions rather than a consequence of a change in the molecular organization of the mitochondrial membrane. Finally, in batch cultures, it is not possible to determine with any reasonable degree of precision the unsaturated fatty acid level at which oxidative phosphorylation becomes uncoupled and other functional changes occur. In the present communication, a technique of cell culture is described which overcomes these difficulties. Cells were grown in a chemostat using glucose-limiting media in such a way that the steadystate glucose level could be maintained below that which induces catabolite repression. Furthermore, cultures were maintained for a number of generations at chosen cellular unsaturated fatty acid levels so that the comparison of cells containing high and low cellular unsaturated fatty acid levels was facilitated. Differences observed were a direct result of the variation in the cellular unsaturated fatty acid content. We found that even small changes in unsaturated fatty acid content below wildtype cell levels resulted in a decrease in the molar growth yield, which is an in vivo determination of the efficiency of oxidative phosphorylation. At a cellular unsaturated fatty acid content of about 45%, the molar growth yield approached that of a respiratory-deficient mutant, indicating that, at this level of unsaturated fatty acid content, mitochondrial oxidative phosphorylation was about 90% uncoupled. The mitochondrial cytochrome levels and whole cell respiration were normal even down to 34% unsaturated fatty acid content but when the steady-state cellular unsaturated fatty
ET AL.
acid content was further decreased to 22-28%, the levels of mitochondrial cytochromes aa3, b, and cc, were also reduced to less than 10% of the normal levels. Cyanide-sensitive respiration approached zero. An investigation into the nature of the changes in the mitochondrial proteinsynthesizing system accompanying unsaturated fatty acid depletion is the topic of the accompanying paper (5) in which it is reported that unsaturated fatty acid-depleted cells do not form functional mitochondrial ribosomes. MATERIALS
AND
METHODS
Strain of Yeast Saccharomyces cereuisiae KD115 (a ole~ p+), an unsaturated fatty acid auxotroph, was obtained from Dr. Resnick (6). A spontaneous revertant, strain KD115-2, was isolated from a culture of KD115 grown in medium lacking unsaturated fatty acids. The petite mutant of KD115 (o( ale- p”) used in this study was selected from an unsaturated fatty acid-depleted culture of KD115 as described previously (7).
Reagents Tween-80 was obtained from H. B. Selby (Nottinghill, Victoria, Australia). The fatty acid composition of the batch of Tween-80 used was 81% oleic acid, 6% palmitoleic acid, 2% myristoleic acid, and 11% saturated fatty acids. Yeast extract was obtained from Difco Laboratories (Detroit MI), ATP, peroxidase (EC 1.11.1.7). glucose oxidase (EC 1.1.3.4), and hexokinase (EC 2.7.1.1) were purchased from the Sigma Chemical Company (St. Louis, MO), and alcohol dehydrogenase (EC 1.1.1.1) from Calbiochem Australia Pty. Ltd. (Sydney, N.S.W. Australia). [32P]Orthophosphate (carrier-free) was purchased from the Australian Atomic Energy Commission, Lucas Heights N.S.W. All other materials used were of reagent grade.
Culturing Procedure Yeast cells were grown in chemostat cultures (8) at 28°C by the following procedure, except when otherwise specified. Cells were grown in 7.5-liter capacity fermenters (New Brunswick) with a working volume of 4 liters. The medium contained a salts mixture as described previously (2) and yeast extract, 10 g/liter. Glucose was autoclaved separately as 75% stock solutions and added to the medium to give a final concentration of 10 g/liter. The medium was supplemented with various concentrations of Tween-80 as a source of unsdurated fatty acids. The inoculum for the 4-liter cultures was cells
YEAST
MITOCHONDRIAL
grown to exponential phase in batch cultures supplemented with an excess of Tween-80 (4000 &ml) and added to the continuous culture to give an initial cell concentration of approximately 0.1 mg cell dry weight/ml. The flow rate of fresh glucose medium into the culture was 400 ml/h (dilution rate D = 0.1 h-l). The cultures were grown under forced aeration (5-8 liters aidmin) and stirred at 380 rpm. Under these conditions, the steady-state cell density of the OLE revertant strain KD115-2 increased linearly with the glucose concentration in the input medium up to a glucose concentration of at least 10 g/liter, indicating that glucose was the growth-limiting factor.
Revertant and Petite Cells in Cultures The reversion rate of KD115 is relatively high and the revertant levels in the cultures were, therefore, routinely determined by plating samples of cultures on solid medium containing a salts mixture (2), yeast extract 2 g/liter, glucose 10 g/liter, and agar 20 g/liter, The frequency of OLE revertants in the cultures was minimized by ensuring a low level of revertants in the inoculum, which was achieved through frequent subcloning of the stock culture. Only those chemostat cultures containing a final level of less than 0.1% OLE revertants were used in the biochemical studies. The frequency of respiratory incompetent (petite) cells was determined as described previously (7).
Preparation of Mitochondria Mitochondria were prepared from glusulasedigested cells as described by Cobon et al. (9). Owing to the fragility of the mitochondria containing low levels of unsaturated fatty acid, these organelles were isolated in 0.9 M sorbitol medium rather than 0.6 M as described previously (9).
Analytical
Methods
The following published analytical procedures were used: cell-density determination by measurement of packed cell volume (3); polarographic measurement of whole-cell respiratory activity (3); enzymatic determination of glucose (10) and ethanol (11) concentrations; and P:O ratios in isolated mitochondria (12). The cytochrome spectra of the whole cells and isolated mitochondria were measured at room temperature or at the temperature of liquid nitrogen as described by Clarke-Walker and Linnane
(13).
Unsaturated Fatty Acid Analyses Whole-cell and mitochondrial samples were saponified in 7 M KOH for 3 h at 100°C and nonsaponifiable material was then extracted into ether. The aqueous fraction was acidified and the fatty acids were extracted into ether which was subsequently
ENERGY
579
METABOLISM
dried over anhydrous sodium removed by evaporation and were methylated in a solution by heating at 60°C for 1 h. extracted into hexane and chromatography in an F and 810 or a Pye Unicam Model
sulfate. The ether was the recovered fatty acids of 14% BF, in methanol The methyl esters were analyzed by gas-liquid M chromatograph Model R.
Phospholipid Analyses Chemostat cultures with 350-ml working volumes were maintained at the desired unsaturated fatty acid level for three to four generations after reaching steady state in the presence of 1.25 PCi [3ZP]orthophosphate/ml of input media. Cells were harvested, washed, and a whole-cell sample was disrupted by passage through a French pressure cell at 1.8 x lo5 k pascals. The remainder of the cells were used for the preparation of mitochondria. The lipid components were extracted from the whole cell and the mitochondrial samples and separated by two-dimensional tic on silica gel as previously described (9). Areas of gel containing lipids were scraped into scintillation vials and the radioactivity determined in a Philips liquid scintillation analyzer.
Calculation of Molar Growth Yields and Phosphorylation Efficiency in Vivo The molar growth yield for glucose cells formed per mole of substrate calculated as follows: Y,l”
= cws,
Yglu (g dry wt of consumed) was
- S,)
where x is the cell density in the steady state (mg cell dry wt/ml), S, is the molar glucose concentration in the inflowingmedium, and S, is the remaining molar glucose concentration in the culture medium. It was found that when the cells were grown in the presence of radioactively labeled glucose, the cells incorporated into the cell mass about 10% of the amount of glucose utilized (unpublished observation). Consequently, the Y,,, calculated was corrected for the proportion of glucose incorporated and thus not available for energy production. The Y,,” was also corrected for the energy used for the maintenance process as described by Pirt
(14). The molar growth yield for ATP, Y,.r, (g dry wt of cells formed per mole of ATP) was calculated as 0.5 x Y *,” of a respiratory incompetent mutant, assuming the following stoichiometry: Glucose
+ 2 ADP
+ 2 P, = 2 ethanol
+ 2 CO,
+ 2 ATP
The phosphorylation efficiency under other conditions was calculated using this determined value for YATP and the experimentally determined Y,,” (15).
580
MARZUKI
ET
AL.
functions. In agreement with these workers, a shift to a fermentative metabolism Mitochondrial samples were prepared for electron was observed as the dilution rate was microscopy as described by Watson et al. (16). increased above 0.2 h-’ (corresponding to RESULTS the maximum specific growth rate of the revertant in ethanol). This change is indiGrowth Characteristics of KDl15 Related cated by a decrease in the cell density to Strains in Glucose-Limited Chemostat about 1.5 mg cell dry wt/ml and the Cultures accumulation of ethanol in the culture To determine the growth conditions medium. The actual glucose concentration under which metabolism was purely fer- in the culture remained below 0.5 mM until mentative and those under which both the dilution rate was about 0.3 h-’ and fermentative and oxidative processes then increased sharply as the critical diluwould occur, the effect of dilution rate on tion rate of about 0.35 h-’ was approached. culture parameters of the OLE revertant For comparison, a respiratory incompewas investigated. Figure la shows the tent strain, which had no mitochondrial growth yield of the OLE revertant plotted DNA, KD115 Po, was used to establish the as a function of the specific growth rate p culture parameters when the metabolism which, in the steady state, equals the was purely fermentative (Fig. lb). This dilution rate D. With a 10 g/liter initial petite mutant grown in an excess of unsatglucose concentration (55.5 mM), the urated fatty acid at a dilution rate of 0.1 steady-state cell density was constant at h-’ attained a steady-state cell density of about 5.5 mg cell dry wt/ml when the about 1.2 mg cell dry wt/ml which was dilution rate was between 0.025 and 0.2 constant over the whole range of dilution h-‘. Over this range of dilution rate, the rate below the critical dilution rate of 0.3 metabolism of the cells was purely oxida- h-‘. tive as shown by the low level of ethanol accumulated in the culture medium. The Manipulation of Membrane Lipid slight decrease in the cell density as the Composition of Strain KD115 in dilution rate was lowered has been attribContinuous Culture uted by Fiechter and von Meyenburg (17) The steady-state cellular unsaturated to an increase in the proportion of the energy required for maintenance of cellular fatty acid content could be varied from the Electron Microscopy
GLUCOSE
CELL e-.-e
i D,U~T,ON RATE D = SPECIFIC
JO
DENSITY -*--
0.1 GROWTH RATE p
7 :
20
:,
10
5 I
r
:
0.2
0.3
t
(hr-‘)
1. The effect of dilution rate on continuous culture parameters of KD115related strains. Cultures of the OLE revertant KD115-2 (Fig. la) in the absence of unsaturated fatty acid supplement or of the respiratory-deficient KD115 p0 (Fig. lb) in the presence of 4000 pg Tween80/ml of input media were grown at different dilution rates in chemostat cultures. The growth-limiting substrate in the inflowing media was glucose (10 g/liter) in both cases. At each dilution rate, after a steady state had been established, samples were taken at intervals over a period of at least 46 h. Cell density, e-0, ethanol, A-A, and glucose O--O concentrations were determined on each sample. Each point represents the average of at least three determinations. FIG.
YEAST
MITOCHONDRIAL
ENERGY
wild-type level of 75% to as low as 15% by altering the level of Tween-80 in the input medium. For example, at input levels of 4000 and 1000 pg of Tween-80 per ml of medium, the steady-state cellular unsaturated fatty acid contents were 75% and 50% of the total fatty acids, respectively (Table I). As in batch cultures (2), an increase in the level of short-chain fatty acids was observed as the unsaturated fatty acid content was lowered (Table I). There was no significant difference between the mitochondrial and the total cellular fatty acid compositions (data not shown). However, there were differences in the phospholipid compositions of whole cells and mitochondria. These differences varied with unsaturated fatty acid composition (Table II). Cells with a low unsaturated fatty acid content were lower in diphosphatidyl glycerol and phosphatidyl serine and higher in phosphatidic acid than cells grown with a high unsaturated fatty acid content. These phospholipid differences were much more pronounced in the mitochondria from such cells where for mitochondria with high and low unsaturated fatty acid contents, respectively, the percentages of diphosphatidyl glycerol were 15.0 and 2.6, phosphatidyl ethanolamine 23.4 and 14.9, phosphatidyl inositol 10.8 and 16.4, and phosphatidic acid 2.0 and 9.8. In all cases, mitochondria were richer in diphosphatidyl glycerol and phosMANIPULATION
Tween-80
OF CELLULAR
4000 2000
1000 500 350 200
100 KD 115-2
phatidyl ethanolamine than the whole cells, a result similar to those previously reported for yeast (9, 18) and rat liver (19) mitochondria. The Nature of Substrate Limitation at Low Levels of Unsaturated Fatty Acid Supplementation At a dilution rate of 0.1 h-l KD115 chemostat cultures supplemented with 4000 pg of Tween-BO/ml were glucose limited; the steady-state concentration of glucose in the culture was about 0.3 mM (Table III). At such glucose levels the cells were not subject to catabolite repression, as the cells were capable of growth by oxidative metabolism as indicated by negligible accumulation of ethanol in the medium and the high cell yield (Fig. la). Since many mitochondrial functions are sensitive to catabolite repression, it was critical to demonstrate that when the level of unsaturated fatty acid supplement was lowered, the unsaturated fatty acid did not become growth limiting, with a consequential increase in the concentration of glucose in the culture and concomitant catabolite repression. Analysis of the medium showed that the remaining glucose concentration was less than 0.5 mM under all conditions even though the residual unsaturated fatty acid concentration in the medium decreased from about 1400 pg/ml to 38 pg/ml as the level of Tween-80 in the input
TABLE I UNSATURATED FATTY ACID LEVELS OF STRAIN KD115 CHEMOSTAT CULTUREP
(fig/ml)
Weight c,:,
581
METABOLISM
14:o
1.7 * 0.2 4.0 * 1.0 5.6 i 0.2 13.1 * 2.8 15.0 * 1.2 12.9 + 0.6 12.0 * 1.3 6.0 z+ 0.3
C 16 0 - LB 0 * 34.9 * 43.5 i 41.7 * 49.9 * 63.0 zt 79.1 * 20.1 * 22.3
1.2
1.0 2.3 2.2 3.0 2.1
1.9 1.2
% of total
fatty
acids Cl,:,
Cl,:, 7.9 * 0.5 5.9 * 0.1 4.0 * 0.2 5.1 * 0.2 3.6 * 0.3 1.6 i- 0.5 1.5 * 0.2 36.0 zt 2.0
IN GLUCOSE-LIMITED
* 1.7 53.1 * 0.2 45.5 * 2.1 38.3 + 2.8 30.0 * 2.1 20.3 ,z 1.8 10.5 * 0.1 36.9 ze 1.8
66.5
Total
UFA
75.2 * i* 44.2 i 34.1 zt 22.0 i 12.8 + 73.3 *
60.3 50.2
1.4 0.1 3.5 3.0 2.3 1.4
1.3 1.2
n KD115 was grown at a dilution rate of 0.1 h-’ to a steady state in chemostat cultures supplemented with the indicated levels of Tween-80 in the input media. Samples were taken at various times after the steady state was reached. The cells were harvested, water washed, and the unsaturated fatty acid composition was determined by gas-liquid chromatography as described in Methods. Results are expressed as molar R of total fatty acids * standard deviations. At least three independent samples were taken from each culture,
582
MARZUKI
PHOSPHOLIPID
Unjatu.ated fatty acid content (% total fatty cids) Whole cells Mitochondria Whole cells Mitochondria
75
15
COMPOSITION
Phosphatidyl inositol
ET AL.
TABLE II OF CELLS AND MITOCHONDRIA OF HIGH AND Low UNSATURATED Phospholipid (Mole % total lipid phosphorus) Phosphatidyl serine
Phosphatidic acid
Phosphatidyl ethanolamine
FATTY ACID CONTENT
T Diphosphatidyl glycerol
Phosphatidyl glycerol
Unidentified
4.6 + 0.6
0.8 * 0.1
5.9 i 1.8
* 0.4
1.0 f il.3
3.0 * 1.1
:
: !l.l
* 1.0
11.4 * 1.1
3.4 zt 1.5
10.6 zt 0.7
1.0.8 * 1.2
4.4 * 0.3
2.0 * 0.2
23.4 e 2.1
2!2.6 z+ 0.1
6.1 I 0.7
9.7 f 3.0
11.7 * 0.3
1.5 * 0.1
0.6 zr 0.1
3.7 * 0.3
16.4 i 0.0
6.8 rt 1.2
9.8 * 0.2
14.9 * 0.5
2.6 ze 0.2
0.3 * 0.1
3.9 * 0.2
I.5.0
1
L
o Cells were grown in chemostat cultures containing the appropriate unsaturated fatty acid supplement and 1.25 HCi [32P]orthophosphate/ml as described in Methods. Lipids were extracted from cells and mitochondria and separated by thin-layer chromatography. Radioactivity in each lipid species was determined and is expressed as mole % of total lipid phosphorus i- standard deviation of four determinations.
medium decreased from 4000 pg/ml to 100 pg/ml (Table III). It was concluded that glucose was the growth-limiting substrate at all levels of unsaturated fatty acid supplementation tested. The Effect of Unsaturated Fatty Acid Depletion on Glucose Utilization Two of the most useful parameters in studying the efficiency of energy metabolism in microorganisms are the molar growth yield Y (g dry wt of cells formed per mole of substrate consumed) and the molar growth yield on ATP, YATp (g dry wt of cells formed per mole of ATP). The molar growth yield on a given substrate is a constant for a given organism. If the metabolic pathway is known, YATp can be calculated from Y. On the other hand, since YATP is also a biological constant, if this value for an organism is known or can be calculated from one set of conditions, it may be used to estimate the ATP yield for metabolic processes carried out under another set of conditions (15).
The Yglu of the culture supplemented with excess Tween-80 (cellular unsaturated fatty acid 75%) was calculated from the steady-state cell density at a dilution rate of 0.1 h-’ as described in Methods. At this level of cellular unsaturated fatty acid, the ym was about 123 g/mole, similar to that of the revertant strain (Table IV). A 22% decrease in Y,,, was observed when the cellular unsaturated fatty acid content was decreased to 50%. AS no accumulation of ethanol in the culture was observed, oxidation of ethanol was less efficient in terms of energy yield. A further comparatively small decrease in the unsaturated fatty acid content to 44% resulted in a pronounced decrease in YgIU and substantial amounts of ethanol accumulated in the culture. In this case, not only was the efficiency of oxidative phosphorylation reduced but the ability of the cells to utilize ethanol was also impaired. Progressive decrease in the unsaturated fatty acid content to 15% resulted in a Y,,, of 21.8 and a large accumulation of ethanol. This Y,,, corresponded to the
YEAST TABLE
MITOCHONDRIAL
III
ANALYSIS OF FAW ACID AND GLUCOSE CONCENTRATIONS IN CULTURE SUPERNATANT OF STRAIN KD115 GROWN WITH VARIOUS CONCENTRATIONS OF TWEEN80 SUPPLEMENT IN THE INPUT MEDIAN Tween-80 (r&l)
input
Steady-state culture supernatant Fatty acid (~cg/ml)
4000 2000 1000 500 350 100
1419 213 109 79 57 38
Glucose (mM) 0.29 0.16 0.26 0.34 0.42 0.37
0 KD115 was grown as described in the legend to Table I. Samples were taken from the culture at various times after reaching the steady state, cells were removed by centrifugation, and the fatty acid and glucose concentrations in the supernatants were determined as described in Methods. The results are representative of at least two determinations.
basic fermentation value obtained for the respiratory deficient p” mutant of KD115 when grown under conditions of either high or low unsaturated fatty acid content (Table IV). We have previously reported that growth at low cellular unsaturated fatty acid levels leads to the induction of cytoplasmic petite mutants (7). However, it is clear that the increase in YBIU at low levels of unsaturated fatty acid content is not attributable to the accumulation of petites. At a cellular unsaturated fatty acid level of 34%, for example, the petite frequency of the culture when harvested was 11.5% of the total cell population; the Y,,, expected from such a culture on the basis of a simple mixture of petite and grande cells would be 111 g/mole whereas the observed Yg,,, was only 38.5 g/mole. Calculation of Oxidative Phosphorylation Efficiency in Vitro and in Vivo We have previously reported that the oxidative phosphorylation efficiency of mitochondria isolated from batch-grown yeast cells decreased with the unsaturated fatty acid content of the mitochondria (2). In the present study, mitochondria isolated
ENERGY
583
METABOLISM
from cells grown in the chemostat to various steady-state levels of unsaturated fatty acid were also examined for their oxidative phosphorylation efficiency, and the results are in a very close agreement to the previous observations (Table V). With 75% of the fatty acids unsaturated, the mitochondria had a P:O ratio of 1.15, which was comparable with the level in the revertant KD115-2 (Table V), and was in the normal range for Saccharomyces cerevisiae mitochondria. With the decreasing unsaturated fatty acid level in the mitochondria, there was a concomitant fall in the P:O ratios, which at the 50% unsaturated fatty acid level was about half that of normal mitochondria, and essentially no oxidative phosphorylation could be detected at about the 34% unsaturated fatty acids level (Table V). TABLE
IV
THE EFFECT OF UNSATURATED ON GLUCOSE METABOLISM Organism
KD115
KD 115-2 KD 115 p”
Cellular unsaturated fatty acid (% total fatty acid) 75 60 50 44 34 22 15 73 75 21
FATTY ACID DEPLETION BY STRAIN KD115”
YglU (g cell dry weight/ mole glucose) 122.6 113.2 95.7 43.2 38.5 22.4 21.8 121.9 22.1 19.8
lo Petite cell level of harvested culture
Ethanol (culture supernatant, mM)
0.8 1.2 2.2 2.2 11.5 32 34 1.0 100 100
2 8 4 44 50 71 84 9 103 99
a The fatty acid desaturase mutant KD115 was grown to steady state as described in the legend to Table I to give the indicated cellular unsaturated fatty acid content. The revertant strain KD115-2 was grown to steady state under identical conditions in the absence of Tween-80 supplement. The respiratorydeficient mutant derived from KD115, KDll5 pO, was grown under identical conditions in the presence of either 4000 pg/ml Tween-80 or 300 pg Tween-BO/ml to give the indicated cellular unsaturated fatty acid content. For each culture the Y,,” was calculated and the petite level and ethanol concentration in the culture supernatants were determined as described in Methods. Results are representative of at least three determinations,
584
MARZUKI TABLE
V
OXIDATNE PHOSPHORYLATION ACTIVITIES OF MITOCHONDRIA ISOLATED FROM KD115 GROWN TO VARIOUS STEADY-STATE CELLULAR UNSATURATED FAT-TV ACID LEVELS~ Cellular unsaturated fatty acid (% total fatty acids) 75 60 50 44 34 22 KD115-2
Substrates ETOH
1.15 0.92 0.56 0.38
