Adaptation of the mitochondrial systems of Rhodotorula gracilis to low oxygen pressure

Cell Difl'erentiation, 4 (1975) 155--165 © North-Holland Publishing Company, Amsterdam

Printed in The Netherlands

ADAPTATION OF THE MITOCHONDRIAL SYSTEMS OF RHODOTOR ULA GRACILIS TO LOW O X Y G E N P R E S S U R E

M. COCUCCI, G.F. ROSSI and T. VANDONI Istituto di Scienze Botaniche, Universit~ di Milano, Centro di studio C.N.R. per la Biologia Cellulare e Molecolare delle Piante, via G. Colombo 60, 20133 Milano, Italy

Accepted 10 March 1975

The obligate aerobic yeast, Rhodotorula gracilis, was grown in a liquid minimal medium at 1 mm Hg partial pressure of oxygen, conditions under which growth (measured as the increase in total protein) is reduced to 30% of the maximum rate. A significant increase in the ratio between mitochondrial oxidative enzymes and total protein occurs rapidly under these conditions. A concurrent increase in the ratio area of mitochondria/area of cytoplasm was also observed. The relative increase in mitochondrial enzymes, oxidase activities and mitochondrial membranes is due to the inhibition affecting the increase in cytoplasmic structures more significantly than the increase in mitochondrial structures at low pO2. The difference between mitochondrial and cytoplasmic syntheses cannot be ascribed to changes in the availability of ATP but it might rest with some other oxygen-utilising process (pyrimidine redox coenzymes, synthesis of sterols). The experimental conditions studied appear to offer a valuable tool for the investigation of the relationships between mitochondrial and cytoplasmic structures.

O x y g e n is an essential f a c t o r in the c o n t r o l o f cell g r o w t h . In a large variety o f organisms, b o t h a e r o b i c and a n a e r o b i c , o x y g e n c o n t r o l s the develo p m e n t o f the oxidative a p p a r a t u s : it specifically affects the synthesis o f c y t o c h r o m e s and o f m a n y d e h y d r o g e n a s e s involved in r e s p i r a t i o n as well as the synthesis o f e n z y m e s o f t h e t r i c a r b o x y l i c acid c y c l e ( L i n d e n m a y e r et al., 1 9 6 4 ; V a r y et al., 1 9 6 9 ; King, 1 9 7 1 ; W i m p e n n y et al., 1 9 7 1 ) . In facultative aerobic yeasts such as S a c c h a r o m y c e s cerevisiae, a n a e r o b i c c o n d i t i o n s i n d u c e a d r o p in t h e level o f c y t o c h r o m e s t o g e t h e r with a large increase in f e r m e n t a t i o n . In the a b s e n c e o f o x y g e n , the m i t o c h o n d r i a o f this y e a s t are r e d u c e d to ' p r o m i t o c h o n d r i a ' , w h i c h are s t r u c t u r a l l y simpler t h a n n o r m a l m i t o c h o n d r i a and lack a n u m b e r o f essential c o m p o n e n t s ( c y t o c h r o m e s and u n s a t u r a t e d f a t t y acids) (Wallace et al., 1 9 6 8 ; Criddle et al., 1 9 6 9 ; P a l t a u f et al., 1 9 6 9 ) . O n c e the n o r m a l level o f o x y g e n is r e s t o r e d , rapid synthesis o f c y t o c h r o m e s and d e v e l o p m e n t o f n o r m a l m i t o c h o n d r i a are i n d u c e d (Wallace et al., 1 9 6 4 ; L i n n a n e , 1 9 6 5 ; C h e n et al., 1 9 6 9 ) . In obligate aerobes, severe a n a e r o b i c c o n d i t i o n s i n d u c e the b l o c k o f synt h e t i c activities in the cell, t h e o c c u r r e n c e o f degenerative processes and e v e n t u a l l y the d e a t h o f the organism. Previous research h a d s h o w n t h a t in the y e a s t R h o d o t o r u l a gracilis, an

156 aerobe obligate, the synthesis of both protein and RNA is completely stopped under anaerobic conditions. The cells appear to survive w i t h o u t showing any sign of degeneration for eight hours in anaerobiosis, though they do not seem to be able to ferment (Cocucci, 1972). At low oxygen pressures, as low as 3 mm Hg and lower, however, growth (measured as increase in total protein) continues linearly for several hours, even at oxygen pressures at which the growth rate is reduced. At 2 mm Hg and 1 mm Hg oxygen pressure (as measured in the culture medium) the growth rate is reduced to respectively 50 and 30% of the growth rate measured under fully aerobic conditions. Moreover, under such growth-limiting conditions, the level of ATP was found to be the same as in the control or higher (Cocucci et al., 1973), a demonstration that oxidative phosphorylation still remains very active in spite of the extremely low oxygen level. In this paper we describe the effect of low oxygen pressures on mitochondria of R. gracilis. MATERIALS AND METHODS Cultures of R. gracilis were set up as described previously (Beffagna et al., 1972). Exponentially growing cells were harvested by filtration on cellulose acetate filters (Sartorious, 1.2 p diameter) at 4°C and resuspended to a density corresponding to 1 0 D 6 6 0 n m , in a minimal medium containing 2% glucose and 2% a m m o n i u m sulphate. The cell suspension was then transferred to a 2-1 glass-stainless steel container, fitted with a Clark polarographic electrode (Radiometer, Copenhagen) and with a teflon membrane for the control of oxygen pressure. Cell suspension, suitably agitated, were grown at 30°C and the desired oxygen pressure was obtained by bubbling appropriate mixtures of nitrogen and oxygen through the culture medium. The gas stream was controlled by means of flow regulators (ASA, Sesto S. Giovanni, Milano) operating by feedback from the electrode.

Preparation of cell-free extracts Cells were harvested as described, washed, resuspended in 0.05 M phosphate buffer, at pH 7 and quickly pelleted. Pellets were frozen at - - 2 0 ° C. The cells were thoroughly ground in a mortar with alumina and extracted in 0.01 M Tris buffer, containing 0.1 M NH4 C1 and 0.01 M MgC12, pH 7. The homogenates were centrifuged at 1000 g for 3 min at 4 ° C. Enzyme activities were assayed in the supematant. Assays were carried out spectrophotometrically at 30°C. C y t o c h r o m e oxidase was assayed according to Chen et al. (1969); succinate dehydrogenase as described by King (1967) and fumarase according to Vary et al. (1969).

Assays of cytochromes in intact cells Cells were harvested and carefully suspended in 0.05 M phosphate buffer, pH 7, containing cycloheximide (25 pg per ml) and antimycin A (1.5 pg per

157 ml). Cytochromes were assayed by monitoring the change in the redox absorbance spectrum with a Dual Wavelength Aminco--Chance spectrophotometer, according to Chance (1957). Oxidising conditions inside the cells were obtained by bubbling 40% oxygen through the suspension; reducing conditions occurred after endogenous respiration or were induced by treatmerit with dithionite. The wavelengths chosen for both references and measurement, as well as the extinction coefficients for cytochromes a3, a and c, were those reported by Linnane (1965). Electron microscopy Fixation and inclusion were performed as reported in a previous paper (Cocucci et al., 1972). We found that fixation with permanganate was more convenient than fixation with OsO4 for evaluating size and membrane distribution of the mitochondria. Therefore we have used permanganate fixation throughout. Evaluation o f number and area of the mitochondria Sections were cut at a thickness as constant as possible and sampled for E.M. observation at intervals sufficiently apart to ensure that no cell was photographed twice. Pictures of mitochondria were cut out of photographic prints. The area of the mitochondria and of the cytoplasm (without nucleus) was determined by weighing the cut-outs. RESULTS The amounts of cytochromes a3, a and c, and the activities of three mitochondrial enzymes, cytochrome oxidase, succinate dehydrogenase and fumarase measured in R. gracilis cells grown under saturating oxygen pressure (100 mm Hg, as measured in the medium) remain virtually the same when pO2 is lowered to 2 mm Hg, a condition under which the m a x i m u m growth rate is reduced by 50% (Table I). When pO2 is further lowered to 1 mm Hg (corresponding to 30% of the m a x i m u m growth rate) both the ratio of c y t o c h r o m e s / t o t a l protein and the specific activities of c y t o c h r o m e oxidase, succinate dehydrogenase and fumarase are significantly increased (Figs. 1 and 2). Fig. 1 (upper) shows the changes in the specific amounts of cytochromes (pmoles/total protein) during a 4-hr period of growth under conditions of saturating oxygen and strongly limiting (1 mm Hg) pO2 respectively. The levels of all three cytochromes significantly increase under conditions of limiting oxygen. The difference between cytochromes a3 and a seems to be a significant one, even when considering that neither the absorbance in the Sorer band can be due to a3 alone nor the absorbance in the visible light to a alone (Morrison, 1965).

158 TABLE I

C y t o c h r o m e levels ( A ) and activities (B) o f three m i t o c h o n d r i a l e n z y m e s in e x p o n e n t i a l l y growing cells ofRhodotorulagracilis (100 m m Hg o x y g e n pressure), and after a 4-hr period o f growth under 2 m m Hg o x y g e n pressure, c o n d i t i o n under which the m a x i m u m g r o w t h rate is reduced by 50%.

A

O x y g e n pressure ( m m Hg) 100

2

Cyt. a3 Cyt. a Cyt. c

157 168 206 212 226 233 Values are expressed as ~ m o l e s / g of protein. C y t o c h r o m e s were assayed s p e c t r o p h o t o m e t r i c a l l y in w h o l e cells.

B

O x y g e n pressure (ram Hg) 100

Cyt. o x i d a s e (1) S u c c i n a t e d e h y d r o g e n a s e (2) F u m a r a s e (2)

2

0.219 0.237 0.0385 0.0415 0.2704 0.2793 Cells were ground in a m o r t a r with alumina and e n z y m e activities were assayed in cell-free extracts. (1) K s e e - l / m g p r o t e i n m l ; ( 2 ) p motes/ min mg p r o t e i n .

Fig. 1 (lower) shows that the increase in the specific amounts of cytochromes is essentially due to the fact that under low pO2 the total increase in cell protein is reduced more than the increase in the level of cytochromes. The increase in the level of cytochrome a3 remains in fact unaffected by the low pO2 and the increase in the level of both cytochrome a and c is reduced to a relatively small extent. Fig. 2 shows (upper and lower respectively) the changes in the specific activities (activity/mg total protein) and in the absolute amounts (activity/ml of culture) of cytochrome oxidase, succinate dehydrogenase and fumarase in cells grown at saturating (100 mm Hg) and low (1 mm Hg) tensions of oxygen. Here too an increase can be observed in the specific activities of all three enzymes, as their development is affected by low pO2 to a much lower extent than is the total synthesis of protein. Good agreement is found when comparing the changes induced by low pO2 on the levels of cytochromes a3 and a measured in vivo, and on the cytochrome oxidase activity measured in cell-free extracts, if the fact that

159

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b o t h c y t o c h r o m e s are involved in the c y t o c h r o m e oxidase activity is taken into account.

Morphological aspects In R. gracilis, low oxygen tension (1 m m Hg) induces a rapid and conspicuous increase in the apparent n u m b e r of m i t o c h o n d r i a per cell. Figs. 5 and 6 as co mp ar e d to Fig. 4 show that there is already an increase in the mitochondrial profiles per cell after 1 hr of partial anaerobiosis and this continues until 5 hr of incubation in low pO2. It is difficult to assess whether the increase in mitochondrial structures per cell section depends on a real increase in distinct m i t o c h o n d r i a or on a higher degree of branching of the existing ones. However, in either case the general meaning of the phen o m e n o n seems to be the same, as it leads to an increase in the active mitochondrial surfaces (outer m e m b r a n e and cristae) per cell. Fig. 3 reports the changes with time in the apparent n u m b e r of organelles observed per cell section. The shape of the curves suggests a greater variability in the apparent n u m b e r of organelles at low 02 tension as com pared with the cells grown under saturating pO2. Here again a shift in the m a x i m u m f r e qu en cy class towards higher values of the apparent n u m b e r of m i t o c h o n . dria is clearly observed from the 1st to the 5th hr.

161

Fig. 4. Logarithmic phase cells of Rhodotorula gracilis grown in saturating oxygen tension (100 mm Hg). N, nucleus; M, mitochondria; Va, vacuole.

Fig. 5. Rhodothorula gracilis cells grown for 1 hr under low oxygen tension (1 mm Hg).

]62

Fig. 6. Rhodotorula gracilis cells g r o w n for 5 hv u n d e r low o x y g e n t e n s i o n .

An increase in the number of mitochondria can frequently be observed in the cells when growth slows down after the exponential phase, following environmental changes apparently not related to the lack of oxygen (Yotsuyanagi, 1962; Elliot et al., 1964; Kitsutani et al., 1970). In such circumT A B L E II C h a n g e s w i t h t i m e o f t h e m i t o c h o n d r i a l a n d c y t o p l a s m i c areas o f Rhodotorula gracilis cells c u l t u r e d at low o x y g e n t e n s i o n (1 m m Hg in t h e m e d i u m ) . M e a s u r e m e n t s were d o n e o n t h e s a m e cell s e c t i o n s c o n s i d e r e d in Fig. 3.

Growth conditions

Aerobiosis (control) Partial. a n a e r o b i o s i s Partial. a n a e r o b i o s i s Partial. a n a e r o b i o s i s Partial. a n a e r o b i o s i s Partial. a n a e r o b i o s i s

A v e r a g e area (t~:) per cell s e c t i o n

1 2 3 4 5

hr hr hr hr hr

Mitochondria

Cytoplasm (without nucleus)

1.28 1.47 1.6 1.59 1.93 1.83

5.94 5.73 6 6.29 6.6 6.74

Mitochondria/ cytoplasm

0.215 0.256 0.266 0.253 0.289 0.271

+- 0 . 0 1 9 +- 0 . 0 1 6 -+ 0 . 0 1 3 -+ 0 . 0 1 7 +- 0 . 0 1 2 +- 0 . 0 1 2

163 stances the number of mitochondria has been reported to increase, but individual mitochondria become smaller in size. In the case of R. gracilis, the observed increase in the number of mitochondria appears to correspond to a true increase in the total mass of the organelles rather than to a simple increase in the extent of subdivision of the existing ones. In fact, as shown in Table II, measurements of the area occupied, per cell section, by the mitochondria and by the remaining cytoplasm (nucleus excluded) show a significant increase in the ratio area of mitochondria/area of cytoplasm during the incubation at low pO2 • CONCLUSION AND DISCUSSION The results reported above can be summarised as follows. In the aerobe obligate R. gracilis a significant and rapid increase in the ratio mitochondrial oxidases/total protein is induced by lowering the pO2 to growth-limiting levels (1 mm Hg in the medium, corresponding to 30% of the m a x i m u m growth rate). From the morphological point of view, a parallel increase in the ratio of active mitochondrial structures (membranes and cristae) to cytoplasmic area is observed. The relative increase in mitochondrial enzymes and mitochondrial membranes is due to the fact that at low pO2 the increase in cytoplasmic protein is inhibited to a higher degree than the increase in mitochondrial components. The data reported here are not only different from those obtained in Saccharomyces cerevisiae, which does possess an alternative anaerobic pathway, but are also in contrast with the results reported from Candida parapsilosis (Kellerman et al., 1969), an obligate aerobe which under limited oxygen availability shows a reduction in the mitochondrial apparatus. This difference might be explained by the fact that C. parapsilosis displays a cyanide-insensitive respiratory pathway, capable of supporting a considerable part of the total cell respiration. It should be noted, however, that the experimental conditions adopted in the oxygen-limited growth of C. parapsilosis are widely different from our experimental conditions. The fast metabolic as well as structural response shown by Rhodotorula gracilis to low pO2 appears to have a clearly adaptive significance. In fact, the greater tolerance shown by the energy-producing mitochondrial system in comparison with the energy-consuming cytoplasm, allows the cell to maintain its metabolic equilibrium even under conditions unfavourable to mitochondrial oxidative phosphorylation. The increase, at low pO2, of the ratio mitochondrial components/cytoplasm is in agreement with the finding that under these conditions the level of ATP is the same or even higher than that observed under saturating pO2 (Cocucci, unpubl, data). The difference between the synthetic activity of the mitochondria and that of the cell as a whole cannot therefore be ascribed to a difference in the availability of ATP, but rather to the sensitivity to changes of pO2 of an oxygen-utilising system, which is not oxidative phos-

164

phorylation. Candidates could be the system which maintains the balance redox of the pyrimidine coenzymes or the system involved in the synthesis of sterols (unpublished data by M.C. Cocucci have shown a marked decrease in the ratio sterols/protein in R. gracilis grown at low pO2. The pO2 -dependent regulatory system could possibly have protein synthesis as its ultimate site of action and could discriminate between cytoplasmic and mitochondrial protein synthesis. The consideration that mitochondrial protein synthesis involves limiting factors altogether different from the cytoplasmic ones, as it depends upon 70 S ribosomes and possibly upon the formulation of methionine (Bianchetti et al., 1971), may be significant in this respect. From the data reported above it therefore appears that under these experimental conditions R. gracilis offers a suitable system for the study of the regulation of the relationship between mitochondrial and cytoplasmic structures. ACKNOWLEDGEMENTS

The authors wish to thank Professor E. Marrb for his help in the preparation of the manuscript and for his useful comments. REFERENCES Beffagna, N., S. Cocucci, M. Cocucci and M.C. Cocucci: Ann. Microbiol. 22, 119--130 (1972). Bianchetti, R., G. Lucchini and M.L. Sartirana: Biochim. Biophys. Res. Commun. 42, 97--102 (1971). Chance, B.: In: Methods in Enzymology, eds. S.P. Colowick and N.O. Kaplan (Academic Press, New York and London) Vol. 4,273--329 (1957). Chen,W. and F.C. Charalampous: J. Biol. Chem. 244, 2767--2776 (1969). Cocucci, M.: Ann. Microbiol. 22, 63--70 (1972). Cocucci, M.C. and G. Rossi: Arch. Mikrobiol. 85,267--279 (1972). Cocucci, M., M.C. Cocucci and E. MarrY: Plant. Science Letters 1,425--431 (1973). Criddle, S. and G. Scats: Biochemistry 8,322--334 (1969). Elliot, A.M. and I.J. Bak: J. Cell Biol. 20, 113--129 (1964) Kellerman, G.M., D.R. Biggs and Linnane: J. Cell Biol. 42,378--391 (1969). King, T.E.: In: Methods in Enzymology, eds. R.W. Estabrook and M.E. Pulman (Academic Press, New York and London) Vol. I0, pp. 322--329 (]967). King, T.E.: In: Metabolic Pathways, ed. H.J. Vogel (Academic Press, New York and London) Vol. 5, pp. 55--76 (1971). Kitsutani, S., K. Sawada, M. Osumi and M. Nagahisa: Plant Cell. Physiol. II, 107--118 (1970). Lindenmayer, A. and L. Smith: Biochim. Biophys. Acta 93,445- 461 (1964). Linnane, A.W.: In: Oxidases and Related Redox Systems, eds. T.E. King, H.S. Mason and M. Morrison (John Wiley and Sons Inc., New York--London--Sydney) Vol. 2, pp. 1102--1121 (1965). Morrison, M.: In: Oxidases and Related Redox Systems, eds. T.E. King, H.S. Mason and M. Morrison (John Wiley and Sons Inc., New York--London--Sidney) Vol. 2, pp. 639--666 (1965).

165 Paltauf, F. and G. Schatz: Biochemistry 8 , 3 3 5 - - 3 3 9 (1969). Vary, M.Y., C.L. Edwards and P.R. Stewart: Arch. Biochem. Biophys. 130, 2 3 5 - 2 4 3 (1969). Wallace, P.G. and A. W. Linnane: Nature 201, 1191--1194 (1964). Wallace, P.G., M. Huang and A.W. Linnane: J. Cell Biol. 37, 207--220 (1968). Wimpenny, J.W.T. and K. Necklen: Biochim. Biophys. Acta 2 5 3 , 3 5 2 - - 3 5 9 (1971). Yotsuyanagi, Y.: J. Ultrastruct. Res. 7, 121 -140 (1962).