synthesis of the inner mitochondrial membrane and … · harmon, hall & crane, 1974; janki,...

J. Cell Sci. 21, 329-340 (1976) 329

Printed in Great Britain

SYNTHESIS OF THE INNER MITOCHONDRIAL

MEMBRANE AND THE INTERCALATION OF

RESPIRATORY ENZYMES DURING THE

CELL CYCLE OF CHLORELLA

B. G. FORDE*, B. E. S. GUNNINGf AND P. C. L. JOHN|Department of Botany, The Queen's University,Belfast BT7 iNN, Northern Ireland

SUMMARY

The ratio of inner to outer mitochondrial membrane area remains close to 1 -8 throughout thecell cycle in synchronized cells of Chlorella ftisca var. vacuolata 2ii-8p. Using estimates of thisratio, together with our previous estimates of mitochondrial surface area, to calculate the absolutearea of inner mitochondrial membrane, it is demonstrated that growth of the inner mitochon-drial membrane during the cell cycle occupies an extended period and parallels the growth of thewhole cell. In contrast, the synthesis of succinate dehydrogenase and cytochrome oxidase isrestricted to the last third of the cell cycle. It is concluded that mitochondrial growth involvesthe intercalation of periodically synthesized respiratory enzymes into membranes made earlierin the cycle, with consequent 5 -fold changes in the density of active enzyme molecules in themembrane. These observations are discussed in relation to the control of mitochondrialmembrane synthesis, membrane assembly and respiration rate during the cell cycle.

INTRODUCTION

Changes in the rate of respiratory enzyme accumulation have been reported duringprogress through the cell cycle of Saccharomyces (Cottrell & Avers, 1970), in Chlorella(Forde & John, 1973), in Euglena (Davis & Merrett, 1974) and in cultured cells ofAcer (King, Cox, Fowler & Street, 1974). These observations raise the possibility thatthe synthesis of enzymes of the inner mitochondrial membrane may be restricted toa period of the cycle when new membrane is being synthesized and is available toaccomodate them, but no corresponding data for growth of the inner mitochondrialmembrane have previously been available.

A hypothesis which could explain the periodicity of membrane growth (Barath &Kiintzel, 1972a, b) proposes that nuclear genes for mitochondrial enzymes are de-repressed when newly synthesized, and in the strain of Chlorella which has been studiedseveral respiratory enzymes are indeed synthesized immediately after the 5-phase(Forde & John, 1973).

• Present addresses: B. G. Forde, Institut fur Biochemie und Experimentelle, Krebsfor-schung, Universitat Innsbruck, Innsbruck, Austria.

•f Present address: B. E. S. Gunning, Research School of Biological Sciences, AustralianNational University, Box 4 P.O., Canberra, A.C.T. 2600, Australia.

% Author to whom requests for reprints should be addressed.

330 B. G. Forde, B. E. S. Gunning and P. C. L. John

We have therefore extended our previous investigations, aimed at correlating struc-tural with biochemical events in the cell cycle, to determine whether the inner mito-chondrial membrane displays a burst of synthesis concurrent with the formation ofrespiratory enzymes.

METHODS

Chlorellafusca var. vacuolata 21 i-8p was grown autotrophically and synchronized by a regimeof 15 h light and 9 h darkness, with dilution at the end of the dark period to maintain a densityof s x io8 cells per ml, as described previously (McCullough & John, 1972). Conditions ofgrowth are therefore identical with those employed by Forde & John (1973) for the study ofrespiratory enzymes and with those employed by Atkinson, John & Gunning (1974) for thestudy of organelle growth during the cell cycle of this strain.

Enzyme assay

The procedure of Forde & John (1973) was followed for the harvesting of cells, preparation ofcell-free extracts and assay of enzyme activity.

Electron microscopy

Cells were fixed with permanganate, because this procedure gives a greater prominence tomitochondrial membranes, as seen in Figs. 2-5, and so allows more rapid stereological analysisof these components than does fixation with glutaraldehyde and post-fixation with osmium(Atkinson, Gunning & John, 1972; Atkinson et al. 1974). The ratio of inner to outer mitochon-drial membrane area, which was observed in cells fixed with permanganate, is not peculiar tothis fixative since the same ratio was observed when an asynchronous sample of cells, whichhad been fixed in glutaraldehyde and then osmium, was analysed in the same way.

Samples of culture containing 1-25 x io8 cells were centrifuged at 2000 g for 2 min and thecells were suspended in 25 ml 2 % (w/v) aqueous potassium permanganate for 2 h at 20 °C.Cells were then freed from permanganate by 2 repetitions of centrifugation and resuspension in25 ml 0-025 M sodium phosphate buffer, pH 7-0 at o °C. The cells were then suspended in thesame buffer containing 2-5 % (w/v) egg albumin. A pellet formed by centrifugation in thealbumin solution was fixed by overlaying with 2-5 % (w/v) glutaraldehyde (Atkinson et al.1972). Small portions of the pellet were dehydrated in acetone, infiltrated with Spurr low-viscosity resin (Spurr, 1969) and sectioned as described previously (Atkinson et al. 1974).

Quantitative stereologyTo avoid sampling bias (Wiebel, 1969), random samples of sections were obtained by making

use of the random orientation of cells in blocks prepared for sectioning and then making micro-graphs of every cell encountered in a regular search of the grid. Lens hysteresis was countered inthe A El EMB microscope by reducing magnification from the maximum setting to a standardvalue of 7500. Micrographs were printed at a final magnification of 32400.

To estimate the ratio of the areas of inner and outer mitochondrial membrane, the followingprocedure was adopted. Only mitochondria in which at least 75 % of the outer membrane wasclearly continuous were measured, and the simple outline of the outer membrane could then becompleted before scoring. A transparent overlay was marked with 3 sets of parallel lines 44 mmapart and each set of parallel lines was oriented at an angle of 120 ° to the others. Each micro-graph was analysed for the number of intersections between lines on the overlay and outer andinner mitochondrial membrane. As an aid to accuracy of scoring, membranes were pencilled inmore prominently before the micrograph was overlaid. Intersections with adjacent inner andouter membrane were scored once and included in totals for both membranes, and intersectionswith the cristae were scored once and doubled to allow for the double membrane. Tangentialintersections with membrane were assessed as either complete intersections and so given a fullscore, or as glancing sections and so given a half score. Scores for cristae are subject to error

Mitochondria! membrane and enzyme synthesis 331

where the membrane was sectioned at shallow angles, and the consequent Holmes effect wascorrected by a factor of 1-5, according to Sitte (1967) and Wiebel (1969). It should therefore benoted that estimates of absolute membrane area are partly determined by the assumption of thisfactor, but the essential observation of constant ratio of inner to outer membrane area is indepen-dent of the assumption. No correction was necessary for the outer membrane, since mito-chondrial profiles in which the outer membrane was indistinct were discarded.

For statistical analysis, a total of 30 mitochondrial profiles were analysed at each time ofsampling. Intersections with inner membrane were calculated as percentages of total mitochon-drial membrane intersections for each profile and, because values are ratios, it is expected thatthe individual estimates will deviate from a normal distribution about the mean for each timeof sample by showing some degree of skewness. If skewness is pronounced then the conventionalexpression for calculation of confidence limits, which assumes a normal distribution of indi-vidual estimates about the mean, is inaccurate if applied to untransformed data (Snedecor &Cochran, 1967). Therefore the estimates in each sample were tested for deviation from a normaldistribution by the method of Snedecor & Cochran (1967). Skewness was slight in all samplesand was only statistically significant in samples taken at o and 24h. In these samples an estimateof confidence limits was obtained by transforming the data to a normal distribution. All thelimits shown in Fig. 1 indicate 95 % confidence levels.

RESULTS

Mitochondrial membrane ratios

The ratio of inner to outer mitochondrial membrane area is shown in Fig. 1, whichalso illustrates in diagrammatic form the timing of some events in the cell cycle.Details of ultrastructural development during the cell cycle have been providedelsewhere (Atkinson et al. 1972; John, McCullough, Atkinson, Forde & Gunning,1973; Atkinson et al. 1974).

Micrographs representative of those used in stereological analysis are seen in Figs.2-5. There is no indication of conformational change in cristae with progress throughthe cycle. The apparently constant relationship between inner and outer mitochon-drial area is confirmed quantitatively in Fig. 1. It is clear that the changing emphasisof structural development which is involved in progress through the cell cycle doesnot disturb the closely co-ordinated growth of the inner and outer membrane, andthese maintain an area ratio close to i-8 throughout the cell cycle.

Mitochondrial membrane growth in the cell cycle

Estimates of total outer mitochondrial membrane area have previously been made byAtkinson et al. (1974) using measurements of organelle volume and diameter atdifFerent stages in the cycle. These values, along with the present estimate of membraneratios (Fig. 1) which were obtained under identical conditions of culture, allow calcu-lation of absolute inner mitochondrial membrane areas during the cell cycle. As seenfrom Fig. 6, growth of the inner and outer membranes occurs during the 15-h periodof illumination. Membrane growth therefore parallels growth in total cell volume(Atkinson et al. 1974) and slows only as total protein synthesis slows with entry intocytokinesis (Forde & John, 1973).


25

20

o1-5

2 10

E 05

I

12Time, h

16 20 24

Fig. i. The ratio of inner to outer mitochondrial membrane area during the cell cyclein a synchronized population of Chlorella. Procedures for cell culture, stereologicaland statistical analysis are detailed in the methods section and 95 % confidence limitsare shown. Some events in the cell cycle are indicated diagrammatically above.Additional information concerning ultrastructural and biochemical events in the cellcycle are given by Atkinson et al. (1972, 1974) and John et al. (1973).

Mitochondrial enzyme synthesis in the cell cycle

Synthesis of succinate dehydrogenase was followed in the culture sampled formeasurement of membrane area. The activity of succinate dehydrogenase increasedfrom 15 h in the cycle (top curve in Fig. 7) and the close correlation between thispattern and the routinely observed pattern (Forde & John, 1973, 1974), which isshown as the middle curve in Fig. 7, therefore indicated that these cells which weresampled for stereological analysis were carrying out the usual periodic accumulationof respiratory enzymes. The pattern of cytochrome oxidase accumulation under thoseconditions of culture is also shown as the lower curve in Fig. 7, so that the accumula-tion of both enzymes can be contrasted with the growth of the inner mitochondrialmembrane (Fig. 6) in which they become located (Tzagoloff, Rubin & Sierra, 1973;Harmon, Hall & Crane, 1974; Janki, Aithal, Tustanoff & Ball, 1975).

The inner and outer mitochondrial membranes (Fig. 6) grow in step with the wholecell (Atkinson et al. 1974) but the synthesis of succinate dehydrogenase and cyto-chrome oxidase (Fig. 7) is restricted to the last third of the cycle, when mitochondrialmembrane synthesis and cell growth pause with the onset of cytokinesis. The conse-quent fluctuation in enzyme activity per unit area of inner membrane is demonstratedin Fig. 8. To derive the estimate of cytochrome oxidase activity at 4 h in this figure it

Mitochondrial membrane and enzyme synthesis 333

Figs. 2-5. The conformation of mitochondria (m) in cells taken from the culture employedfor stereological analysis at 4, io, 16 and 24 h, respectively. The figures show areastaken from micrographs typical of those employed for the quantitative stereologicalanalysis presented in Fig. 1. No change in the conformation of cristae is apparent withprogress through the cell cycle. Magnifications for Figs. 2-5, respectively are:x 25 800, x 26800, x 22 500, and x 28500.


150

100

50

o E

112 16

Time, h20 24

Fig. 6. The increase in inner (O) and outer (A) mitochondrial membrane area percell, during the cell cycle. Data for outer mitochondrial membrane area are taken fromAtkinson et al. (1974) and the area of inner membrane is calculated by using theseand the mean values for ratio shown in Fig. 1. The area shown at 24 h is distributedbetween 4 unreleased daughter cells.

4)h 4

§§ *DO U P

S S V

•S 1 .3

« £•-£.

~ 4

S I12u a* OJ« c u

s ° 2•S -5 3 8

i 1 s 4

U

.o—o—0-0

-t.-"'

8 12 16Time, h

20 24

Fig. 7. The activities of succinate dehydrogenase and cytochrome oxidase during thecell cycle under the conditions of culture employed for stereological analysis. Theactivity of succinate dehydrogenase in the cells employed in this investigation (O).and the pattern of accumulation of succinate dehydrogenase (A) and cytochromeoxidase ( • ) which is normally observed under these conditions of culture (Forde &John, 1973) are shown.


was necessary to interpolate a value from the lower curve in Fig. 7. Because of theslight decline in enzyme activity, and particularly in cytochrome oxidase activity,which occurs during the phase when enzyme is not being synthesized, the dilutingeffect of membrane growth in this period is supplemented, and enzyme activity perunit of inner mitochondrial membrane area falls to about a fifth of the peak levelpresent at the end of enzyme synthesis.

10

0 8

0-6

o uM c

u O« E

| = 0 4C CID O

O w

a:

0-2

12Time, h

16 20

Fig. 8. The activity of succinate dehydrogenase (O)an<l cytochrome oxidase (A) Pe r

unit of inner mitochondrial membrane area during the cell cycle. Data for enzymeactivity are taken from Fig. 7 and for membrane area from Fig. 6.

DISCUSSION

During the cell cycle of Chlorella, the ratio of inner to outer mitochondrial membranearea does not deviate significantly from i-8. There is therefore a close parallel betweenthe rates of growth of the 2 membranes and this is not disturbed by the oscillation ofmean mitochondrial radius from 240 nm to a minimum of 174 nm in mid cell cycle,or by the process of cleavage by the developing septum during cytokinesis (Atkinsonet al. 1974). The close co-ordination of inner and outer membrane growth is reflectedin a constant ratio of inner membrane area to total mitochondrial volume, since theestimates of area presented in Fig. 6 can be compared with previous estimates ofmitochondrial volume (Atkinson et al. 1974) and show that the inner membrane isheld at an area of 30-40 /tin8 per /tm3 of mitochondrial volume throughout the cellcycle.

There is evidence that, as well as being balanced against each other, the growth ofthe 2 mitochondrial membranes keeps pace smoothly with the growth of the cell. Pre-vious estimates of organelle volume through the cell cycle of Chlorella have indicatedthat the major organelles in general grow in proportion to growth of the whole cell.Comparison of whole cell volume (Atkinson et al. 1974) with the area of inner mito-chondrial membrane presented in Fig. 6 shows that this co-ordination also extends tothe inner membrane. The beginning of the cell cycle is marked by slight expansion ofwhole cell volume and this is paralleled by an expansion of inner membrane area,


as well as an expansion of the other cell organelles (Atkinson et al. 1974). This isprobably a recovery from the slight concentration of cytoplasm which occurs atcytokinesis, when appreciable intercellular spaces are formed together with daughtercells inside the persisting mother cell wall (Atkinson et al. 1972). During growth of thecell there is a smooth proportionate growth of the inner membrane, which does notdeviate significantly from values between 0-7 and I-I /im2 per /im3 of cell volume.

In some cultured animal cells (Schnedl, 1974) as in Chlorella (Atkinson et al. 1974),growth of a single mitochondrial continuum extends throughout interphase, but dataconcerning growth of the inner mitochondrial membrane through the cell cycle havenot previously been available, although these are valuable for understanding thebalance between organelle growth and whole cell growth. Some effects of inhibitorsof mitochondrial activity have been interpreted to indicate that mitochondrial productsrepress the activity of nuclear genes which code for mitochondrial enzymes, and soa balance of organelle growth and cell growth may be achieved. The hypothesis isattractive because when further mitochondrial growth is required, either because the cellhas grown or because nuclear gene products have begun to limit growth of theorganelle, a lower repressor concentration is predicted and consequent nuclear geneexpression would initiate the required growth by the organelle. Two publishedhypotheses, based upon this mechanism, propose that mitochondrial growth anddivision are synchronized with the nuclear cycle. Both hypotheses envisage thatnewly replicated nuclear genes which code for mitochondrial enzymes are de-repressedwhen first formed and then later in the cycle become repressed again by mitochondrialproducts following a phase of mitochondrial growth and division. Evidence of mito-chondrial division in Tetrahymena (Lloyd, Turner, Poole, Nicoll & Roach, 1971)has led to the proposal that new copies of nuclear genes code for proteins which promotemitochondrial division. Evidence of enzyme de-repression in Neurospora (Barath &Kiintzel, 1972 a, b) has led to the economical proposal that new de-repressed copiesof nuclear genes code for both mitochondrial respiratory enzymes and also for enzymesof the organelle's biosynthetic equipment, which are envisaged as initiating a burst ofmitochondrial membrane growth to acommodate the new respiratory enzymes. Theevidence presented in this paper indicates, however, that growth of the inner mito-chondrial membrane resembles that of the whole organelle and closely follows thegrowth of the cell, being at a minimum after the «S-phase (Fig. 6) when inner membranerespiratory enzymes accumulate (Fig. 7). There is no evidence for a constraint uponmembrane synthesis at this time, because amino acid labelling shows that proteinsynthesis is continuing (Forde & John, 1974) and other membranes are seen to pro-liferate during cytokinesis at this time (Atkinson et al. 1972). From the presentevidence in Chlorella we therefore propose that interaction between organelle andnucleus to adjust mitochondrial growth is not necessarily restricted to the periodafter the 5-phase in the cell cycle.

The availability of information concerning inner mitochondrial membrane growthduring the cell cycle also throws light on the mechanism of membrane assembly.It is clear that enzyme activities can accumulate at a time when inner membranegrowth is minimal, yet there is extensive and consistent evidence that succinate

Mitochondria! membrane and enzyme synthesis 337

dehydrogenase and cytochrome oxidase do become located in the inner membrane(Tzagoloff et al. 1973; Harmon et al. 1974; Janki et al. 1975) and we have reportedearlier that, although the increase in succinate dehydrogenase activity representscytoplasmic synthesis of new enzyme molecules, the enzyme is incorporated intoa particulate form before gaining catalytic activity (Forde & John, 1974). We thereforeconclude that the structural development of inner mitochondrial membrane, whichoccurs in parallel with cell growth, is not dependent upon the concurrent synthesisof its respiratory enzyme complement and also that the cytochrome oxidase andsuccinate dehydrogenase molecules can later be inserted into the existing membranewithout a requirement for concurrent synthesis of membrane.

We have also investigated the possibility that the mitochondrion itself must synthe-size proteins of the inner membrane to act as sites for the attachment and maturationof the cytoplasmically synthesized succinate dehydrogenase. The organelle certainlycontributes some proteins to the membrane, since the identified products of mito-chondrial protein synthesis now include components of cytochrome b (Weiss, 1972),ATPase (Tzagoloff et al. 1973) and cytochrome oxidase (Mason & Schatz, 1973),all of which are proteins located in the inner membrane. However, the effect of D-f/treo-chloramphenicol, which at o-8 mg per ml selectively inhibits organelle proteinsynthesis in our strain of Chlorella (Forde & John, 1974), argues against a contribu-tion of the mitochondrion to succinate dehydrogenase incorporation. When the in-hibitor was present from 11 h in the cycle the enzyme was still incorporated into themembrane, since the amount of enzyme present in the soluble fraction, after 30 mincentrifugation at 10 000 g, was no greater at the end of the period of enyzme accumu-lation in inhibited cells than the 5 % which is detected in this fraction in control cells.Therefore, if the organelle does synthesize proteins necessary for the incorporationand activation of succinate dehydrogenase it does so more than 4 h before the enzymebegins to accumulate. Certainly no concurrent synthesis of proteins by the organelleis necessary for the incorporation of succinate dehydrogenase into the mitochondrion.

The demonstration that membrane growth and respiratory enzyme synthesis arenot temporarily co-ordinated in the cell cycle, points to the unexpected conclusionthat enzyme density in the membrane changes considerably during the cell cycle.Although the growth of membrane during the period when enzyme synthesis doesnot occur is 4-fold, the decline in active enzyme density approaches 5-fold, becausethere is commonly a slight decline in enzyme levels during the period when theseenzymes are not accumulated. This fluctuation in enzyme density would not bepredicted from the surveys of Srere (1972) and others (Kistler & Weber, 1975), whonoted a correlation between inner membrane area and respiratory enzyme activity inseveral investigations of animal tissues. It is clear from the present study that sucha correlation may represent only an average value for the population and if respiratoryenzyme synthesis is discontinuous, then enzyme-to-membrane ratio in each individualcell can change several-fold with progress through the cell cycle, as it does in Chlorella.Since there is evidence for periodic accumulation of succinate dehydrogenase insynchronized cultures of mouse cells (Bosmann, 1971), the same fluctuation of enzymedensity may occur in mammalian cells during the cell cycle.


There are precedents for the phenomenon of enzyme intercalation into existingmembrane. The process is readily reconciled with the general model of a fluidmosaic for membrane structure (Singer & Nicholson, 1972) and there are someinstances of changes of enzyme composition in mitochondria, in response to changesof metabolism. The situation which has been most intensively studied is the initiationof aerobic respiration in mitochondria of yeast. This profound change in respiratorymetabolism is accompanied by considerable synthesis of both new respiratoryenzymes and inner mitochondrial membrane and it is reasonable to suppose that muchnew enzyme is incorporated into new membrane. Recent elegant evidence from tran-sition temperatures for activation energy is consistent with this view and indicates thatcytochrome oxidase synthesized after transfer to aerobic growth is influenced byproximity to fatty acids which are also absorbed into the inner membrane aftertransfer, but it is not clear whether synthesis of totally new membrane is an absolutenecessity for the accomodation of new enzyme (Janki et al. 1975). Under conditionsof milder metabolic change there is clearer evidence that the enzyme content of theinner mitochondrial membrane can be modified by insertion of enzymes. In heartmuscle and liver cells of the rat, thyroxine deficiency reveals an independent regulationof inner mitochondrial membrane synthesis and of respiratory enzyme synthesis(Reith, Brdicka, Nolte & Staudte, 1973). The mitochondria of rice plant coleoptiles,when exposed to an increase in oxygen tension, can increase cytochrome oxidaseactivity without increase of inner membrane area (Opik, 1973). A capacity for thegeneral insertion of new material into the inner mitochondrial membrane is alsoindicated by the even dilution of cytochrome oxidase activity throughout the membraneof HeLa cells when further synthesis of the enzyme is inhibited by D-^reo-chlor-amphenicol (Storrie & Attardi, 1973). The present report has novelty in identifyingchanges of enzyme density, brought about by insertion of new enzyme molecules, asa recurring part of mitochondrial biogenesis during the cell cycle of synchronizedChlorella cells.

The identification of up to 5-fold changes in the density of respiratory enzymes inthe inner mitochondrial membrane during the cell cycle has implications for theorganization and function of these enzymes. The possibility that respiratory enzymesmight form functional complexes organized on the surface of the inner membrane hasbeen evaluated by Srere (1972). Some of the evidence considered in favour of thehypothesis is a constant ratio between inner membrane area and respiratory enzymelevel in several mammalian tissues, with the implication that synthesis of membrane isco-ordinated with synthesis of enzyme to allow organization of the enzymes. Thepresent investigation makes it clear that such a co-ordination may not exist in anindividual cell in which respiratory enzyme synthesis is periodic. However, the hypo-thesis that matrix enzymes are organized into functional aggregates remains anattractive explanation for the efficient working of the tricarboxylic acid cycle at lowsubstrate concentrations (Srere, 1972) and for the transition in activation energy withphase change of the inner membrane, which is shown by enzymes not themselvesincorporated into the membrane (Matlib & O'Brien, 1975). The hypothesis remainsconsistent with the available evidence in Chlorella and may provide an explanation for


the capacity of the cell to increase its rate of aerobic respiration by 4-fold in the first15 h of the cycle, in proportion to the growth of the cell (McCullough & John, 1972).The increase in rate of respiration occurs in spite of a concurrent 5-fold decline inrespiratory enzyme density at the inner mitochondrial membrane. It is thereforepossible that the respiratory enzymes are organized in functional aggregates, in whichtheir spatial inter-relationships are not disturbed by growth of the surrounding mem-brane. We shall show elsewhere (E. Cole & P. C. L. John, unpublished observations)that several tricarboxylic acid cycle enzymes show a degree of co-ordinate regulation inthe normal cell cycle and under conditions of adaptive synthesis and therefore couldmaintain the relative proportions required for assembly of functional aggregates.

We therefore conclude that mitochondrial biogenesis in the cell cycle of Chlorellainvolves a capacity for growth of the inner membrane in proportion to growth of thecell, with the subsequent insertion of periodically-synthesized respiratory enzymes.Further investigation is required to understand the control of these temporallyseparate processes.

We thank the Science Research Council for support and Mr G. McCartney and Mr D.Kernoghan for expert technical assistance with electron microscopy and photography. We areindebted to Dr A. C. Butcher for statistical analysis. Dr B. G. Forde thanks the Department ofEducation, Northern Ireland, for a post-graduate research studentship and The Queen'sUniversity of Belfast, for a Foundation Studentship.

REFERENCESATKINSON, A. W., JR., GUNNING, B. E. S. & JOHN, P. C. L. (1972). Sporopollenin in the celt

wall of Chlorella and other algae: ultrastructure, chemistry, and incorporation of C14-acetate,studied in synchronous cultures. Planta 107, 1-32.

ATKINSON, A. W. JR., JOHN, P. C. L. & GUNNING, B. E. S. (1974). Growth and division of thesingle mitochondrion and other organelles during the cell cycle of Chlorella, studied byquantitative stereology and three-dimensionsal reconstruction. Protoplasma 81, 77-109.

BARATH, Z. & KUNTZEL, H. (1972a). Co-operation of mitochondrial and nuclear genes speci-fying the mitochondrial genetic apparatus in Netirospora crassa. Proc. natn. Acad. Sci. U.S.A.69, I37I-I374-

BARATH, Z. & KUNTZEL, H. (19726). Induction of mitochondrial RNA polymerase in Neuro-spora crassa. Nature, New Biol. 240, 195-197.

BOSMAN, H. B. (1971). Mitochondrial biochemical events in a synchronized mammalian cellpopulation. J. biol. Chem. 246, 817-823.

COTTRELL, S. F. & AVERS, C. J. (1970). Evidence of mitochondrial synchrony in synchronouscell cultures of yeast. Biochem. biophys. Res. Commun. 38, 973-980.

DAVIS, B. & MERRETT, M. J. (1974). The effect of light on the synthesis of mitochondrialenzymes in division synchronized Euglena cultures. PI. Physiol., Lancaster 53, 575-580.

FORDE, B. G. & JOHN, P. C. L. (1973). Stepwise accumulation of autoregulated enzyme activi-ties during the cell cycle of the eucaryote Chlorella. Expl Cell Res. 79, 127-135.

FORDE, B. G. & JOHN, P. C. L. (1974). Transcription translation and maturation of succinatedehydrogenase during cell cycle. Nature, Lond. 252, 410-412.

HARMON, H. J., HALL, J. D. & CRANE, F. L. (1974). Structure of mitochondrial cristae mem-branes. Biochim. biophys. Acta 344, 119-155.

JANKI, R. M., AITHAL, H. N., TUSTANOFF, E. R. & BALL, A. J. S. (1975). The biogenesis ofmitochondrial membranes in the yeast Saccharomyces cerevisiae. Biochim. biophys. Acta 375,446-461.


JOHN, P. C. L., MCCULLOUGH, W., ATKINSON, A. W. Jr., FORDE, B. G. & GUNNING, B. E. S.(1973). The cell cycle in Chlorella. In The Cell Cycle in Development and Differentiation,vol. 1 (ed. M. Balls & F. Billett), Symp. Br. Soc. dev. Biol. pp. 61-76. Cambridge UniversityPress.

KING, P. J., Cox, B. J., FOWLER, M. W. & STREET, H. E. (1974). Metabolic events in synchro-nised cell cultures of Acer pseudoplatanus L. Planta 117, 109-122.

KISTLER, A. & WEBER, R. (197s). A morphometric analysis of inner membranes related tobiochemical characteristics of mitochondria from heart muscle and liver in mice. Expl Cell Res.91, 326-332.

LLOYD, D., TURNER, G., POOLE, R. K., NICHOLL, W. G. & ROACH, G. I. (1971). A hypothesisof nuclear-mitochondrial interactions for the control of mitochondrial biogenesis based onexperiments with Tetrahymena pyriformis. Sub-Cell. Biochem. 1, 91-95.

MASON, T. L. & SCHATZ, G. (1973). Cytochrome oxidase from baker's yeast. II. Site of transla-tion of the protein components. J. biol. Chem. 248, 1355-1360.

MATLIB, M. A. & O'BRIEN, P. J. (1975). Compartmentation of enzymes in the rat liver mito-chondrial matrix. Archs Biochem. Biophys. 167, 193-202.

MCCULLOUGH, W. & JOHN, P. C. L. (1972). Temporal control of the de novo synthesis ofisocitrate lyasc during the cell cycle of the eucaryote Chlorella pyrenoidosa. Biochim. biophys.Acta 269, 287-296.

OPIK, H. (1973). Effect of anaerobiosis on respiratory rate, cytochrome oxidase activity andmitochondrial structures in coleoptiles of rice (Oryza sativa L.). J. Cell Set. 12, 725-739.

REITH, A., BRDICKA, D., NOLTE, J. & STAUDTE, H. W. (1973). The inner membrane of mito-chondria under influence of triiodothyronine and riboflavin deficiency in rat heart muscle andliver. Expl Cell Res. 77, 1-14.

SCHNEDL, W. (1974). Veriinderungen des Mitochondrienbestandes wShrend des Zellzyklus.Cytobiologie 8, 403-411.

SINGER, S. J. & NICHOLSON, G. L. (1972). The fluid mosaic model of the structure of cellmembranes. Science, N.Y. 175, 720-731.

SITTE, H. (1967). Morphometrische Unterschungen an Zellen. In Quantitative Methods inMorphology (ed. E. R. Wiebel and H. Elias), pp 167-198. Berlin, Heidelberg and New York:Springer Verlag.

SNEDECOR, G. W. & COCHRAN, W. G. (1967). Statistical Methods. Iowa State University Press.SPURR, A. J. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy.

J. Ultrastruct Res. 26, 31-43.SRERE, P. A. (1972). Is there an organisation of Krebs cycle enzymes in the mitochondrial

matrix? In Energy Metabolism and the Regulation of Metabolic Processes in Mitochondria(ed. M. A. Mehlman & R. W. Hanson), pp. 79-91. New York and London: Academic Press.

STORRIE, B. & ATTARDI, G. (1973). Mode of mitochondrial formation in HeLa cells. J. Cell Biol.S6, 833-838.

TZAGOLOFF, A., RUBIN, M. S. & SIERRA, M. F. (1973). Biosynthesis of mitochondrial enzymes.Biochim. biophys. Acta 301, 71-104.

WEISS, H. (1972). Cytochrome b in Neurospora crassa mitochondria. A membrane proteincontaining subunits of cytoplasmic and mitochondrial origin. Eur. J. Biochem. 30, 469—478.

WIEBEL, E. R. (1969). Stereological principles for morphometry in electron microscopiccytology. Int. Rev. Cytol. 25, 235-302.

(Received 26 August 1975)

synthesis of the inner mitochondrial membrane and … · harmon, hall & crane, 1974; janki,...

Documents