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Bioehimica et Biophysics Actu, 489 (1977) 343-347 0 Elsevier/North-Holland Biomedical Press

BBA Report

BBA 51213

POLYPRENYLPYR~PHOSP~ATE:~-HYDROXY3ENZOATE- POLYPRENYLTRANSFERASE ACTIVITY IN YEAST

RELATIONSHIP TO ~ITOCHONDRIAL DEVELOPMENT

JOHN CASEY and DAVID R. THRELFALL

Department of Plant Biology, University of Hull, Hull HU6 7RX (U.K.)

(Received May 16th, 1977)

Summary

Polyprenylpyrophosphate: 4-hydroxybenzoate-polyprenyltransf~rase activity is highest in aerobically grown yeast cells and lowest in anaerobi- cally grown cells. Its production is subject to catabolite repression in aerobic cells and the increase in its activity in anaerobic cells undergoing respiratory adaptation is inhibited by cycloheximide.

It is generally accepted that octaprenylpyrophosphate 4-hydroxyben- zoate polyprenyltransferase is the first enzyme on the biosynthetic pathway leading from 4-hydroxybenzoate to ubiquinone-8 in the bacterium Esche- richia coli [ 11. Polyprenylpyrophosphate:4-hydroxybenzoate-polyprenyl- transferase (hydroxy benzoate polyprenyltransfer~e} activity has been shown to be associated with the inner membrane of rat liver mitochondria [Z] and the mitochondrial membranes of Succ~a~o~yces cereuisicae, S. curls- bergensis and broad bean seeds [ 3,4] . As yet, however, there is no clearcut evidence to support the invoIvement of hydroxybenzoate-polyprenyltrans- ferases in the biosynthesis of ubiquinone by animals and plants, since it has not been possible to demonstrate the presence in these organisms of any mito- chondrial enzymes that are capable of using 4-hydroxy-3-polyprenylben- zoates as substrates. Indeed Olson and his colleagues [ 51 have presented a substantial body of indirect evidence which indicates that in rat liver mito- chondria the first enzyme on the pathway is a hydroxybenzoate-CoA-nona- {and deca) prenyltransferase. This group of workers have also suggested that mitochondri~ hydroxybenzoate-polyprenyltransferase activity is not due to a single enzyme but is attributable to the sequential action of a hydroxy- benzoate-CoA ligase, a hydroxybenzoate-CoA-polyprenyltransferase and a non-specific CoA deacylase [5], However, we have been unable to obtain

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evidence for the operation of this in vitro sequence of reactions in yeast mitochondria and we are of the opinion that the first enzyme on the path- way is either a hydroxybenzoate-polyprenyltransferase or a hydroxyben- zoate-CoA-polyprenyltransferase which can act as a hydroxybenzoate-poly- prenyltransferase (Casey, J. and Threlfall, D.R., unpublished). Clearly a knowledge of the precise details of the reactions catalysed by the first enzyme on the animal and plant pathways of ubiquinone biosynthesis is important as far as a full understanding of the pathways is concerned, however, even without this knowledge the hydroxybenzoate-polyprenyl- transferase activity exhibited by mitochondrial membranes can be used in the study of the biosynthesis of polyprenyl units by mitochondria [ 21 and the factors affecting the in vivo synthesis of the polyprenyltransferase itself, be it a hydroxybenzoate-polyprenyltransferase or a hydroxyben- zoate-CoA-polyprenyltransferase with in vitro hydroxybenzoate-polyprenyl- transferase activity.

In this communication we report the effects of anaerobiosis, catab- olite repression and antibiotic inhibitors of protein synthesis on the hydroxy- benzoate-polyprenyltransferase activity of the mitochondrial membranes of S. carlsbergensis NCYC 14.

To investigate the effects of anaerobiosis and catabolite repression, the organism was grown (a) anaerobically at 30°C in 1800 ml of 2% (w/v) glucose-containing medium [6] supplemented with 0.5% (w/v) Tween 80 and 0.002% (w/v) ergosterol, (b) aerobically at 30°C in 700 ml of 2% (v/v) ethanol, 0.5% (w/v) mycological peptone and 0.3% (w/v) yeast extract in water and (c) aerobically at 30°C in 700 ml of 10% (w/v) glucose, 0.5% (w/v) mycological peptone and 0.3% (w/v) yeast extract in water. Each of the cultures was inoculated by adding 10 ml of aerobic culture that had been grown overnight on a 1% (w/v) glucose-containing medium. The aerobic cultures were grown in 2-1 flasks with constant agitation (200 rev./min) in an orbital shaker.

Early- to mid-exponential aerobic ceils and 48 h old anaerobic cells were harvested by centrifugation, and resuspended in 0.3 M mannitol/ 0.05 M phosphate buffer (2 vol./g fresh wt. of ceils), pH 7.5. The cells were then broken by one passage through a pressure cell (10 000 lb/inch’ ) and the resultant homogenate centrifuged at 1500 X g for 10 min to remove whole cells and cell debris followed by 100 000 X g for 1 h to sediment mito- chondrial membranes. The 100 000 X g membrane preparation was resus- pended in 2 ml of 0.05 M phosphate buffer, pH 7.5, and assayed for protein content, succinate dehydrogenase (EC 1.3.99.1) [7] and hydroxybenzoate- polyprenyltransferase activities. The latter activity was assayed by meas- uring the incorporation of 14C from 4-hydroxy[ U-r4C] benzoate into 4-hy- droxy-3-polyprenylbenzoates. The incubation mixture for the assay con- sisted of 12 nmol of 4-hydroxy[U-14C] benzoate (7.7 Ci/mol), 0.5 ml of Micrococcus luteus extract that had been preincubated with 2.5 pmoles of Liz IPP (this preparation acts as a course of polyprenylpyrophosphates [4], 50 pmol of MgCl, , 3-11 mg of protein and 2.5 ml of 0.05 M phos- phate buffer, pH 7.5. The mixture was incubated in air for 30 min at 30°C

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with gentle agitation, and then the 14C present in 4-hydroxy-3-polyprenyl- benzoates was determined [4] .

The highest succinate dehydrogenase (87 nmol. min-’ - mg-’ protein) and hydroxybenzoate polyprenyltransferase (7.1 pmol of product. min-’ l mg-’ protein) activities were obtained from the cells grown aerobic- ally on a non-fermentable substrate (ethanol). The two activities were sub- ject to catabolite repression in the presence of a readily fermentable sub- strate (glucose) (succinate dehydrogenase, 5.1 nmol. min-’ - mg-’ protein; hydroxybenzoate-polyprenyltransferase, 4.1 pmol of product*min-’ - mg-’ protein), the very low succinate dehydrogenase activity providing con- firmation that the cells were fully catabolite repressed. In anaerobically grown cells succinate dehydrogenase activity (0.4 nmol. min-’ - mg-’ protein) was almost fully repressed but the hydroxybenzoate-polyprenyltransferase activity (0.76 pmol of product.min-’ *mg-’ of protein), although markedly repressed, was still significantly high.

In view of the above findings we went on to investigate the effects of cycloheximide, an inhibitor of cytoplasmic protein synthesis, and chlor- amphenicol, an inhibitor of mitochondrial protein synthesis, on hydroxy- benzoate-polyprenyltransferase, succinate dehydrogenase and cytochrome oxidase (EC 1.9.3.1) activities in yeast cells undergoing respiratory devel- opment. The activities of the mitochondrial enzymes succinate dehydro-

I I I I 1 I I I

I 1

2 4 6 0 2

Time of adaptation (h)

Fig.1. Yeast cells were grown anaerobically under conditions identical to those described in the text. The cells were harvested after 48 h and then resuspended in 100 ml of an antibiotic-free growth medium containing 1% (w/v) galactose, 0.05% (w/v) mycological peptone. 0.3% (w/v) yeast extract and 0.05 M phosphate buffer, pH 7.5, or 700 ml of growth medium containing 4 mg-ml-’ of chlor- amphenicol or 700 ml of growth medium containing 100 Irgoml* of cycloheximide. The cell sus- pensions (A 6,,o nm = 8.5) were incubated in 2 1 conical flasks at 3O’C with constant agitation (200 rev./min). Samples of the cells were harvested at appropriate time intervals and assayed for succinate dehydrogenase [71 (0 ---u), cytochrome oxidase (EC 1.9.3.1) [Sl ( a-4) and hydroxybenzoate (HBA)-polyprenyltransferase (see text) (0-I) activities.

346

genase and cytochrome oxidase were determined because it had been shown that in yeast cells undergoing respiratory development the induction of cytochrome oxidase is inhibited by chloramphenicol and cycloheximide [ 91 whilst the induction of succinate dehydrogenase is inhibited only by cyclo- heximide [ 91.

In the absence of antibiotics the levels of the three enzyme activities increased throughout the 8 h period of adaptation (Fig. 1). The succinate dehydrogenase and hydroxybenzoate-polyprenyltransferase activities reach- ing 65% and 82% respectively of the activities previously recorded for ethanol grown cells. The addition of chloramphenicol completely sup- pressed the formation of cytochrome oxidase, but had no effect on the production of the two other enzymes. On addition of cycloheximide, how- ever, the de novo synthesis of all three enzymes was inhibited.

The results of these studies show that hydroxybenzoate-polyprenyl- transferase activity, an activity that is associated with the mitochondrial membranes of aerobically grown cells [4], is highest in yeast cells grown aerobically on a non-fermentable substrate. The activity is subject to catab- olite repression in cells grown aerobically on a readily fermentable substrate and is lowest in anaerobically grown cells. The presence of activity in an- aerobic cells and the inhibition by cycloheximide of the increase in the level of this activity in anaerobic cells undergoing respiratory adaptation establish that hydroxybenzoate-polyprenyltransferase is a constitutive enzyme that is synthesised on cytoplasmic ribosomes.

In view of the probable involvement of this enzyme in the biosynthesis of ubiquinone, a non-protein component essential to the activity of the mitochondrial electron transport chain, it is instructive to compare our find- ings with those obtained in similar studies concerned with the formation of ubiquinone in yeast cells. Thus, the highest levels of ubiquinone-6 are found in aerobically grown cells [lo]. The levels are markedly reduced in catab- olite repressed cells and the quinone is barely detectable in anaerobic cells [ 11,12 ] . In anaerobically grown cells undergoing respiratory adaptation in the presence of antibiotics, ubiquinone-6 formation is unaffected by chlor- amphenicol but is 50% reduced in the presence of cycloheximide [ 121. These findings show that all of the enzymes involved in the biosynthesis of ubiquinone-6 are constitutive to the organism, and are present in the anaero- bic cell at levels sufficiently high to carry out the synthesis of significant amounts of ubiquinone-6, even though the synthesis cannot proceed in the anaerobic cell due to the O2 -requirement of some of the terminal steps on the pathway.

Clearly the amounts of ubiquinone-6 synthesised in aerobic cells grown under a variety of physiological conditions and the latent potential of an- aerobic cells to synthesise ubiquinone-6 are qu~itatively related to the hydroxybenzoate-polyprenyltransferase activities of the cells. The relation- ship is not quantitative, however, since the level of transferase activity in the anaerobic cells (10% of the level in aerobic cells) is sufficient to allow the formation of 50% of the amount of ubiquinone-6 found in aerobic cells, whilst the level of transferase activity in catabolite repressed cells (50% of the level in unrepressed cells) is in excess of the requirement for

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the synthesis of the small amounts of ubiquinone-6 (<lo% of the amount in unrepressed cells) found in such cells. These comparisons show that ubi- quinone biosynthesis is not controlled directly by the amounts of hydroxy- benzoate-polyprenyltransferase present in the cells, although the trans- ferase levels may contribute to the overall regulation of ubiquinone bio- synthesis.

We thank Mrs. Susan Swetez for technical assistance. One of us (JC) was in receipt of a grant from the Science Research Council.

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