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Intracellular and Extracellular Levels of Cyclic AMP During the Cell Cycle of Saccharomyces cerevisiae MAXINE E. SMITH*?, J. RICHARD DICKINSON* A N D ALAN E. WHEALS?$ *School of' Pure and Applied Biology, University of Wales College of Cardif, Main Building, Museum Avenue, P.O. 30.y 915, CardifCFl3TL, U.K. t Microbiology Group, School of Biological Sciences, University of Bath, Bath BA2 7A Y , U .K.

Received 24 May 1989; revised 4 August 1989

Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Succharomyces cerevisiae there are stage-specific fluctuations in the intracellular concentration of adenosine 3',5'cyclic monophosphate (CAMP). Results shown here indicate that the intracellular concentration of cAMP is at its highest during the division cycle, and at its lowest immediately prior to and just after cell separation. Results also show the extrusion of extracellular cAMP into the medium by Saccharomyces cerevisiae, extracellular cAMP levels being ten to one hundred times higher than intracellular levels. During the cell cycle of Saccharomyces cerevisiae the extracellular level of CAMP does not fluctuate. KEY WORDS - Cyclic

AMP, Cell Cycle, Saccharomyces cerevisiae

INTRODUCTION Early in the cell cycle of the budding yeast Saccharomyces cerevisiae there is a major regulatory step

called 'start' (Hartwell et al., 1974), when the cell assesses its environmental status. Presence of adequate carbon sources for energy production will lead to commitment of the cell to a series of developmental changes culminating in cell division (Wheals, 1987). Adenosine 3',5'-cyclic monophosphate (CAMP)was first implicated as having a role in the yeast cell division cycle by Matsumoto et al. ( 1982a). Low levels of cAMP were associated with starvation arrest in G1 at 'start' and high levels with growth and proliferation. An entire cAMP generation and degradation system has since been found (Matsumoto et al., 1985; Tatchell, 1986). The membrane-bound CDC25 gene product regulates the activity of the RASl and RAS2 gene products. They in turn control the activity of adenylate cyclase (the CDC35 or CYRZ gene product) which forms CAMP from ATP. The sole function ascribed to cAMP so far is to activate CAMP-dependent protein kinases (protein kinase A) via control of a regulatory protein (the BCYI gene product). Cyclic AMP can be destroyed by two phosphodiesterases (encoded by the genes PDEl and PDE2). The levels :Addressee for correspondence. 0749-503X ;90/01005348 $05.00 0 I990 by John Wiley & Sons Ltd

of cAMP seem to be highly regulated by a complex feedback control mechanism (Nikawa et al., 1987). Both growing cultures and cells containing a hyperactive RAS2"a'19 mutation have high levels of intracellular CAMP; stationary phase cultures and cells with disruptions to RAS2 contain low levels (Nikawa et al., 1987). Abolition of function of the regulatory gene of protein kinase A releases the cell from G I arrest. It is clear that cAMP seems to act as a second messenger, transducing information about the potential growth status of the cell. However, it is not known whether this intracellular CAMP-dependent pathway is (i) a conditional control, allowing proliferation to occur but with the actualcontrol exerted elsewhere, or (ii) whether cAMP levels directly regulate onset of cell proliferation by fluctuating in amount during thecell division cycle, reachinga high level for cell cycle initiation in G1. Consequently, cAMP levels have been measured during a synchronous cell cycle using the technique of centrifugal elutriation (Creanor and Mitchison, 1979), which is currently the least perturbing technique available (Mitchison, 1988). An earlier report on fluctuations in intracellular levels through the cell cycle used what is now considered to be an inappropriate synchronizing procedure with no asynchronous control (Watson and Berry, 1977). We report

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INTRACELLULAR AND EXTRACELLULAR LEVELS OF CYCLIC AMP

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here that intracellular cAMP concentration shows an oscillatory pattern during the cell division cycle, being at its highest during the cycle and its lowest at cell separation. Most of the cAMP is secreted from the cell into the environment.

For both intracellular and extracellular samples. cyclic AMP was assayed as described previously (Brown et al., 1971, 1974). Material that was assayed as cAMP was destroyed in the same way as bonafide cAMP by beef heart phosphodiesterase.

MATERIALS A N D METHODS

RESULTS

The results from a representative experiment are shown in Figure 1 . The synchronous culture was made by collecting cells in the same medium in A Succhuromjsces cererisiae prototrophic diploid which they had been grown. The culture took 40 niin strain D1 was used in this work. It was grown in glucose minimal medium containing (per litre): to load into the elutriator and the unbudded fracglucose (20 g); ammonium sulphate ( 5 g) and yeast tion for the synchronous culture took 20 min to be nitrogen base (without amino acids and ammonium collected. The asynchronous control was made by inoculating the remainder of the cells into fresh sulphate. 1.67 g). medium, which took 2 min. Zero time was when the first measurements were taken occurring 5 min Elutriution after unloading. At zero time the cells in the synAn early exponentially growing culture (OD600nm chronous culture were unbudded and remained so 0.3) of DI was loaded into a Beckman JElOX for approximately 30 min, after which the budded elutriator rotor in a Beckman JE-6M centrifuge, index rapidly increased. During this time the level of maintained at 25°C. The synchronous and asyn- cAMP was maintained at a high level in the cell. As chronous cultures were prepared as described pre- the budding increased to its maximum and the cells viously (Creanor and Mitchison, 1979; White et a f . , approached the end of the first division cycle. the 1986). Cell numbers were obtained using a Coulter level of cAMP decreased rapidly. By the time half Counter model ZM and a Coulter Channelyzer the cells had divided and new cell cycles were being CIOOO. Percentage budding was determined by initiated in the parent cells, the intracellular CAMP concentration was transiently zero. In our hands this microscopic inspection of several hundred cells. means less than 0.05 pmol cAMP per sample. The magnitude of this fluctuation varied between exper.4.s.suj.,fbrC AM P iments depending upon the degree of synchrony. On Intrucellulur C A M P .Samples were taken at specific completion of cell separation. cellular CAMP was time intervals and filtered on Sartorius cellulose ace- restored to a high level. The next synchronous cell tate filters (0.45 pm pore size), and washed once with separation also resulted in a decrease in cAMP but distilled water. Cyclic AMP was extracted by sus- to a different level than previously. The (asynchronpending cells in ice-cold 8 % trichloroacetic acid ous) control culture, which comprised the rest of the (TCA). The TCA was subsequently removed by cells loaded in the centrifuge. showed no significant washing five times with water-saturated diethyl- fluctuations (Figure 2). ether. Extracts were lyophilized and reconstituted in The extracellular level of CAMP was also assay buffer. measured in both asynchronous and synchronous culture (Figure 3) where levels decreased slightly or Estracellulur C A M P .The filtrate, after cell harvest- remained constant throughout the cycle. In both ing and immediately prior to washing with distilled cases there was an extremely high level ofextracelluwater, was collected and boiled for 3 min to destroy lar cAMP which exceeded intracellular levels any phosphodiesterase activity. Samples were then between ten- and one hundred-fold. The extracellustored at - 20°C. lar cAMP appears within minutes of putting cells Struin undgro\c,tli conditions

Figure I . Analysis of a synchronous culture. Cell number, budded index and intracellular cAMPconccntration were measurcd i n ii synchronous culture obtained by centrifugal elutriation. (a) Intracellular cAMP concentration; (b) budded index; (c) cell number; ( d ) diagram of principal cell cycle events. IB, duration of initiation of budding in the population; CS. duration of cell separation in the population; D, daughter cells: P, parent cells.

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Figure 2. Analysis of an asynchronous culture. Cell number, budded index and intracellular cAMP concentration were measured in asynchronous culture obtained by elutriation. For details see legend to Figure 1.

into fresh medium (Figure 4). Figure 4 is expressed on a per cell basis so as to enable a direct comparison to be made between extracellular levels of cAMP and intracellular levels of synchronous and asynchronous CAMP,which are shown in Figures 1 and 2. The three-fold decline observed in Figure 5, which represents the concentration of cAMP per cell from the original asynchronous culture, is due to the increase in cell number as seen in Figure 4, which also showed similar initial reductions.

DISCUSSION Any interpretation of these data must relate to the pronounced asymmetry of yeast cell cycles. Cell

division is asymmetric into larger parents and smaller daughters. Since there is a size control over traverse of start, daughter cells take longer to complete a cycle than their parents (Hartwell and Unger, 1977). This has a number of important consequences for understanding fluctuations in cAMP levels. (i) The initial high level is contributed by a population consisting of very small unbudded daughter cells which were uniform in size (data not shown). High levels of cAMP might be expected at this stage (Matsumoto et al., 1985). (ii) The decrease was associated with the end of one cycle and the onset of a new cycle in parent cells. New bud formations are a cytological manifestation of a cell having traversed start approximately 10 min previously

cAMP PER ML

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Figure 3. Levels of extracellular cAMP in synchronous ( A ) and asynchronous (A)cultures obtained by elutriation.

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Figure 4. Levels of extracellular CAMPin batch culture, taken from exponentially growing cells (A)and stationary cells ( O ) ,after transfer to fresh medium

(Hartwell and Unger, 1977). Low levels of cAMP were not expected at the start of any cell cycle. (iii) The next plateau was substantially (seven-fold) lower than the initial level. The differences between the plateaux of cAMP can be attributed to two factors. First, the daughters are asynchronous with respect to parent cell cycles as described above and consequently the rise may only be due to parent cells which constitute 50% of the population. Secondly, over a period of time, cAMP in batch culture decreases exponentially in concentration, reflecting the decreasing concentration of glucose (Frangois et al., 1987) as seen in the asynchronous control. However the data are not obviously compatible with current proposals on the role of cAMP in growth control. In particular we see both a high

and a low cAMP level associated with cells about to traverse start. The only identifiable differences between the populations involved were that the first high level was associated with small unbudded daughters recently taken from the elutriation chamber, and the second high level was associated with larger first generation parent cells. Since cells were proliferating under both circumstances it is likely that the fluctuations are not causal in regulating the cycle. Furthermore, since very low levels have been associated with GI arrest, it must be presumed that the low levels that have been observed here are still above threshold. One possibility is that the cell uses very low levels of CAMP as a ‘gate’ into the next cell division cycle to enable an assessment of nutrient status.

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Levels of extracellular cAMP in asynchronous culture obtained by elutriation.

Cyclic AMP exerts its effects through a CAMPdependent protein kinase (protein kinase A) activating or inactivating cell proteins. The stage-specific peaks and troughs in cAMP match up to those found in the specific activities of trehalase, phosphofructokinase, hexokinase and pyruvate kinase, but are opposite to those of fructose 1,6-biophosphatase (Van Doorn et al., 1988a,b; Purwin et al., 1982). Both trehalase and fructose 1,6-bisphosphatase are activated by protein kinase A and thus the data presented here provide a causal explanation of the fluctuations in activity of some of these enzymes involved in carbohydrate metabolism. It was important to discover the cause of these changes in intracellular cAMP levels. The most obvious candidates for control were the enzymes of synthesis and degradation but we were unable to detect sufficient activity in synchronous culture to ascertain whether their activities fluctuated in parallel with CAMP. Strains containing both pdel and pdeZ mutations (and hence lacking both the low- and high-affinity phosphodiesterases) are viable (Nikawa et al., 1987). Thus if these decreases in CAMPare vital, then control of CAMP cannot be through this system of degradation. The alternative involves switching adenylyl cyclase on and off. Adenylyl cyclase appears to be regulated by the activity of protein kinase A and by the phosphorylation of the RASZ-encoded protein. As the level of

intracellular cAMP increases, protein kinase A will switch off adenylyl cyclase and thus lower the level of CAMP. Alternatively, another method of control may be operating and evidence has been obtained here that the cell can export cAMP rapidly and in large quantities into the environment. The intracellular fluctuations could thus be ascribed to differential export rather than degradation. Since at all times there was a large extracellular excess, no cell cycle stagespecific change in extracellular cAMP could be detected. What causes the cells to secrete cAMP into the medium? It is not due to the elutriation procedure because it is also found in batch culture (Figure 4 and unpublished results). The level of cAMP in the asynchronous and synchronous cultures is comparable even though the synchronous culture was prepared by maintaining the cells in the same medium and the asynchronous control was made by putting cells into fresh medium. Unlike the relationship between intracellular levels of cAMP and glucose concentration in the medium (FranCois et al., 1987), extracellular levels of cAMP do not depend on this. Figure 3 shows that the level of extracellular cAMP in the asynchronous culture, which has been transferred to fresh medium, is lower than that in the synchronous culture, which has been growing in the original medium. The role of extracellular cAMP could therefore be as a sink.

60 Alternatively a more unusual role could be that of signalling to other cells as a pheromone, with control of cAMP levels involving a decrease in the rate of extrusion or a disposal mechanism for extracellular cAMP possibly in the form of a phosphodiesterase enzyme. Such a role is not without precedent in other microbial eukaryotes. Indeed recent work on sporulation of Saccharomyces cerevisiue suggests there is intercellular communication via purine nucleotides (Jakubowski and Goldman, 1988). However, Saccharomyces cerevisiae is thought to be largely impermeable to cAMP and requires mutational changes to become permeable (Matsumoto e t ul., 1982b). ACKNOWLEDGEMENTS We thank Paul Nurse (ICRF Cell Cycle Control Laboratory, University of Oxford) for the use of the elutriator rotor, and Viesturs Simanis for help with making some of the synchronous cultures. This work was supported in part by the SERC. MES is in receipt of a SERC studentship. REFERENCES Brown, B. L., Albano ,J. D. M., Ekins, R. P. and Sgherzi, A. M. (1971). A simple and sensitive saturation assay method for the measurement of adenosine 3’,5’-cyclic monophosphate. Biochem. J. 121,561-562. Brown, B. L., Albano, J. D. M., Barnes, G. D. and Ekins, R. P. (1974). The saturation assay of adenosine 3’3cyclic monophosphate in tissues and body fluids. Biochem. SOC.Trans. 2,388-390. Creanor, J. and Mitchison, J. M. (1979). Reduction of perturbations in leucine incorporation in synchronous cultures of Schizosaccharomyces pombe. J . Gen. Microbiol. 112,385-388. Frangois, J., Eraso, P. and Gancedo, C. (1987). Changes in the concentrations of CAMP, fructose 2,6-bisphosphate and related metabolites and enzymes in Saccharomyces cerevisiae during growth on glucose. Eur. J. Biochem. 164,369-373. Hartwell, L. H., Culotti, J., Pringle, J. R. and Reid, B. J. (1974). Genetic control of the cell division cycle in yeast. Science 183,46-51. Hartwell, L. H. and Unger, M. W. (1977). Unequal division in Sacchuromyces cerevisiae and its implications for the control of cell division. J. Cell Biol. 75, 422435.

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Jakubowski, H. and Goldman, E. (1988). Evidence for cooperation between cells during sporulation of the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 8, 5 166-5 178. Matsumoto, K., Uno, I., Oshima, Y. and Ishikawa, T. (1982a). Isolation and characterization of yeast mutants deficient in adenylate cyclase and CAMPdependent protein kinase. Proc. Natl. Acad. Sci. USA 79,2355-2359. Matsumoto, K., Uno, I., Toh-e, A,, Ishikawa, T. and Oshima, Y. (1982b). cAMP may not be involved in catabolite repression in Sacchuromyces cerevisiae: Evidence from mutants capable of utilizing it as an adenine source. J . Bacteriol. 150,277-285. Matsumoto, K., Uno, I. and Ishikawa, T. (1985). Genetic analysis of the role of CAMP in yeast. Yeast 1, 15-25. Mitchison, J. M. (1988). Synchronous cultures and age fractionation. In Campbell, I. and Duffus, J. H. (Eds), Yeast: A Practical Approach. IRL Press Ltd, Oxford, pp. 51-63. Nikawa, J., Cameron, S., Toda, T., Ferguson, K. M. and Wigler, M. (1987). Rigorous feedback control of cAMP levels in Saccharomyces cerevisiae. Genes and Development 1,931-937. Purwin, C., Leiding, F. and Holzer, H. (1982). Cyclic AMP-dependent phosphorylation of fructose- 1,6bisphosphate in yeast. Biochem. Biophys. Res. Commun. 107,1482-1489. Tatchell, K. (1986). RAS genes and growth control in Saccharomyces cerevisiae. J . Bacteriol. 166,364367. Van Doorn, J., Scholte, M. E., Postma, P. W., Van Driel, R. and Van Dam, K. (1988a). Regulation of trehalase activity during the cell cycle of Saccharomyces cerevisiae. J . Gen. Microbiol. 134,785-790. Van Doorn, J., Valkenburg, J. A. C., Scholte, M. E., Oehlen, L. J. W. M., Van Driel, R., Postma, P. W., Nanninga, N. and Van Dam, K. (1988b). Changes in activities of several enzymes involved in carbohydrate metabolism during the cell cycle of Sacchuromyces cerevisiae. J . Bacteriol. 170,48084315 . Watson, C. D. and Berry, D. R. (1977). Fluctuations in cAMP levels during the cell cycle of Saccharomyces cerevisiae. FEMS Microbiology Letters 1, 175-178. Wheals, A. E. (1987). Biology of the cell cycle in yeasts. In Rose, A. H. and Harrison, S . E. (Eds), The Yeasts, vol. 1, 2nd ed. Academic Press Inc. (London) Ltd, pp. 283-390. White, J. H. M., Barker, D. G., Nurse, P. and Johnston, L. H. (1986). Periodic transcription as a means of regulating gene expression during the cell cycle. Contrasting modes of expression of DNA ligase genes in budding and fission yeast, EMBO J. 5,1705-1709.

Intracellular and extracellular levels of cyclic AMP during the cell cycle of Saccharomyces cerevisiae.

Using the technique of centrifugal elutriation it was demonstrated that during the cell division cycle of the budding yeast Saccharomyces cerevisiae t...
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