Planta

Planta 141, 205-209 (1978)

9 by Springer-Verlag 1978

Inhibition of Meiosis in Saccharomyces cerevisiae by Ammonium Ions: Interference of Ammonia with Protein Metabolism A.F. Croes, J.M.J.M. Steijns, Geertrui J.M.L. De Vries, and Tonneke M.J.A. van der Putte Department of Botany, University, Nijmegen, Netherlands

A b s t r a c t . Meiosis and sporogenesis in yeast are completely blocked by ammonia added in low concentration (10 mM) to the sporulation medium. Premeiotic D N A synthesis is not initiated in the presence of ammonium ions. The inhibitor interferes with protein turnover by reducing both synthesis and breakdown. The in vitro activities of proteinases A and B in sporulation medium supplemented with ammonia a r e much lower than in the control. This may partially explain the effect of ammonium ions on protein metabolism in vivo. Key words: Ammonia

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lism - Proteinases -

Saccharomyces

Meiosis

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Protein metaboYeast.

Introduction

Yeast cells switch from mitosis to meiosis when transferred from a rich presporulation medium (PSP) into a poor sporulation medium (SPM). During mitotic growth, the meiotic pathway is considered to be blocked by constituents of the PSP. Meiosis is initiated once these substances are no longer present. A number of nitrogenous compounds including ammonia support growth in PSP but inhibit meiosis and ascus formation when added to SPM (Miller, 1963 a). If, for instance, ammonium ions (10 mM) are present in SPM, meiosis is arrested at a very early stage (Miller, 1963b; Pifion, 1977). Meiosis and sporogenesis depend on continuous synthesis of new proteins (Esposito et al., 1969; Magee and Hopper, 1974). As there is no external source of nitrogen, the pool of free amino acids becomes depleted very soon (Ramirez and Miller, 1964; Abbreviations: PSP=presporulation medium; SPM=sporulation

medium

Croes, 1967). Any further synthesis must occur at the expense of pre-existing proteins. During mitotic growth, there is virtually no protein turnover (Halvorson, 1958) as protein breakdown is at a low level in the presence of ammonia (Johnston et al., 1977). Proteolysis begins as soon as cells are incubated in the sporulation medium (Hopper et al., 1974; Klar and Halvorson, 1975; Betz and Weiser, 1976). The process is accompanied by a rise in proteinase activity (Klar and Halvorson, 1975; Betz and Weiser, 1976). This rise seems to be sporulation-specific as it only occurs in sporulating diploids (Klar and Halvorson, 1975). Addition of ammonia to the sporulation medium might interfere with the development of protein turnover. This suggests a possible mechanism for the inhibition of meiosis by ammonium ions. A low level of protein degradation would deprive the cells from precursors for synthesis of new proteins if the yeast is unable to synthesize amino acids from N H 2 and acetate at a sufficient rate. It therefore looked worthwhile to study protein synthesis and breakdown, and proteinase activities in cells incubated in SPM in the presence of N H +. The present study shows that these processes are greatly affected by ammonia.

Material and Methods

The wild type (K7) and mutant (K28-10) yeast strains and all experimental procedures have been described previously (Croes et al., 1978) except for the following. DNA Synthesis

Premeiotic DNA synthesis was monitored according to Simchen ai. (1972). Cells were prelabelled with [3H]uracil, 1.gCi/rnl, during growth in presporulation medium.. :.

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Chase Experiments For measuring protein breakdown in SPM, cells were prelabelled with [3HI arginine, 1 gCi/ml during growth and subsequently incubated in SPM supplemented with 2 mM unlabelled arginine. To avoid effects of increasing pH on cellular permeability for arginine, the pH was kept constant at 8.5 by refreshing the medium every 2 h. The repeated medium change does not interfere with normal development.

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Ceils were collected at intervals from SPM by centrifugation, resuspended in ice-cold phosphate buffer, 0.01 M, pH 7.0, and passed through the orifice of a French press cell at 8.3 x 107 N/m 2. A crude enzyme extract was prepared by centrifuging the suspension of broken cells for 10 min at 1.2 x 10s N/kg. Proteinases A and B were activated by incubation at pH 5 and 25~ C for 24 h (Saheki and Holzer, 1975) and assayed at pH 3 and pH 9 respectively. The procedure of Betz and Weiser (1976) was used with 3Hlabelled, acid-denatured yeast protein as the substrate. The specific activity is expressed as the acid-soluble count liberated from the substrate per mg of protein in the cude extract. Protein content was determined according to Lowry et al. (1951).

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[5,6-3H]uracil, 44.5Ci/mmol, L-[5-3H]arginine, 8.8 Ci/mmol, L[4,5-3H]leucine, 58 Ci/mmol, and L-[4,5-3H]lysine, 40Ci/mmol, were purchased from the Radiochemical Centre, Amersham, U.K.

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Effect of N H 2 on Meiosis and Sporogenesis T h e wild type (K7) a n d m u t a n t (K28-10) strains display different sensitivities t o w a r d the a m m o n i u m c o n c e n t r a t i o n in S P M (Fig. 1). C o n c e n t r a t i o n s of 6 m M a n d 10 m M completely b l o c k sporogenesis in m u t a n t a n d wild type respectively. Therefore, these c o n c e n t r a t i o n s have been used to i n h i b i t meiosis in the f o r t h c o m i n g experiments. T o d e t e r m i n e at what time sporogenesis is affected b y N H 2 , we followed the premeiotic D N A ~synthesis in cultures with a n d w i t h o u t a m m o n i a (Fig. 2). The results show that the i n h i b i t o r blocks meiosis at a very early stage as D N A replication is completely abolished in its presence. C o n t r o l cells start to replicate D N A at a b o u t Th. I n cultures with NH~-, on the contrary, D N A begins to be b r o k e n d o w n by that time.

Fig. 2. Effect of ammonia on premeiotic DNA synthesis in strain K7. Cells were prelabelled with [3H]uracil prior to incubation in SPM. SPM +NH~ contained NH4C1 at a concentration of 10 raM. /, cpm in DNA: cpm in DNA at Tx minus cpm in DNA at To cprn

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T h e d e g r a d a t i o n of p r o t e i n d u r i n g early meiosis a n d the effect of a m m o n i u m ions u p o n it were studied in cells prelabelled with [3H] a r g i n i n e d u r i n g growth (Fig. 3A). W h e n n o a m m o n i a is present, proteolysis

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It may be concluded that N H 2 considerably reduces protein breakdown during early meiosis.

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Fig. 4 A and B. Effect of a m m o n i a on amino acid transport and incorporation during early meiosis. At the times indicated, cells were transferred to fresh SPM and pulse-labelled with 3H-lysine, 2 pCi/ml, 1 m M for 10 rain. A Wild type; B m u t a n t ; o - - o uptake and A - - A incorporation by ammonia-inhibited ceils, o - - o uptake and z x - - z x incorporation in the control

begins at T2 and proceeds at a rate of 3% per h for about 3 h. After T 5 the process continues at a slightly lower rate. Addition of ammonia to SPM causes a delay in the onset of protein degradation of about 2 h. Later on, protein is broken down at approximately the same rate as in the control. This means that the ammonia effect is, relatively, most severe in the period immediately preceding the onset of premeiotic D N A replication. The possibility exists, however, that from T~ on the extent of proteolysis is underestimated because of the steady decrease in the permeability of the cell to amino acid (Fig. 4A). If limited entrance of unlabelled arginine causes [3H]arginine to be re-used for protein synthesis, the extent of this recycling would largely depend on the activities of the argininetransporting systems. As these activities are lower in the presence of ammonia than in its absence, the differences in conservation of 3H-counts in protein could be related to differences in the effectivity of the chase rather than to the extent of protein breakdown. To exclude this possibility, we repeated the experiment using the mutant strain which is characterized by an enhanced rate of amino acid transport (Fig. 4B). It is seen that NH~- causes a considerable reduction in cellular permeability to amino acid. Nevertheless, the uptake rate in the presence of ammonia is much higher than in wild-type cultures without inhibitor at any time beyond T 3. The negative effect of N H 2 on protein breakdown turns out to be more pronounced in the mutant than in the wild type (Fig. 3B). It is also remarkable that the extent of protein degradation in uninhibited mutant cells is of the same order of magnitude as in the corresponding wild-type culture. This means that a greater influx of unlabelled arginine in the cell does not lead to an increase in count loss from cellular protein.

The reduction of proteolysis in ammonia-inhibited cells might lead to a shortage of precursors for synthesis of new protein. It is also possible, however, that amino acids are synthesized from NH~- and acetate at a rate sufficient to compensate for the decline in supply from protein breakdown. To discriminate between these possibilities, we followed protein synthesis during early meiosis by measuring the incorporation of [3H]lysine in inhibited and uninhibited cells. The results are presented ~n Fig. 4A. At first glance, a reduction in protein synthesis due to the presence of ammonia seems obvious. Here again, however, we have to reckon with the effect of ammonium ions on amino acid uptake. That uptake of amino acid could be a limiting factor in its incorporation, is suggested by the fact that a major part of the lysine transported into the cell is immediately incorporated into protein at all times but T2. This could mean that the incorporation patterns reflect differences in amino acid uptake rather than changes in the rate of protein synthesis. As this problem can not be solved unambiguously with the wild-type strain, the experiment was repeated with the mutant (Fig. 4B). The behavior of the latter differs from that of the former in two particular points: a) amino acid transport proceeds much faster than incorporation even in the presence of ammonia, and b) incorporation does not change with time in the same fashion as uptake. Both features make it unlikely that lysine incorporation is restricted by its uptake. Consequently, the lower incorporation rate in ammoniacontaining culture is indicative of a lower rate of protein synthesis. Proteinase Activities

During early meiosis in yeast, the onset of proteolysis is accompanied by an increase in the activities of proteinases A and B (Klar and Halvorson, 1975; Betz and Weiser, 1976). If these activities are repressed when ammonia is added to SPM, a possible regulation mechanism for protein breakdown would be at hand. Yeast proteinases are localized in the vacuole whereas substrate and proteinase inhibitors are present in the cytoplasm (Lenney et al., 1974; Matern et al., 1974). Upon disruption of the cell, cytoplasmic and vacuolar contents are mixed up and the proteinases become complexed with their inhibitors. The enzymes can be activated by removal of the inhibitors (Saheki and Holzer, 1975).

208

A.F. Croes et al. : Inhibition of Meiosis by A m m o n i a

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Proteinase activities were assayed at intervals during incubation of cells in sporulation media with and without ammonium chloride (Fig. 5). Both enzyme activities which are low at the moment of the medium change, start to rise upon incubation in SPM. The rise is most conspicuous in activated extracts but is also observed with the enzyme inhibitors present. In media supplemented with NH4- the activities remain low for 4-6 h and begin to rise afterwards. The picture is more or less analogous in the mutant (Fig. 6) but the ammonia effect is weaker than in the wild type. No N H 2 effect at all is found in non-activated extracts. It should, however, be recalled that a complete accessibility of the proteinases for their respective inhibitors is an artificial situation brought about by rupture of the vacuoles during preparation of the cell-free extract. These inhlbitors which seem to be

Ammonia completely blocks meiosis in yeast at a concentration eight times lower than the concentration in presporulation medium. Comparable data have been presented by Miller (1963a) and Pifion (1977). Premeiotic D N A synthesis, a specific event in yeast meiosis (Croes, 1966; Roth and Lusnak, 1970), is completely inhibited by N H 2 . This suggests that the inhibitor interferes with meiotic development at a very early stage, presumably at or around the time of induction. Our data do not confirm the observation of Pifion (1977) that in the presence of ammonium ions premeiotic D N A replication is initiated but not terminated. The rise in proteinase activities during early meiosis has been shown to be sporulation-specific as no increase is observed in non-sporulating strains (Klar and Halvorson, 1975). The interference of ammonia with the development of these enzyme activities may, therefore, be considered a direct effect on meiosis and sporogenesis. A number of other enzymes functioning in nitrogen catabolism have been described as sensitive to ammonium repression (Grenson and Hou, 1972; Dubois et al., 1973). Our data on proteinase activities fit well into this scheme. There is a lag of some hours between the rise in proteinase activity and the onset of protein degradation in vivo. This makes clear that activation of these enzymes is presumably necessary but not sufficient for proteolysis to take place. Protein breakdown requires an interaction between substrate and enzyme localized in different compartments of the cell. One could speculate that these molecules interact at the vacuolar membrane. In that case, the extensive fragmentation of the vacuole during the first hours of incubationin SPM (Svihla et al., 1964; Croes, 1967) might be a factor in the regulation of proteolysis as contact of enzyme and substrate is facilitated by an increase of the membrane surface. In NH2-inhibited cells, fragmentation is much less obvious than in the control. The onset of protein breakdown in vivo roughly coincides with the exhaustion of the pools of free amino acids. A coupling of these events as suggested by this coincidence would be physiologically meaningful.

A,F. Croes et al. : Inhibition of Meiosis by Ammonia

The effect of ammonium ions on protein synthesis can be partially explained as a result of reduced proteolysis. However, this explanation does not hold for the T~-T2 period when protein breakdown is negligible in the control. During this period, N H 2 must exert its action via another, unknown effect on cellular metabolism. The switch from mitosis to meiosis is made somewhere between To and Ts. During that period, metabolic processes take place which are required for starting D N A replication and for entering meiosis (Croes, 1967). It is in this early period that a severe disturbance of protein metabolism by ammonia becomes apparent. One can only speculate about the relation between this disturbance and the inability of the cell to undergo meiosis. An attractive hypothesis is that one or more proteins required for the initiation of meiosis is not present in sufficient amounts. Alternatively, one or more vegetative proteins which must be removed before the cell can enter meiosis may be retained in a too high concentration. These hypotheses can be tested by a qualitative analysis of newly synthesized and conserved proteins in normal and ammonia-inhibited cells.

References Betz, H., Weiser, U. : Protein degradation and proteinases during yeast sporulation. Eur. J. Biochem. 62, 65-76 (1976) Croes, A.F. : Duplication of DNA during meiosis in baker's yeast. Exp. Cell Res. 41,452-454 (1966) Croes, A.F. : Induction of meiosis in yeast. I. Timing of cytological and biochemical events. Planta 76, 209-226 (1967) Croes, A.F., De Vries, G.J.M.L., Steijns, J,M.J.M.: Amino acid uptake and protein synthesis during early meiosis in Saccharornyces cerevisiae. Planta 139, 93-96 (1978) Dubois, E., Grenson, M., Wiame, J.M. : Release of the " a m m o n i a " effect on three catabolic enzymes by NADPH-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 50~ 967 972 (1973) Esposito, M.S., Esposito, R.E., Arnaud, M., Halvorson, H.O.: Acetate utilization and macromolecular synthesis during sporulation of yeast. J. Bacteriol. 100, 180 186 (1969)

209 GreI~son, M., Hou, C.: Ammonia inhibition of the general amino acid permease and its suppression in NADP-specific glutamate dehydrogenaseless mutants of Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 48, 749-756 (1972) Halvorson, H.: Studies on protein and nucleic acid turnover in growing cultures of yeast. Biochim. Biophys. Acta 27, 26%276 (1958) Hopper, A.K., Magee, P.T., Welch, S.K., Friedman, M., Hall, B.D.: Macromolecular synthesis and breakdown in relation to sporulation and meiosis in yeast. J. Bacteriol. 119, 619-628 (1974) Johnston, G.C., Singer, R.A., McFarlane, E.S.: Growth and cell division during nitrogen starvation of the yeast Saccharomyces cerevisiae. J. Bacteriol. 132, 723-730 (1977) Klar, A.J.S., Halvorson, H.O.: Proteinase activities of Saccharomyces eerevisiae. J. Bacteriol. 124, 863-869 (1975) Lenney, J.F., Matile, Ph., Wiemken, A., Schellenberg, M., Meyer, J.: Activities and cellular localization of yeast proteases and their inhibitors. Biochem. Biophys. Res. Commun. 60, 1378-1383 (1974) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenoI reagent. J. Biol. Chem. 193, 265-275 (1951) Magee, P.T., Hopper, A,K. : Protein synthesis in relation to sporulation and meiosis in yeast. J. Bacteriol. 119, 952-960 (1974) Matern, H., Betz, H., Holzer, H. : Compartmentation of inhibitors of proteinases A and B and carboxypeptidase Y in yeast. Biochem. Biophys, Res. Commun. 60, 1051-1057 (1974) Miller, J.J.: The metabolism of yeast sporulation. V. Stimulation and inhibition of sporulation and growth by nitrogen compounds. Can. J. Microbiol. 9, 259-277 (1963a) Miller, J.J.: Determination by ammonium of the manner of yeast nuclear division. Nature 198, 214-215 (1963b) Pifion, R. : Effects of ammonium ions on sporulation of Saccharomyces eerevisiae. Exp. Cell Res. 105, 367-378 (1977) Ramirez, C., Miller, J.J.: The metabolism of yeast sporulation. VI. Changes in amino acid content during sporogenesis. Can. J. Microbiol. 10, 623-63l (1964) Roth, R., Lusnak, K.: DNA synthesis during yeast sporutation: Genetic control of an early developmental event. Science 168, 493-494 (1970) Saheki, T., Holzer, H.: Proteolytic activities in yeast. Biochim. Biophys. Acta 384, 203-214 (I975) Simchen, G., Pifion, R., Salts, Y.: Sporulation in Saccharornyces cerevisiae: Premeiotic DNA synthesis, readiness and commitment. Exp. Cell Res. 75, 207-218 (1972) Svihla, G., Dainko, J.L., Schlenk, F.: Ultraviolet microscopy of the vacuole of Saccharomyces cerevisiae during sporulation, J. Bacteriol. 88, 449-456 (1964) Received 7 March; accepted 21 March 1978

Inhibition of meiosis in Saccharomyces cerevisiae by ammonium ions: Interference of ammonia with protein metabolism.

Meiosis and sporogenesis in yeast are completely blocked by ammonia added in low concentration (10 mM) to the sporulation medium. Premeiotic DNA synth...
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