Assimilation of Ammonia During Sporogenesis of Saccharomyces cerevisiae: Effect of Ammonia and Glutamine

Abstract

~Comparison of the pools of glutamic acid and glutamine and of the specific activities of glutamine synthetase and glutamate dehydrogenases in sporulating a/cc and non-sporulating a/cc cells of Saccharomyces cerevisiae revealed a difference in their nitrogen metabolism.Glutamhe synthetase and glutamine appeared to be necessary for the sporulation process, glutamine playing, at least, a catabolic role. However, exogenous glutamine as well as ammoniainhibited sporulationwhile glutamic acid did not. Glutamine seemed to act through its amino group. Both inhibitors had at least two sites of action, one effective early in sporulation and related to DNA synthesis and the other acting later and not related to it. I N T R O D U C T I O NStarvation of the yeast Saccharomyces eerevisiae induces a differentiation process leading to meiosis and sporulation of diploid ( a / a ) cells. This process is inhibited by ammonia and some other nitrogen compounds (Miller, 1963; Piiion, 1977) but very little is known about the metabolism of ammonia in sporulating cells. In this paper, we report our studies on the activities of enzymes involved in ammonia assimilation (glutamhe synthetase, NAD-and NADP-linked glutamate dehydrogenases), the effect of the substrates of these enzymes on the sporulation process, and the variation in the sizes of pools of free glutamine and glutsmic acid during the first hours after the transfer of cells to the sporulation medium with or without ammonia. METHODS Yeast strains, growth and sporulation. Diploid cells of Saccharomyces cerevisiae 532 a / a and 533 ala,isogenic except for their mating type (Pellucuer, 19731, were used. The a / a cells were grown and allowed to sporulate as described by Durieu-Trautmann & Delavier-Klutchko (1977). The alee cells, which are unable to sporulate, were used as a control.Enzyme activities. Extracts were prepared at 4 "C by passing suspensions of cells in 100 mwpotassium phosphate buffer pH 7-8, containing 1 mhl-2-mercaptoethano1, through a French press at 100 MPa. Cell debris was removed by centrifuging for 30 min at 30000 g and the supernatant was dialysed overnight at 4 "C against buffer similar to that used for extraction.Activities of NAD-linked glutamate dehydrogenase (NAD-Gdh; EC 1 .4.1.2) and of NADP-linked glutamate dehydrogenase (NADP-Gdh; EC 1.4.1.4) were estimated by determining the rate of NADH or NADPH oxidation at 25 "C (Boehringer, 1968). The rate of reaction with NAD-Gdh was corrected by subtracting the rate of NADH oxidation observed in the absence of 2-oxoglutarate.The activity of glutamine synthetase (GS; EC 6.3.1 .2) was measured by assaying transferase activity (Woolfolk et al., 1966).Protein was determined by the Folin method of Lowry, using bovine serum albumin as standard.

Ammonium Ion Repression of Sporulation in Saccharomyces cerevisiae

“…The mechanism of this effect is unknown for there is a lack of agreement on whether NH,+ itself participates directly, or whether it is due to derivatives (e.g. glutamine) synthesized from it (Piiion, 1977;Dubois et al, 19'77;DurieuTrautmann & Delavier-Klutchko, 1977 ;Delavier-Klutchko et al, 1980). Ammonium ion and glutamine are reported to have at least two sites of action: one quite early preventing RNA, protein and glycogen synthesis, and affecting pre-meiotic DNA synthesis; the ,other later when DNA synthesis is completed (Durieu-Trautmann & Delavier-Klutchko, 1977;DelavierKlutchko & Durieu-Trautmann, 1978;Delavier-Klutchko et al, 1980;Piiion, 1977;Croes et al, 1978).…”

“…glutamine) synthesized from it (Piiion, 1977;Dubois et al, 19'77;DurieuTrautmann & Delavier-Klutchko, 1977 ;Delavier-Klutchko et al, 1980). Ammonium ion and glutamine are reported to have at least two sites of action: one quite early preventing RNA, protein and glycogen synthesis, and affecting pre-meiotic DNA synthesis; the ,other later when DNA synthesis is completed (Durieu-Trautmann & Delavier-Klutchko, 1977;DelavierKlutchko & Durieu-Trautmann, 1978;Delavier-Klutchko et al, 1980;Piiion, 1977;Croes et al, 1978). NH: does not inhibit the initiation of pre-meiotic DNA synthesis (Piiion, 1977), but it is reported that DNA synthesis (post initiation) is arrested by NH,+ (Piiion, 1977), or at least reduced and slowed down by NH,+ or glutamine (Durieu-Trautmann & Delavier-Klutchko, 1977).…”

Co-ordinate control of ammonium-scavenging enzymes in the fruit body cap of Coprinus cinereus avoids inhibition of sporulation by ammonium

“…meiosis and ascospore formation) in Saccharomyces cerevisiae is induced by transferring diploid vegetative yeast cells from a rich medium (usually with high levels of glucose and complex nitrogen sources) to a nitrogen-free medium [9][10][11][12]. Within 2 h of the transfer, NAD-GDH activity in the yeast cells in repressed to about 5% of its level in vegetative cells, but NADP-GDH activity remains unchanged [13]. The net result is a change in the ratio of the specific activities of the two enzymes from 2.04 : 1 (NAD-GDH : NADP-GDH) to 0.10 : 1.…”

ULTRASTRUCTURAL DISTRIBUTION OF GLUTAMATE DEHYDROGENASES DURING FRUIT BODY DEVELOPMENT IN COPRINUS CINEREUS

“…Conversely, therefore, the primary function of the NADP-GDH/glutamine synthetase ammonium scavenger in C. cinereus may be to remove ammonium from the environment of some process which is sensitive to ammonium inhibition. As NADP-GDH has been localized to basidia -the meiocytes -the most relevant comparison may be with meiosis and sporulation in yeast, which are inhibited by ammonium (Pinon, 1977) and dependent on glutamine synthetase activity (Delavier-Klutchko et al, 1980).…”