Botanieal Laboratory, University of Bergen, Bergen, Norway

Control by Ammonium of Intercompartmental Guanine Transport in Chlorella

RUNE PETTERSEN

With 6 figures

Reeeived February 7, 1975

Summary

1. Guanine taken up in darkness by autotropieally grown synchronous Chlorella jusca passed through a "metabolie eompartment" eontaining guanine metabolizing enzymes, before it was stored in a "nonmetabolie eompartment", probably the vaeuoles.

2. Stored guanine was metabolized only at growth eonditions, i.e. at eonditions with high nitrogen demand.

3. Ammonium (1 mM) did not influenee the rate of guanine metabolism during guanine uptake, but in presenee of rhis N-souree the utilization of stored guanine was inhibited.

4. The results are interpreted as evidence that ammonium is able to control the metabolism of stored guanine by controlling the transport of guanine from the "nonmetabolie" to the "metabolie" eompartment.

Key words: Ammonium, Compartmentalization, Guanine, Transport.

Introduction

Autospores of Chlorella fusca are able to aeeumulate exogenously supplied guanine in a stable pool (PETTERSEN and KNUTSEN, 1974). Preliminary experiments revealed that irrespeetively of the amount of guanine added to eells kept in darkness, guanine was metabolized mainly during influx thus suggesting that it passed through a «metabolie eompartment» before it aeeumulated in a <<llonmetabolie eompartment». In the present paper evidenee supporting this suggestion is presented, and so is a hypothesis on guanine eompartmentalization whieh imply that the utilization of stored guanine depends on its transport from one eompartment to another. To understand how this intercompartmental transport is eontrolled, I have studied the prerequisites for the utilization of stored guanine.

Compartmentalization of enzymes and metabolites is well doeumented and its possible partieipation in controlling eell metabolism has reeently been paid in­ereasingly attention to (HEFENDEHL, 1969; OAKS and BIDWELL, 1970; WEISS and DAVIS, 1973; NUR SE and WIEMKEN, 1974; STEBBING, 1974). The understanding of this level of metabolie contral depends on knowledge of the transport systems of the membranes separating the enzymes from their substrates. The present study

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214 R. PETTERSEN

dealing with the control of guanine transport between compartments is thus a con­tribution to this knowledge.

Material and Methods

The strain of Chlorella fusca used, the method of synchronization and the conditions of culture were the same as described previously (MoBERG et al., 1968; LIEN et al., 1971). The present investigation was performed with autospores, i.e. the cells harvested after sporulation at the end of the dark period. The cell number (cells per ml) was determined with a Coulter counter Model ZB (Coulter Electronics, Ltd, England).

[8-14C] guanine was supplied by the Radiochemical Centre, Amersham, England, and unlabelled guanine by Sigma Chem. Co., USA. In nitrogen free medium (-N medium) nitrate was substituted by equivalent amounts of chloride. However, 22 nM ammonium was present due to the ammonium molybdate of the medium. The effect of nitrate and ammonium was studied by adding solutions of potassium nitrate and ammonium sulfate, respectively, to the cells kept in -N medium.

Autospores harvested by sentrifugation were nitrogen starved by keeping them in the -N medium under the same photosynthetic conditions as used for cultivation.

The radioactivity of aquous sampies, of cells harvested on membrane filters (pore size 0.45 ,uM), and of sections of thin layer chromatograms, were measured with a Tri Carb liquid scintillation counter using "Insta -gel" as scintillation liquid (both from Packard Instrument Co., USA).

The uptake experiments were carried out as described previously (PETTERSEN and KNUTSEN, 1974). At the conditions called "standard conditions" 5· 106 cells per ml were incubated in nitrate medium (containing 16 mM nitrate) at 30 C and darkness and aerated with air containing 2.5 Ofo CO2• The CO2 added was shown to minimize the photoreassimilation of the labelled CO2 which was liberated by complete degradation of [14C] guanine to CO2, NHs and glyoxylic acid. Hence (higher rates of) complete degradation could be measured in light as well as in darkness by recording the decrease in total radioactivity of the cell suspension. Moreover, the radioactivity accumulated in the trichloroacetic acid insoluble (TCAI) fraction also in illuminated cells is an approximate measure of the incorporation of guanine into polymeres. The "pool", i.e. the content of nonpolymere 14C-Iabelled compounds, was calculated as the difference between the total radioactivity of the cells and the radioactivity of the TCAI fraction.

The utilization of stored guanine at different conditions was studied as follows: Autospores incubated with [14C] guanine (1 IlM) at standard conditions were harvested by membrane filtration after a stable pool was established. The cells were resuspended to the original volume in prewarmed -N-medium and then kept at 30 C with aeration (2.5 0/0 CO2) under the conditons which effect was to be tested (darkness, presence of light, nitrate, ammonium or glucose, respectively). In addition to recording the time courses, the pool composition prior to and after 4 hours under the conditions studied, was determined in order to detect a possible effect on the guanine meta bol i sm not revealed by the in­corporation or the complete degradation. For studies of the pool composition the cells were extracted with hot water (about 90 C for 2 min). The extracts were separated from the cells by filtration, freezedried and the residue dissolved in 0.05 N NH40H. SampIes were chromatographed on cellulose thin layers with destilled water as solvent (PETTERSEN and KNUTSEN, 1974).

Non-standard abbreviations:

TCAI = Trichloroacetic acid insoluble (fraction of the 14C-Iabelled compounds 111

the cells).

Z. Pflanzenphysiol. Bd. 76. S. 213-223. 1975.

Intercompartmental Guanine Transport in Chlorella 215

vTCAI = The rate of the incorporation of [14C] guanine into the TCAI fraction. VC02 = The rate of complete degradation of guanine to NH3, CO2 and glyoxylic acid. i.c. = Intercompartmental.

Results

Compartmentalization 0/ exogenously supplied guanine

To study the guanine compartmentalization autospores were incubated with 1 ,uM[14CJguanine at standard conditions. The time courses of uptake, incorporation and of the total radioactivity per ml ceII suspension were recorded and the pool composition was analyzed. I found that the uptake ceased after about 15 min due to depletion of guanine in the medium, and the incorporation after about 20 min. Then a stable (constant) pool was established which consisted of 92 Ofo guanine, the remaining 8 Ofo was anabolie and catabolic intermediates. The stored guanine could remain unmetabolized either due to depletion of endogenous compounds necessary for its metabolism or due to compartmentalization.

The depletion alternative was disproved since labelIed guanine added to cells containing a stable pool was incorporated into the TCAI fraction during its influx (figure 1). When such repeated additions were performed with unlabelled guanine, none of the stored labelIed guanine was incorporated. Thus the guanine being incorporated during influx was not derived from the stored guanine.

As I found that the rate of complete degradation (vC02) was very low at standard conditions (2-3 % of the uptake rate) more accurate time courses of CO2 evolution were gained from continous monitoring the radioactivity of the gas phase of a closed system (RAA and GOKS0YR, 1965). Assuming that the time courses of uptake, incorporation and degradation, respectively, of cells incubated

18

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L I~

<~~ "~ l o 6 f r C'>------<~ ~ ,_.~. I

o I

ur n "0

0.6 (]I

3 "-0.3 3

o r 0 o 2 3 4 h

time

Fig. 1: Uptake and incorporation of guanine prior to and after a stable pool is established. The time course of the uptake (0) left ordinate, and of the incorporation into the TCAI fraction 0.) right ordinate, after three additions of guanine (ab out 1 ,uM) to cells incubated at standard conditions. Readditions shown by arrows.

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216 R. PETTERSEN

in the closed reaetion ehambers were not different from those found with aerated eells, these experiments revealed that guanine was eompletely degraded mainly during influx and not after the ineorporation had stopped. The presented experi­ments on guanine compartmentalization suggest the following hypothesis: «Ex­ogenously supplied guanine is transported into a <metabolie eompartment> where eatabolie enzymes, anabolie enzymes and a system transporting the guanine into a <llonmetabolie eompartment> compete for it. In order to be metabolized stored guanine has to be transported baek to the <metabolie eompartment>. Thus its metabolism can be controlled on the level of intercompartmental transport». The hypothesis is visualized in the simple model of figure 2. However, each of the two postulated compartments may consist of subcompartments. Moreover, experi­ments showed that the stored amount increased with increasing initial concentration in the medium, and that the fr action stored was higher the higher the influx was. This suggests that the system transporting guanine into the <<llonmetabolic compartment» has lower aHinity to guanine, but higher capacity than the guanine metabolizing enzymes have. - As the rate limiting step in the metabolism of stored guanine very likely is its intercompartmental (i.c.) transport, information on the control of this transport can be gained by studying the eHect of various conditions on the utilization of stored guanine.

metabolie I nanmetabalic campartment campartment

C02 NH3

~ ~:?1 ~ , Gua~ Gua ~ Gua

~ N.A.

~ biosynthesis ~ transport

----»- catabolism ~ inhibited

Fig. 2: Model of guanine transport, metabolism and compartmentalization pertinent for the present investigation. N. A. = Nuc1eic acids.

What are the metabolie and the nonmetabolie compartments?

In Candida large amounts of urie acid can be stored in the vaeuoles as shown by treating the cells with AgN03 followed by Na2S (ROUSH, 1961). Using this method I could not demonstrate where guanine was stored in Chlorella cells. But the vacuoles of cells which had accumulated an amount of guanine equal to 4 0/0

of their dry weight, seemed to be larger and more refractile, as ROUSH also found with Candida. Thus I hold that the <<llonmetabolic compartment» is made up

Z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

Intercompartmental Guanine Transport in Chlorella 217

of the vacuoles. Since some of the enzymes of the guanine metabolizing pathways are confined to organelles and other to the cytoplasm (see e.g. MÜLLER and M0LLER, 1969; THEIMER and BEEVERS, 1971; Rurs, 1972), the «metabolie com­partment» is a complex of subsystems.

The eHeet of light and glucose on the utilization of stored guanine

At standard conditions stored guanine remains unmetabolized because it is not transported to the «metabolie compartment». The lack of such transport can be due to:

a) Lack of necessary transport system. b) Lack of energy necessary for the transport. c) Inhibition of the transport.

The eHect of an exogenous energy supply was studied by incubating preloaded cells in darkness and without any supplement (control cells), in darkness and with 10 mM glucose, and with illumination (18 klux), respectively. While the pool was retained in the control cells, illumination eaused an immediate initiation of in­corporation as well as of eomplete degradation (figure 3). Provided the hypothesis is right this shows that the eells possess a constitutive system for the transport of stored guanine to the «metabolie compartment». Thereby alternative a above is excluded. Also in the glucose supplemented cells stored guanine was metabolized, but after a lag and at a lower rate than with illumination. The lag was probably due to the time needed for glucose uptake and meta bol i sm, whereas the lower rate could be due to less eHective energy supply from glucose assimilation than from photosynthesis.

These results apparently favours the energy alternative (b) above. However, both light and glucose addition increase the metabolism and may thereby aet

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Fig. 3: The effect of light on the utilization of the established stable pool In presencc of nitrate. Pool size at 0 h was 0.85 nmoles per 5· 106 cells (93 Ofo of the label was in guanine). Incubation in darkness (O,e) and in light (Li, .) was pcrformed in nitrate medium (16 mM). Open symbols: Total radioactivity of the cell suspension. Closed symbols: Radioactivity of thc TCAI fraction.

z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

218 R. PETTERSEN

by removing a possible inhibitor of t:he i.c. transport. To distinguish between the two alternatives (b and c above) one could either see if it was possible to in­crease the utilization of guanine without the mentioned treatments (i.e. by lowering the inhibitor concentration), or by reducing the utilization in presence of glucose or light (i.e. by increasing the level of inhibitor). Since the endogenous energy supply did not limit uptake and incorporation of guanine added to cells containing a stable pool (figure 1), alternative b seem to be the less probable. As an apriori guess, ammonium would be a potential candidate as inhibitor since it is an end product of the complete guanine degradation and plays a sentral role in the nitrogen metabolism. In the following I therefore tested the influence of ammonium on the i.c. transport of guanine (by recording effects on the metabolism of stored guanine).

The eifect 0/ lowering the endogenous ammonium level

The cells of the experiments with light and glucose had nitrate as nitrogen source, thus attaining a certain level of endogenous ammonium. To reduce this level in order to try to release inhibition, cells with stable pools were transferred to -N medium and one culture kept in darkness and another kept in light. Thus two low levels of ammonium was expected, that in illuminated cells being the lowest due to higher protein synthesis in light (increased demand for ammonium in aminating reactions).

Both the pool size and its composition of the cells kept in darkness were un­changed, while illumination caused pool to decrease at a rate comparable to that of cells illuminated in presence of nitrate (compare fig. 3 and 4). Provided that the inhibitor alternative is right, these results can mean that the endogenous ammonium supply at low nitrogen demand was sufficient to sustain the inhibition, whereas the inhibition at high demand was abolished even in presence of nitrate. The cessation of the incorporation within half an hour (after the nitrate removal)

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2 i ' .j.=-'--'_. ~ • ==---",LI. .;=====---;&.0.. . " •

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Fig. 4: The effect of nitrogen removal on the utilization of the established stable pool. Autospores with such a pool (0.85 nmoles per 5· 106 cells) were harvested and resuspended in -N-medium. Two suspensions were incubated with (D, .6.) and without (0, e) light, respectively. Open and closed symbols as in figure 3.

z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

Intercompartmental Guanine Transport in Chlorella 219

may be due to depletion of other nucleotides necessary for the incorporation of the guanine nucleotides formed. That the utilization rate did not decrease when the incorporation ceased, verify that the utilization rate is a measure of the i.c. transport rate (i.e. the transport rate limits the rate at which stored guanine is utilized).

The effeet 0/ ammonium addition

Next I studied the eHect of exogenously supplied ammonium (S mM) on the guanine utilization at different levels of nitrogen demand. This was done by incubating guanine preloaded cells in presence of light and in darkness without and with glucose (10 mM) present.

As was the case in presence of nitrate the pool of cells kept in darkness without glucose was unchanged. Photosynthesis and glucose assimilation both caused utiliza­tion of the stored guanine, but at rates being lower than in the corresponding ex­periments with nitrate (compare Eg. S with Eg. 3). Since nitrate is reduced to ammonium in the cells, but ammonium not oxidized to nitrate, the lower utilization rates in presence of ammonium show that nitrate is not the inhibitor. The lower inhibitior level in presence of nitrate may be ascribed to the ability of ammonium or an ammonium assimilate to control the nitrate reduction (SYRETT and MORRIS, 1963; LOSADA et al., 1970).

Furthermore these experiments showed that guanine was anabolized, but not catabolized in presence of ammonium. Two alternative explanations for this are:

1. The eHect of ammonium is solelyon the i.c. transport. In this case the anabolie pathway must have much higher affinity to guanine (in the metabolie compartment) than the catabolic has.

2. The concentration of ammonium is large enough to inhibit the catabolic pathway. In this case the utilization rate is lower than the i.c. transport rate studied.

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o

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Fig. 5: The effcct of ammonium addition in presencc of light or glucose, on the utilizatioll of the established stable pool. Autospores with such a pool (0.85 nmol per 5 . 106 cells) were harvested and resuspcnded in a medium with 5 mM ammonium. Threc suspensions wcre incubated in darkness (- - - - -, the control), in darkness with 10 mM glucose (0, e), and in light (6, j.), rcspectivcly. Open and closed symbols as in figure 3.

Z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

220 R. PETTERSEN

According to the model (fig. 2) it was possible to distinguish between these alternatives by comparing the effect of ammonium on guanine metabolism during guanine influx with its effect after influx cessation. To be able to evaluate the results the effect of ammonium on the uptake had also to be recorded. In order to record VC02 significantly during influx as the rate of decreased radioactivity of the incubation suspension, vC02 was increased by nitrogen starving the cells for 4 hours prior to the addition of guanine. The starvation also increased the uptake rate. To obtain accurate time courses higher guanine concentration (30,uM) was used than in the previous experiments. The ammonium solution was added to the illuminated cell suspensions 6 min prior to the addition of labelled guanine.

I mM NH 4' 100 pM NH:

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" ... I 0 4

2

...

r~ 0

0

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10 pM N H4' -N

10

8

6

4

2

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Fig. 6: The effect of the ammonium concentration on the metabolism of guanine during and after its uptake in illuminated cells. The autospores (107 cells per ml) were nitrogen starved for 4 hours and the suspension divided into four. Ammonium was added to the given concentrations 6 min. before the addition of guanine to an initial concentration of 30 11M. The radioactivity of the ceIl suspension (e) and of the TCAI fraction (.) was recorded by two parallel measurements, the range of which are given by the vertical bars through the points. The time course of the radioactivity accumulated in the cells is given by Q. The broken lines show the hypothetical time courses which the complete degradation would have if it had continued with the same rate as during the uptake.

Z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

Intercompartmental Guanine Transport in Chlorella 221

Carefully performed experiments with initial eoneentrations of 1 mM, 100,uM and 10,uM ammonium, respeetively, revealed that neither the uptake rate nor vC02 and VTCIA were signifieantly influeneed during the uptake of guanine. However, 1 mM ammonium did inhibit the utilization of stored guanine by about 90 Ofo during the two hours of reeording, while the inhibition eaused by 100,uM was released after one hour and 10,uM did not inhibit at all (figure 6). A rough estimation of the amount of nitrogen neeessary to support the protein synthesis of unstarved eells, revealed that the inhibition was released due to eonsumption oE the ammonium added.

That the metabolism of guanine passing through the «metabolie eompartment» was uninflueneed by ammonium present at concentrations high enough to inhibit the utilization of stored guanine, favours the alternative 1 above, and verify that ammonium (directly or indirectly) is able to limit the utilization of stored guanine solely by inhibiting its i.c. transport from the «nonmetabolic» to the «metabolic compartment».

In absence of ammonium VTCAI but not vC02 decreased concomitantly with the influx cessation. This can be explained by compartmentalization assuming that guanine is incorporated, but not completely degraded in the chloroplast: The parietal localization of the large cup shaped chloroplast favours the transport into this organelle (where it is incorporated into the TCAI fraction) of guanine passing plasmalemma. The guanine not being withhold is transported to more central regions (containing catabolic enzymes, nucleus and vacuoles) responsible for degrada­tion, incorporation and storage. However, when only stored guanine is utilized it is metabolized before reaching the chloroplast.

Discussion

The present investigation suggests that Chlorella cells compartmentalize exogen­ously supplied guanine between a small cytoplasmic pool and a large vacuolar pool. This is analogous to the compartmentalization of amino acids in Candida (WIEMKEN and NURSE, 1973) and Neurospora (SUBRAMANIAN et al., 1973). The vacuolar pool is a storage which may be utilized by transport of pool content to the compartment containing the metabolic enzymes. This intercompartmental transport is obviously controlled by ammonium itself or by a metabolite accumulated in presence of ammonium. This metabolite might be an ammonium assimilate or an intermediate guanine metabolite. However, the release of inhibition at conditions favouring the assimilation of ammonium, and apparently no inhibition of the guanine metabolizing pathways in presence of ammonium, suggest that ammonium itself is able to control the transport.

Since neither the uptake nor the incorporation of guanine, both being energy dependent prosesses taking pi ace at different sites in the cells, were inhibited in the presence of ammonium, ammonium seems to act by inhibition rather than by

z. Pjlanzenphysiol. Bd. 76. S. 213-223. 1975.

222 R. PETTERSEN

lowering the energy supply to the transport system studied. This is also in agree­ment with the finding that ammonium added to photosynthesizing Chlorella cells did not decrease the ATP level (KANAZAWA et al., 1970).

Ammonium has previously been shown to inhibit the system transporting amino acids into yeast (GRENsON et al., 1970). And PATEMAN and coworkers (1973) have shown that the uptake of L-glutamate and thiourea in Aspergillus is regulated by ammonium. Their hypothesis for ammonium regulation of various uptake and enzyme systems includes that of purine uptake, and may suggest a possible way for the control of the intercompartmental guanine transport wh ich I have studied. Control by ammonium of guanine compartmentalization in addition to a higher affinity of the anabolie than of the catabolic pathway to guanine, secures the stored guanine to be anabolized rather than wasted by degradation when the supply of simple nitrogen sources (nitrate or ammonium) is sufficient to meet the nitrogen demand. When the nitrogen supply is insufficient, this valuable nucleic acid precursor is degraded and may be utilized as a nitrogen source.

Control by ammonium of intercompartmental transport may be a general way of regulating the utilization of stored complex nitrogen sourees. Such control may explain the low metabolie turnover of purines accumulated in yeast (COWIE and BOLTON, 1957; RouSH et al., 1959). This being the case it adds another way at wh ich ammonium is able to control the nitrogen metabolism and thereby state the key position which ammonium is known to possess in this control.

Acknowledgements

I thank D. DAGESTAD for discussions and Dr. G. KNUTSEN for criticizing the manuscript.

References

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RUNE PETTERSEN, Botanical Laboratory, Allegt. 70, N-5014 Bergen-Universitetet, Norway.

Z. Pflanzenphysiol. Bd. 76. S. 213-223. 1975.