Characterization of an Ammonium Transport System in Filamentous Fungi with Methylammonium-14C As the Substrate*

SUMMARY Penicillium chrysogenum will grow on The amine enters the mycelium by means of a transport system (permease) that satisfies several criteria for its iden-tity as an ammonia (or ammonium ion) permease. Transport is temperature-dependent (Q10 of 2.1 between 20’ and SO”), pH-dependent (optimum between pH 4.5 and pH 8.5), and ionic strength-dependent (optimum and relatively constant between 0 and 10-s M KCl). Methylamine transport by nitrogen-sufficient mycelium is extremely low and obeys first order kinetics up to lop3 M external substrate. Nitrogen starvation causes the derepression or deinhibition (or both) of a saturable transport system (K,,, N 10m6 M, V,,, N 10 pmoles per g-min). This results in an 800-fold increase in the rate of methylamine transport at external substrate concentrations below low6 M. Ethylamine transport has the same Vmax, but a much higher K,,, value (approximately 10e4 M). Methylamine not

that satisfies several criteria for its identity as an ammonia (or ammonium ion) permease. Transport is temperature-dependent (Q10 of 2.1 between 20' and SO"), pH-dependent (optimum between pH 4.5 and pH 8.5), and ionic strength-dependent (optimum and relatively constant between 0 and 10-s M KCl). Methylamine transport by nitrogen-sufficient mycelium is extremely low and obeys first order kinetics up to lop3 M external substrate.
This results in an 800-fold increase in the rate of methylamine transport at external substrate concentrations below low6 M. Ethylamine transport has the same Vmax, but a much higher K,,, value (approximately 10e4 M).
Methylamine transport is not inhibited significantly by other amines, amino alcohols, or ammo acids previously shown to be susbtrates of a nitrogen-regulated amino acid permease (BENKO, P. V., WOOD, T. C., AND SEGEL, I. H., Arch. Biochem. Biophys., 129, 498 (1968) virtually nothing is known about the transport processes by which the ammonium ion enters living cells. This is not too surprising as most studies on transport phenomena are designed for use with radioactive substrates and no long lived radioactive isotope of nitrogen exists. Transport experiments with 16NH3 would be rather expensive and time-consuming. We became interested in ammonia transport as a result of our investigation of the multiplicity and regulation of amino acid permeases in several filamentous fungi (1, 2). We found that Penicillium chrysogenum and Aspergillus nidulans possess a relatively nonspecific amino acid permease that develops as a result of nitrogen deprivation.
Ammonium was the only nonn-a-neutral amino acid compound that seemed to inhibit the permease (2). This result suggested that ammonia might be a regulator of the amino acid permease. However, it was also possible that ammonia was being rapidly transported into the mycelium and converted intracellularly to a feedback inhibitor of the amino acid permease.
(Other results had indeed established that a brief preliminary incubation of nitrogen-starved mycelium with ammonium chloride or amino acids caused a marked decrease in subsequent amino acid transport activity.) It was important then to establish the properties of the ammonia transport system, if one existed. We decided to determine whether methylamine-r4C could be used as an ammonia analogue and substrate for an ammonia permease.
The literature on alkyl amine metabolism is not extensive.
Zarlengo and Abrams (3) presented evidence that ammonia, methylamine, dimethylamine, and trimethylamine penetrated cells of Streptococcusfaecalis as the free bases at pH 7.0 or below. Arst and Cove (4) reported that methylamine could be used as a nitrogen source by A. nidulans if the amine were present at 1 mM in the growth medium, but that it was toxic at 100 mM. Kung and Wagner (5) identified y-glutamylmethylamide as a product of methylamine metabolism by Pseudomonas strain MS, while earlier Shaw,Tsai,and Stadtman (6) identified N-methylglutamate and alanine as methylamine metabolites. Amine oxidases from Pseudomonas AM1 (7) and A. nidulans (8) have been studied.
An amine oxidase would produce NH,+ from methylamine and this might be the first step in methyla-mine utilization. However, an equivalent amount of formaldehyde would also be produced, and this is likely to be toxic.
In this paper we present evidence for the existence of a relatively specific, nitrogen-regulated ammonia permease in mycelium of P. chrysogenum.
The permease will accept methylamine as a substrate, but with a greatly reduced affinity. MATERIALS AND METHODS ~.Growth of Mycelia-Most of the experiments described in this paper were carried out with P. chrysogenum, wild type strain PS75.
The pH of the medium was about 7 at the start and remained above 5 throughout the usual l-or 2-day growth period.
The cells were grown in 500-ml Erlenmeyer flasks containing 100 ml of medium, on a New Brunswick gyrotary shaker operating at a speed of 250 rpm and describing a l-inch circle. Only fine, "hairy," microfilamentous mycelium was used for transport studies.
The maintenance of the organism as spore suspensions and the production of mycelia from the spores have been described earlier (1). Nutrient Starvation-After 1 to 2 days of growth in complete medium, the mycelium was harvested by suction filtration, washed several times with deionized water, and reincubated aerobically (as described above) for 12 to 16 hours in the desired nutrient-deficient medium.
The carbon-deficient (glucosedeficient) medium was the usual citrate No. 3 medium without the glucose.
(The mycelium cannot utilize the citrate as sole carbon source,) Similarly, the sulfur-deficient medium was citrate No. 3 medium minus the Na2S04. Nitrogen-deficient medium  contained: Na3 citrate. 2Hz0 (14.7 g per liter), Na2S04 (1 g per liter), glucose (40 g per liter), and trace metals solution (10 ml per liter), all in 0.1 M potassium phosphate buffer, pH 7.0. The glucose was sterilized separately.
The mycelia were starved at a density of 2 g, wet weight, per 100 ml of medium.
Mycelium in nitrogen-deficient and in sulfur-deficient medium generally doubled in wet weight over a 12-hour period.
Permease Assay-After the 12-to 16.hour starvation period, the mycelia were removed from the nutrient-deficient media, washed, and resuspended at a density of 0.3 to 0.5 g, wet weight, per 25 ml of assay buffer (5 MM potassium phosphate buffer, pH 6.2, except for the experiment described in Table II in which 0.05 M pH 6.0 buffer was used and the pH optimum experiment in which 0.01 M buffers were used).
The suspensions were aerated for 15 min prior to adding the labeled substrate (generally 0.25 ml of a 2 mM solution).
Transport rates were calculated from four 5-ml aliquots taken at 30.set intervals within the first 2 min after adding the labeled substrate.
The aliquots were filtered rapidly with suction and the resulting mycelial pad was immediately washed with about 10 ml of ice-cold deionized water.
The mycelial pad was then peeled off the filter and placed in a scintillation vial containing 0.5 ml of water and 5 ml of scintillation fluid. The scintillation fluid contained 6 g of 2,5-diphenyloxazole and 100 g of naphthalene per liter of dioxane.
After counting the radioactivity, the mycelial samples were removed from t,he scintillation vials, washed with acetone and then water, and then dried overnight at 100" for dry weight determinations.
All transport rates are reported on a dry weight basis.
The material was dissolved in 1 ml of water to yield a stock solution that contained 1 mCi per ml and about 50 pmoles per ml.
For routine assays, 10 to 30 ~1 of this stock were added to 10 ml of 2 mM unlabeled methylamine.HCI (Eastman). Except for saturation studies, in which low substrate concentrations were used, the amount of mass contributed by the labeled methylamineJ4C was ignored. L-Methionine-3% (specific activity >200 mCi per mmole) and cholineJ4C (54 mCi per mmole) were obtained from Amersham-Searle.
L-Phenylalanine-14C and n-leucine-W (specific activity >200 mCi per mmole) were obtained from New England Nu-clear. Ethylamine

AND CONCLUSIONS
Growth of Mycelium on Methylamine-P. chrysogenum will utilize methylamine as sole nitrogen source, but not as well as NH,+ and only after a lag period as shown in Fig. 1. A. nidu-Zans behaved similarly.
The lag was present even when the inoculum was taken from a culture of mycelium growing linearly on methylamine.
Consequently, it seems unlikely that the lag represents an induction period.
There are several possible explanations for the lag period.
(a) Utilization of methylamine requires something that is present in dense mycelial cultures, but is not present or is insufficient in light ones. This factor could be low pH or COZ, or both. ). This inhibition manifests itself as a lag period in young cultures (in which the initial increase in mycelial mass is exponential), and as a reduced linear growth rate in older cultures.
(c) The methylamine contains a contaminant that is inhibitory to fungi.
(d) The utilization of methylamine produces a toxic substance (e.g. formaldehyde).
(e) Mycelial growth is limited by the rate at which the nitrogen can be extracted from methylamine (or the rate at which methylamine can be converted to a more readily metabolizable compound). As in b, this ratelimiting step shows up as a lag in young cultures and as a reduced linear growth rate in older ones.
Linearity of Methylamine Transport- Figs 2 (right). Transport of methylamine-W as a function of time.
The incubation mixture contained 0.3 g, wet weight, of nitrogen-starved mycelium in 25 ml of 5 mM potassium phosphate buffer, pH 6.2. At zero time, 0.25 ml of 1OV M methylamine-'4C was added.
Five-milliliter aliquots of the suspension were taken at 30.see intervals and filtered rapidly with suction. The resulting mycelial pads were t,hen immediately washed with ice-cold water, and then peeled off the filter paper and placed in a scintillation vial containing 0.5 ml of water and 5.0 ml of scintillation fluid.  Table  I shows the effect of varying the nitrogen source used for growth on the levels of the methylamine, methionine, and leucine transport activities of the mycelium.
All of the nitrogen sources used supported good growth.
The amino acid transport activities varied considerably with different nitrogen sources. However, the ratio of leucine transport activity to methionine transport activity was relatively constant at about 1.5. These results suggest that most of the methionine and leucine transport by the mycelium is mediated by a single transport system. &Llethylamine transport also varied with different nitrogen sources.
The ratio of transport rates (methylamine-methionine and methylamineleucine) varied from 0.05 to 27. The results strongly suggested that methylamine transport was not mediated by the same system involved in amino acid transport.
However, it is apparent that the activities of both transport systems are markedly influenced by the nitrogen sufficiency of the mycelium.

Effect of Nutrient
Starvation on Transport- Table  II shows the effect of nutrient starvation on the transport rates of several compounds by three fungal species. The first two columns of figures show the usual variation in transport activity observed in nutrient-sufficient mycelia of different ages. As reported earlier, sulfur starvation resulted in the development (derepression or deinhibition) of specific transport systems for methionine (l), inorganic sulfate (9, lo), and choline-O-sulfate (11). Nitrogen starvation (2) or carbon starvation (12) resulted in the development of a relatively nonspecific transport system for L-a-  The organisms were grown in citrate No. 3 synthetic medium in aerobic, submerged cultured at 25". After 24 hours, each mycelium was removed from the growth medium, washed with deionized water, and divided into five parts.
One part was assayed for transport activity immediately. A second part was reincubated in complete (nutrient-sufficient) medium for 12 hours. A third part was sulfurstarved for 12 hours in citrate No. 3 medium minus a sulfur source. A fourth part was nitrogen-starved for 12 hours in citrate No. 3-N medium.
The fifth part was incubated in citrate No. 3 medium minus glucose for 12 hours. The mycelia were incubated in the indicated media at a density of 2 g, wet weight, per 100 ml of medium.
After the la-hour incubation period, the mycelia were removed from the various media. washed. and resusnended at a density of 0.5 g, wet weight, per 25 ml of assay buffer. All assays were run in 0.05 M potassium phosphate buffer, pH 6.6. c All myceha except that in the carbon-deficient medium increased 2.5-to a-fold in mass during the la-hour reincubation period. The carbon-deficient mycelia remained around 2 g/100 ml.
nitrogen starvation, again suggesting that the methylamine and the amino acid transport systems are not identical.
Ethylamine transport also increased after nitrogen starvation, but not to the same extent as methylamine.
This was encouraging because, if the methylamine permease were in fact an ammonium permease, then we would expect longer chained alkyl amines to be inferior to methylamine as substrates.
It is also noteworthy that choline transport remained constant or decreased as a result of nutrient starvation. This result eliminated a choline permease as the mediator of methylamine transport.

Development of Methylamine
Transport Activity during Nitrogen Starvation- Fig.  4 shows the development of transport activity for methylamine and three amino acids during nitrogen starvation.
Within experimental error, transport activity for the three amino acids developed coincidentally during nitrogen starvation.
Methylamine transport activity started lower and increased more rapidly during the first few hours of nitrogen starvation.
The results confirmed the nonidentity of the methylamine and general amino acid transport systems.  This result might indicate that the actual substrate of the transport system is the free (uncharged) amine, or that the free amine penetrates the mycelium by a noncarrier-mediated process. Fig. 6 shows the temperature dependence of methylamine transport.
The high Q10 value (2.1) and denaturation at temperatures above 30" are characteristic of a carrier-mediated transport process. Fig. 7 shows the effect, of ionic strength on the transport of methylamine, L-methionine, and sulfate. L-Methionine, which is a dipolar ion at the assay pH, is transported at a relatively constant rate over a wide range of ionic strength of the incubation medium.
Transport of choline-O-sulfate (also a dipolar ion) showed the same ionic strength profile (11). Sulfate transport was barely detectable at very low ionic strength, but, increased markedly as the ionic strength increased. Methylamine transport was maximal in suspensions of mycelium in deionized water and decreased slightly as the ionic strength increased. The significance of these differences is not clear, but they might be related to the mechanisms of transport. For example, the transport of the negatively charged sulfate ion may be coupled to the passive, simultaneous uptake of positively charged ions. A membrane-bound binding protein, with a high affinity for sulfate, might concentrate sulfate at the cell surface from a very dilute external environment (13). In order to provide an equivalent surface concentration of positively charged ions, a much higher external concentration of K+ (etc.) might be required.
The mycelium may possess binding proteins and transport systems for cations, but, these may be repressed to a FIG. 5 (left). Effect of pH on methylamine-'4C transport by nitrogen-starved mycelium.
In one set of experiments the mycelium was previously incubated in the 0.01 M buffer for 15 min before adding the substrate (2 X 10+ M).
In the other set of experiments, the mycelium was previously incubated in deionized water for 15 min.
The buffers (2.5 ml of 0.1 M) were added immediately before the substrate.
The pH values indicated were those measured after the 2-min assay period. The buffers used were: pH 2, phosphate; pH 3 to 5, citrate; pH 6 to 7, phosphate; pH 8 to 9, Tris.
The mycelium was added to previously warmed or previously cooled buffer and the transport of methylamine-14C (2 X 10m6 M) was immediately measured.
The arrows indicate the variation in the temperature of the incubation suspension over the 2-min assay period.  9. Distribution of 14C from methylamine-14C (initial concentration 3 X 10m5 M) in a pulse-labeled suspension of nitrogendeficient mycelium.
At zero time the suspension contained 3 Fmoles of methylamine-W and 2 g, wet weight, of mycelium in 100 ml of 5 mM potassium phosphate buffer, pH 6.2. Extractable (intracellular soluble) W was determined by Assay Method I (1). Insoluble mycelial (protein) 1% was determined by directly counting the extracted mycelium.
The inclusion of lo-* M actidione in the assay medium had no significant effect on the rate of transport or the distribution of label.
but rather that active transport and energy production may be intimately coupled within the cell membrane. Arsenate at 10e3 M (added simultaneously with the substrate or incubated with the mycelium before adding the substrate) had no inhibitory effect.
These results are similar to those that we have observed earlier with other transport systems (1, 2, 11). Accumulation and Retention of Methylamine against Concentration Gradient- Fig.  9 shows the distribution of a pulse of 3 pmoles of methylamine-W over a 50-min incubation period. By 10 min, virtually all of the methylamine was removed from the incubation medium.
More than 90% of the transported label could be removed from the mycelium with hot water. Chromatography and electrophoresis of the 50.min extract showed that more than 90% of the 1% was still present as unchanged methylamine-1%.
The remaining 14C had RF values very similar to those of glycine and serine, although positive identification of the metabolites was not made. By 5 hours, most of the methylamine had disappeared and at least three other major 1% peaks appeared.
The ability of the mycelium to retain methylamine and its metabolites against a concentration gradient is shown by the data in Table  IV. Furthermore, the results show that the transport system acts in a unidirectional manner; i.e. there is no apparent exchange between accumulated methylamine-% and unlabeled external methylamine.
Label is lost in the presence of azide or 2,4-dinitrophenol, but at an extremely slow rate compared to the original transport rate (Figs. 2 and 9). The cumulative results suggest that transport is energy-dependent, but that retention is not (except for the energy required for maintaining the integrity of the cell membrane). After 30 min, the mycelium was removed from the incubation medium, washed, and resuspended at a density of 2 g, wet weight, per 100 ml in the various media indicated below.
All media contained lO+ M actidione. Periodically the total W content of the mycelium was determined. After 5 hours a set of samples was extracted in boiling water and the insoluble 1% was determined.  Fig.  10 shows the effect of external methylamine concentration on the initial t.ransport rate.
In nitrogen-starved mycelium, methylamine transport obeys normal hyperbolic (Michaelis-Menten) kinetics with a K, of about lop5 M and a V,,, of about 10 hmoles per g-min on a dry weight basis.
Ethylamine transport in nitrogen-starved mycelium had the same V,,, value, but the K, value was about 1O-4 M. This result was further evidence that we were not observing a general amine transport system.
Methylamine transport by nitrogen-sufficient mycelium was extremely low at low external concentrations and difficult to measure.
Transport seemed to be first order with respect to external concentration up to at least 10m3 M methylamine. Accurate transport rate measurements could not be made at higher concentrations because of high blank values (methylamine-14C nonspecifically adsorbed to or trapped within the mycelial mat). Methylamine uptake by nitrogen-sufficient mycelium seems to be at least partly a result of active transport rather than diffusion, as it is temperature-dependent and decreased by azide.
The data in Fig. 10 suggest that the development of transport activity after nitrogen starvation (Table II and Fig. 3) results from an increase in the affinity of the mycelium for methylamine (i.e. a decrease in the K,,, value).
It is not clear as to exactly how the decrease in K, could arise.
Two possibilities are (a) the utilization of an intracellular nitrogen-containing regulator that competes with external methylamine for a (hypothetical) mobile intramembrane carrier, and (5) the derepression of a binding protein component of the transport system. Possibility b presupposes that in the absence of a binding protein external substrate could still combine with the hypothetical carrier but that a much higher external substrate concentration is required to achieve a given substrate-carrier concentration.
It is also possible that both explanations a and b may contribute to the shift in the K, value.
Effect of Potential Inhibitors on Methylamine Transport-The specificity of the methylamine transport system was investigated by observing the effects of potential inhibition on methylamine uptake.
For routine assays, methylamine-14C (10V4 M) and the potential inhibitor (low3 M) were added simultaneously to the suspension of nitrogen-starved mycelium.
Methylamine transport rates were determined as usual from four aliquots taken at 30-set intervals during the first 2 min of incubation.
These results strongly suggested that we were not dealing with a general amine or amino alcohol transport system. Other simple lcarbon and l-nitrogen compounds such as KHC03, KN03, and urea had little or no effect.
The most potent inhibitor was NH4+ ( >97%). The inhibition by glycine, asparagine, and glutamine was readily explained by the contamination of these compounds by small amounts of NH4+.
The effect of NH4+ on methylamine transport is illustrated in Fig. 11. The release of inhibition FIG. 10. Effect of external methylamine-'% concentration on initial transport rates by nitrogen-starved and nitrogen-sufficient mycelium.
The different symbols refer to separate experiments.
after a few minutes of incubation must result from removal of NH4+ from the incubation medium by the mycelium. (At pH 6.2 it is highly improbable that NH3 was volatilizing.) The length of the lag period before methylamine transport commences is almost proportional to the initial NH4+ concentration at low NH4+ concentrations.
If we assume that the length of the lag period is a measure of the time required to transport NH4+ into the mycelium, we can make a rough estimate of the NH4+ transport rates. For example, it takes approximately 3 min to overcome the lag produced by 3 x low5 M NH4+.
Thus, 0.15 pmole of NH4+ (in a 5-ml aliquot) is transported by about 5 mg, dry weight, of mycelium (i.e. that amount in a 5-ml aliquot) in 3 min.
This corresponds to a transport rate of about 10 pmoles per g-min, which is identical with the V,,, value for methylamine.
This estimate assumes (a) that the lo-' 51 methylamine has negligible effect on the transport of NH4+ at 3 x low5 M NH,+ (i.e. that the K, value for NH4+ is much lower than the Ki value for methylamine) and (b) that the rate of NH4+ transport is constant over the 3-min interval (i.e. that the transport of >90% of the NH4+ starting at 3 x lo+ 11 obeys zero order kinetics).
Both assumptions are confirmed by the results described below.
Because NH4f is so rapidly transported by the mycelium, it was impossible to make accurate measurements of methylamine-i4C transport in the presence of a low and constant concentration of NH,+.
On the other hand, because NH,+ is so powerful an inhibitor of methylamine transport, it was also very difficult to measure methylamine-W transport rates at high external NH4+ concentrations (at which the external NH4+ concentration would remain relatively constant over the assay period), unless the substrate was also quite high in concentration. An attempt to determine the Ki value for NH,+ is shown in Transport Xystem of Fungi Vol. 245,No. 17 FIG. 11. The effect of NHb+ on methylamine-r4C transport by nitrogen-starved mycelium.
(The control rate would actually be 91% of the estimated V,,,.) Fig. 12. The reciprocal plot for the "plus NH4+" rates yielded a straight line with a y-axis intercept at the experimentally determined value for the "minus NHh+" Tr,,, . The apparent K, (reciprocal of z-axis intercept) was 4 X low3 M. If we use this value and the previously determined K, value of 10V5 M, the Ki for NH4+ can be calculated as 2.5 x 1W7 M. These re-    Transport System- Table  VI shows the effect of refeeding substrate amounts of various nitrogen compounds on the methylamine-14C and n-methionine-%l transport rates of nitrogen-starved mycelium.
A number of compounds caused a reduction in both transport activities but asparagine and glutamine had the most striking effect on methylamine transport. Leucine, methionine, cr-aminobutyrat.e, and phenylalanine, on the other hand, were more effective in reducing methionine transport.
A time study established that asparagine and glutamine would "turn off" methylamine transport within a few minutes after their addition to the mycelial suspension.
The reduction in transport activity could result from either a feedback inhibition of the transport system or a denaturation of one or more components of the system.
The rapidity of turn off by glutamine and asparagine suggests the former.
It was interesting to note that glutamine and asparagine were far more effective than NH4+ itself in reducing methylamine transport. Thus, it seems unlikely then that the amides were acting by producing NH&+ intracellularly.
Effect of Actidione on Stability of Methylamine (Ammonium) Transport System-As noted earlier, the addition of actidione to mycelial cultures before nitrogen starvation prevents the development of transport activity. Fig. 13 shows that the addition of actidione after a period of nitrogen starvation stimulates a decrease in previously existing transport activity. The decrease could result from a rapid turnover of a prot.ein component of the transport system, as suggested by Wiley and Matchett (14) for the tryptophan permease.
The decrease might also result from an increase in the intracellular concentration of feedback inhibitors (e.g. glutamine, asparagine) as a result of protein degradation. Grenson et al. (15) have observed a similar phenomenon in yeast and support the latter suggestion.

DISCUSSION
The results presented in this paper confirm that P. chrysogenum possesses a highly specific membrane transport system for NH4+. It is likely that most microorganisms possess such a system. The ammonium transport system is under metabolic control. Glutamine and asparagine seem to be regulators.
Glutamine plays a central role in the nitrogen metabolism of microorganisms, and, as such, its synthesis by glutamine synthetase is under feedback control by multiple products (16). During nitrogen deprivation, the cumulative feedback inhibition of glutamine synthetase is relieved. However, the steady state level of glutamine will decrease as the amide is utilized for the biosynthesis of various nitrogen-containing compounds. Consequently, the intracellular level of glutamine is a reflection of the over-all nitrogen sufficiency of the organism.
It is not surprising then that glutamine should be a regulator of the ammonium transport system.