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A new gene which affects uptake of neutral and acidic amino acids in Neurospora crassa
140
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
25918
A N E W GENE W H I C H AFFECTS U P T A K E OF N E U T R A L AND ACIDIC AMINO ACIDS IN N E U R O S P O R A C R A S S A E R I C S. J A C O B S O N AND R O B E R T L. M E T Z E N B E R G
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison, Wisc. (U.S.A.) (Received S e p t e m b e r 25th,i967)
SUMMARY
By selection on a medium containing both ethionine and p-fluorophenylalanine a mutant strain of Neurospora has been isolated. This strain, nap, shows greatly decreased transport of neutral and acidic amino acids. The mutation also confers resistance to 4-methyltryptophan, aminopterin and glycine. The physiological defect appears to be specific since the mutant showed a normal doubling time for protein, normal oxygen consumption and normal transport of glucose, sulfate and basic amino acids. This gene represents a third locus which governs neutral and acidic amino acid transport in Neurospora, the two previously described being u~t-I (55 yoI) and mtr.
INTRODUCTION
Several genes are already known to control amino acid permeability in Neurospora. By selection of strains resistant to 4-methyltryptophan LESTEI~1 and STADLER~ have obtained aconsiderable number of mutants which all map at one locus in linkage group IV. STADLER has called this locus mtr. These strains are characterized by a markedly decreased uptake of neutral amino acids and by a high degree of resistance to ethionine and to p-fluorophenylalanine, as well as to 4-methyltryptophan. ST:XDLER~ has found suppressors of mtr; these map in linkage group I. ST. LaWI~E~'CEet al. 4 have described a gene which increases permeability to amino acids and peptides; this gene has been located in linkage group VI and called rood-5. Mutant un-t (55 7oz), henceforth called simply 55701, has been shown by KAPPY AND METZENBERG5,6 to have a decreased rate of transport of neutral and acidic amino acids, in addition to other defects that appear to be related to the plasma membrane. This gene maps in linkage group I. The following properties of mutant 557 ol were intriguing to us: (I) It maps very close (about o.I recombination units) to the mating type locus. (2) Its growth shows the same temperature sensitivity as does the formation of female fruiting bodies (properithecia) in the wild type; both are abolished above 31.5 °. (3) It is resistant to ethionine and to p-fluorophenylalanine, apparently because of an altered membrane. This last property suggests that additional mutants near the mating type locus might be obtained by selecting for simultaneous resistance to ethionine and p-fluorophenylBiochim. Biophys. Acta, 156 (1968) 14o-147
U P T A K E OF N E U T R A L AND ACIDIC AMINO ACIDS
141
alanine. Study of such mutants might clarify the relationship of membrane structure and function to mating activity in Neurospora. Mutants were accordingly obtained and mapped. We found that none was closely linked to the mating type locus in linkage group I. One strain, however, showed clear linkage to markers known to be in linkage group V, and since no locus in linkage group V has heretofore been implicated in amino acid transport, we have tried to measure the extent of its function. Since we find that this mutation reduces the permeability of the cell membrane to neutral and acidic amino acids, we have called the locus nap, for neutral and acidic (amino acid) permeability. MATERIALS AND METHODS
14C-Labelled compounds were purchased from New England Nuclear Corp. H23~SO4was obtained from Oak Ridge National Laboratory. The Sigma Chemical Co. was the source of L-ethionine, p-fluoro-L-phenylalanine and 4-methyl-DL-tryptophan. Azetidine-2-carboxylic acid and cycloheximide were from Calbiochem Corp. Aminopterin, 5-aminouracil and actinomycin D were purchased from Gallard, Schlesinger, Eastman and Merck, respectively. All of the 'natural' amino acids used (except, of course, glycine) were the L-enantiomorphs. STA4A is one of the standard wild-type strains, while Wa is derived from the Emerson genetic backgroundL The inos strain used was allele No. 89601, the cot strain No. CIo2t, and the alcoy linkage tester was the standard strain furnished by the Fungal Genetics Stock Center. Selection and scoring of mutants was done on solid Fries' salts medium s, using 1.5 °/o sucrose, 0. 3 mM ethionine and 0.02 mM p-fluoro-Lphenylalanine. Vegetative colonies were maintained and crosses made on slants of crossing medium 9. This same medium, enriched as described b y FROST 1°, wa s used for production of conidia. Dry weights of mycelia in samples of liquid cultures were obtained by filtration on 1.2-~ Millipore membranes, washing with water, and drying of mycelial pads overnight at IiO °. Uptake experiments were performed as descibed by KAPPY AND METZENBERG 5, the incubation temperature being Io ° for reasons that have been previously discussed. The Warburg manometric technique 11 was used to measure oxygen consumption b y conidia which had been allowed to germinate for 5.5 h and resuspended in fresh medium. Growth experiments were performed on 25-ml suspensions of conidia in siliconized flasks which were shaken rapidly at 25 °. The initial inoculum was made to give an absorbance at 420 m/~ of 0.2. Samples were periodically withdrawn and assayed for protein by the method of LOWRY et al. 12. RESULTS
Isolation of mutants Approx. io 7 conidia of strain Wa were suspended in IOO ml of distilled water and irradiated with a ultraviolet germicidal lamp to approx. 50 % killing. One-ml portions were then plated on Fries medium plus ethionine and p-fluorophenylalanine. 24 Resistant colonies were picked after 3 days growth at 22 °. The resulting cultures were then outcrossed twice to the wild-type strain STA4A, and progeny from each cross were plated on Fries plus ethionine and p-fluorophenylalanine agar and scored Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
142
E . S . JACOBSON, R. L. METZENBERG
for resistance after 36 h at room temperature. Four strains showed a I" I segregation of the resistant trait in the progeny, which suggested that resistance in these strains was attributable to a single gene change.
Genetic linkage of mutants A small number of resistant colonies were isolated from each cross and their mating types determined. From each cross resistant progeny of both mating types were found, indicating that none of the mutations was closely linked to the mating type locus. However, we decided to continue studies on the strain showing the greatest degree of resistance, nap. In order to determine its linkage group it was crossed to Perkins' alcoy stock. (This derived strain contains three translocations which ioin linkage groups I and II, I I I and VI, and IV and V. Near each junction are marker genes that are easily scored: albino, yellow, and cot (colonial temperaturesensitive), respectively. The remaining linkage group, vii, is not marked). Testing of progeny from this cross indicated that the nap gene was situated either in IV of V. A cross to a non-translocation cot strain (linkage group IV) showed random segregation, suggesting that the gene was probably not on this chromosome, but instead on V. This was confirmed by crossing to a strain carrying the inos marker (V), requiring inositol for growth. Of 75 viable ascospores isolated from this cross, only I I , or 15 %, were recombinants (Table I). 95 % confidence limits are from 7 to 23 units in either direction from the inos locus but the nap gene is probably to the left of inos, since it showed no significant linkage to the asp locus. Asp (asparagine) is about 25 units to the right of inos, on chromosome V (ref. 13).
Resistance to noxious substances Growth in the presence of various inhibitors was followed in several ways. In one type of experiment, conidial suspensions were spotted on solid medium and the TABLE I C L A S S I F I C A T I O N OF PROG]~NY F R O M T H E CROSS,
n a p a )< inos A
G e r m i n a t i o n rate : 5 6 %.
inos + inos --
nap + (sensitive)
n a p - - (resistant)
8 (Recombinant) 32 (Parental)
32 (Parental) 3 (Recombinant)
TABLE II GROWTH
OF COLONIES
ON
SOLID
FRIES
MEDIUM
CONTAINING
Inhibitor
None (24 h) o. 3 mM ethionine a n d o.o2 mM p-fluorophenylalanine (24 h) I mM 4 - m e t h y l - D L - t r y p t o p h a n (48 h) 0.5 ° mM a m i n o p t e r i n (48 h)
Biochim. Biophys. Acts, 156 (1968) 14o-147
THE
FOLLOWING
INHIBITORS
Growth (mm) nap
55 7 or
W i l d type
16 8 34 20
29
51
II
o
13 :24
3 4
143
U P T A K E OF N E U T R A L A N D A C I D I C A M I N O A C I D S
growth of the colonies was measured after 24 or 48 h at room temperature. Results in Table II show that the nap gene confers resistance to ethionine-p-fluorophenylalanine, 4-methyltryptophan and aminopterin. It did not protect against actidione, 2-azetidine-carboxylic acid (a proline antagonist), 5-aminouracil or actinomycin D. 55 7 °1, which protected against ethionine and p-fluorophenylalanine, also appeared to give protection against aminopterin and 4-methyltryptophan. These results were confirmed by measurement of dry weights after 18 h growth in liquid culture. These are listed in Table III. A surprising finding was that the wild type was partially inhibited by glycine, and that neutral amino acid permease mutations appeared to abolish this effect. Plates of Fries minimal medium supplemented with increasing amounts of glycine i
I
o
i
1.5
I.E
o
o
o
o E
i.o
o
I.C
o
i
13
o o o
ildtype
tlA 55701"~
~ z~~
z~
212 0 0.5 Q~ (.9
fn"_2 I
i0-7
•
•
~
u
•
o
0.5
I
I
I
i0-5
i0-3
lO-i
GLYGINE CONCENTRATION
[M)
I0"7
I
I
I
lO-S
i0-~
lO-I
GLYCINE C O N C E N T R A T I O N
(M)
Fig, I. R a t e of c h a n g e of r a d i u s of c o l o n i e s g r o w n a t 25 ° o n s o l i d F r i e s m e d i u m s u p p l e m e n t e d w i t h t h e i n d i c a t e d c o n c e n t r a t i o n s of g l y c i n e .
TABLE
III
DRY WEIGHT OF MYCELIUM PER m l OF CULTURE MEDIUM AFTER 18 h GROWTH
Inhibitor
Dry wt. (mg/ml)
Wild type
nap
55 ro7
3-7 1.2
0.4 0. 7
5.3 o. 4
1.37 0.46 0.48
0.42 o.2o o. 12
2.2 o.15 o.09
Expt. I None 0.3 m M e t h i o n i n e a n d 0.02 m M p - t i u o r o p h e n y l a l a n i n e
Expt. I f None 0.25 m g / m l a m i n o p t e r i n i .o m M m e t h y l t r y p t o p h a n
Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
144
E.S.
JACOBSON, R. L. M E T Z E N B E R G
were spotted with conidial suspensions. Fig. I shows a plot of growth rate against glycine concentration. It is apparent that the wild type is inhibited 5o % by I mM glycine, but this effect is not seen in either nap or 55 7 ol.
Peremeability properties Fig. 2 shows that the nap mutation reduced by 25-fold the rate at which germinated conidia concentrate ethionine. This experiment was performed on conidia that had been allowed to germinate by incubating for 5.5 h. Essentially the same results were obtained after 3-5, 7.5 and lO.5 h of incubation. Table IV summarizes I
t
8¢
C
E ,c
/
t / _ 0
~\ 30
.
•
T 60
I 90
TIME (min) Fig. 2. U p t a k e of ~14C]ethionine b y g e r m i n a t e d e o n i d i a of t h e i n d i c a t e d s t r a i n s . TABLE
IX:
ACCUMULATION OF DIFFERENT COMPOUNDS
Substance
Uptake (mFmoles/mg protein) nap
nap__× Ioo %
557oz
557oz × zoo % W a ~Va
5.6 6. 4 6. 3 14.7 3.2 1.6 51.o 290.0 i.o 0.97 -5.4
15 22 IO 9 26 17 300 17o 26 50 80 ioo
Wa Ethionine Methionine Fluorophenylalanine Phenylalanine Aspartate Glutamate Arginine Lysine Proline ~-Aminoisobutyrate Glucose S u l f a t e ( i o rain)
1.4 2.6 I. I 2.8 1.6 1.5 16.o 97.0 2.0 o.37 5.5
4 9 2 2 13 16 94 56 53 18 72 lO4
Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
38.4 29.1 65.o 159.o 12.6 9.3 17.o 172.o 3.8 2.0 -5-3
145
UPTAKE OF NEUTRAL AND ACIDIC AMINO ACIDS
the results of twelve such experiments, which compare the amount of accumulation of 12 substances in 30 min (when the uptake curve is still more or less linear). It is evident that only the neutral and acidic amino acids are transported significantly less rapidly in nap than in the wild type. It is possible to suppose that any mutation that resulted in a general impairment of metabolism would manifest itself in many non-specific ways, which could include reduced transport. In order to estimate 'general metabolic rates' we have measured oxygen consumption and protein doubling times during growth in liquid medium. Oxygen consumption is expressed in Table V as the average of three separate experiments; in neither the nap strain nor in 55 7 ol was it dramatically less than that of the wild type, and the same can be said of the growth rates (same table). TABLE V OXYGEN CONSUMPTION AND PROTEIN DOUBLING TIMES nap
nap
x Ioo %
557°z
Wa Oxygen c o n s u p t i o n in #1 per h per m g protein (average of 3 experiments) Protein doubling time (h)
89 2. 9
69 %
557°z × zoo % W a Wa
lO7 4.o
83 %
129 2.6
DISCUSSION
From the data obtained it is apparent that the nap strain derives its resistance from a decreased permeability to neutral and acidic amino acids. Uptake of basic amino acids is not affected, and neither is the uptake of glucose or of sulfate. Since in the mutant strain oxygen consumption and growth rate are not greatly different from normal, it is concluded that the physiological defect is limited to the transport function. These data together with those of others suggest that the products of at least three genetic loci (mtr, 55 7 ol and nap) are required for normal uptake of neutral amino acids from the medium. Both 55 7 ol and nap show greatly decreased uptakes of acidic amino acids, but this has apparently not been measured in the mtr strain. In all three strains the uptake of basic amino acids appears to be unaffected. In nap, as in 55 7 ol, proline transport is reduced by about 50 % ; this is equivocal and probably is not a significant change from the normal. This would be consistent with the finding by DEBUSK AND DEBUSK21 that proline, in contrast to the other neutral amino acids, does not compete with phenylalanine for uptake, and with our own observation that neither mutation confers resistance to azetidine-2-carboxylic acid. KINSEY~° has described an p-fluorophenylalanine-resistant mutant in Neurospora, having an impaired ability to concentrate tryptophan and leucine, but a normal ability to concentrate lysine. This strain, fpr-I, maps in linkage group V and could be allelic with nap. In Sacchararomyces cerevisiae both very general and very specific amino acid transport genes have been found. SURDIN et al. 14 have described a mutant which they call aap; this strain shows a Io-fold reduction of uptake rates of glutamate, aspartate, Biochim. Biophys. Mcta, 156 (1968) 14o-147
I4 6
E. S. JACOBSON, R. L. METZENBERG
threonine and methionine. In addition it is resistant to canavanine and reduces the ability of lysine auxotrophs to grow on added lysine. This locus is thought to be involved in uptake of all amino acids, and m a y be the one that has been studied by SORSOLI, SPENCE AND PARKS1~. The latter group had described an ethionine-resistant strain which showed decreased uptake of randomly labeled algal hydrolysate as well as of labeled ethionine and methionine, and also was resistant to p-fluoromethylalanine 15. On the other hand GRENSON eta/. 16,17 have presented very compelling evidence, both genetic and kinetic, for separate and specific arginine and lysine permeases in Saccharomyces. This appears different from the case in Neurospora, where these basic amino acids reciprocally inhibit each others uptake, presumably because they share a common transport site is. The phenomenon of glycine inhibition has been studied by BEAUD19 in Agro-
bacterium tumefaciens. From the data it can be concluded that there are at least two cistrons which specify machinery common to both acidic and neutral amino acid transport, namely 55701 and nap, and a third, mtr, involved in neutral amino acid transport. Whether the mtr locus is involved in acidic amino acid transport remains to be determined. Neurospora apparently contains two systems for uptake of histidine, one which is subject to competition by neutral amino acids, the other by basic amino acids. WOODWARD, READ AND WOODWARD 22 have obtained mutants for each of these systems. Those for the basic amino acid permease map in linkage group V, in the same general region as does nap. It appears possible, then, that a gene or genes in this part of chromosome V m a y have an important role in determining membrane function; the exact facet seen would then depend on the method used to select mutants. ACKNOWLEDGEMENTS
This work was supported in part by a research grant (GM-o8995-o6) from the U.S. Public Health Service. Support for one of us (E. S. J.) was kindly provided by a Fellowship from tile American Cancer Society, No. PRE-I2, and for the other of us (R. L. M.) by a U.S. Public Health Service Career Development Award (K3-GM19416).
REFERENCES G. LESTER, J. Bacteriol., 91 (1966) 677. D. R. STADLER, Genetics, 54 (1966) 677D. R. STAOLER, Science, 15o (1965) 385 . p. ST. LAWRENCE, B. D. MALING, L. ALTWERGER AND M. RACHMELER, Genetics, 5° (1964) 1383. M. S. NAPPY AND R. L. METZENBERG, Bioehim. Biophys. Acta, lO 7 (1965) 425 . M. S. NAPPY AND R. L. METZENBERG, J. Bacteriol., in t h e press. R. W. BARRATT, Neurospora Newsletter, 2 (1962) 24. G. W. BEADLE AND E. L. TATUM, Am. J. Botany, 32 (1945) 678. M. WESTERGAARD AND H. H. MITCHELL, Am. J. Botany, 34 (1947) 573. C. C. FROST, Neurospora Newsletter, i (1962) i i (2). W. W. UMBREIT, R. H. BURRIS AND J. F. STAUFFER, Manometric Techniques, 3rd ed. Burgess, Minneapolis, Minn., 1959, p. i. 12 O. H. LOWRY, N. J. ROSEBROUGH, A. L. EARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 13 W. STRICKLAND, D. PERKINS AND C. VEACH, Genetics, 44 (1959) 1221. I 2 3 4 5 6 7 8 9 IO it
Biochim. Biophys. Acta, 156 (1968) I 4 O - I 4 7
UPTAKE OF NEUTRAL AND ACIDIC AMINO ACIDS
147
14 Y. SURDIN, W. SLY, J. SIRE, A. M. BORDES AND H. D~ ROBICHON-SZULMAJSTER, Biochim. Biophys. Acta, lO 7 (1965) 546. 15 W. A. SORSOLI, K. D. SPENCE AND L. W. PARKS, J. Bacteriol., 88 (1964) 20. 16 M. GRENSON, M. MOUSSET, J. M. WIAME AND J. BECHET, Bioehim. Biophys. Acta, 127 (1966) 325 • 17 M. GRENSON, Biochim. Biophys. Acta, 127 (1966) 339. 18 R. H. BAUERLE AND H. R. GARNER, Biochim. Biophys. Acta, 93 (1964) 32o. 19 G. BEAUD, Biochim. Biophys. Acta, 129 (1966) 563 . 2o J. A. KINSEY, Genetics, 56 (1967) 57 °. 21 B. G. DEBUSK AND A. G. DEBUSK, Biochim. Biophys. Acta, lO 4 (1965) 139. 22 C. K. WOODWARD, C. P. READ AND V. W. WOODWARD, Genetics, 56 (1967) 598.
Biochim. Biophys. Acta, 156 (1968) I 4 o - I 4 7
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
25918
A N E W GENE W H I C H AFFECTS U P T A K E OF N E U T R A L AND ACIDIC AMINO ACIDS IN N E U R O S P O R A C R A S S A E R I C S. J A C O B S O N AND R O B E R T L. M E T Z E N B E R G
Department of Physiological Chemistry, University of Wisconsin Medical School, Madison, Wisc. (U.S.A.) (Received S e p t e m b e r 25th,i967)
SUMMARY
By selection on a medium containing both ethionine and p-fluorophenylalanine a mutant strain of Neurospora has been isolated. This strain, nap, shows greatly decreased transport of neutral and acidic amino acids. The mutation also confers resistance to 4-methyltryptophan, aminopterin and glycine. The physiological defect appears to be specific since the mutant showed a normal doubling time for protein, normal oxygen consumption and normal transport of glucose, sulfate and basic amino acids. This gene represents a third locus which governs neutral and acidic amino acid transport in Neurospora, the two previously described being u~t-I (55 yoI) and mtr.
INTRODUCTION
Several genes are already known to control amino acid permeability in Neurospora. By selection of strains resistant to 4-methyltryptophan LESTEI~1 and STADLER~ have obtained aconsiderable number of mutants which all map at one locus in linkage group IV. STADLER has called this locus mtr. These strains are characterized by a markedly decreased uptake of neutral amino acids and by a high degree of resistance to ethionine and to p-fluorophenylalanine, as well as to 4-methyltryptophan. ST:XDLER~ has found suppressors of mtr; these map in linkage group I. ST. LaWI~E~'CEet al. 4 have described a gene which increases permeability to amino acids and peptides; this gene has been located in linkage group VI and called rood-5. Mutant un-t (55 7oz), henceforth called simply 55701, has been shown by KAPPY AND METZENBERG5,6 to have a decreased rate of transport of neutral and acidic amino acids, in addition to other defects that appear to be related to the plasma membrane. This gene maps in linkage group I. The following properties of mutant 557 ol were intriguing to us: (I) It maps very close (about o.I recombination units) to the mating type locus. (2) Its growth shows the same temperature sensitivity as does the formation of female fruiting bodies (properithecia) in the wild type; both are abolished above 31.5 °. (3) It is resistant to ethionine and to p-fluorophenylalanine, apparently because of an altered membrane. This last property suggests that additional mutants near the mating type locus might be obtained by selecting for simultaneous resistance to ethionine and p-fluorophenylBiochim. Biophys. Acta, 156 (1968) 14o-147
U P T A K E OF N E U T R A L AND ACIDIC AMINO ACIDS
141
alanine. Study of such mutants might clarify the relationship of membrane structure and function to mating activity in Neurospora. Mutants were accordingly obtained and mapped. We found that none was closely linked to the mating type locus in linkage group I. One strain, however, showed clear linkage to markers known to be in linkage group V, and since no locus in linkage group V has heretofore been implicated in amino acid transport, we have tried to measure the extent of its function. Since we find that this mutation reduces the permeability of the cell membrane to neutral and acidic amino acids, we have called the locus nap, for neutral and acidic (amino acid) permeability. MATERIALS AND METHODS
14C-Labelled compounds were purchased from New England Nuclear Corp. H23~SO4was obtained from Oak Ridge National Laboratory. The Sigma Chemical Co. was the source of L-ethionine, p-fluoro-L-phenylalanine and 4-methyl-DL-tryptophan. Azetidine-2-carboxylic acid and cycloheximide were from Calbiochem Corp. Aminopterin, 5-aminouracil and actinomycin D were purchased from Gallard, Schlesinger, Eastman and Merck, respectively. All of the 'natural' amino acids used (except, of course, glycine) were the L-enantiomorphs. STA4A is one of the standard wild-type strains, while Wa is derived from the Emerson genetic backgroundL The inos strain used was allele No. 89601, the cot strain No. CIo2t, and the alcoy linkage tester was the standard strain furnished by the Fungal Genetics Stock Center. Selection and scoring of mutants was done on solid Fries' salts medium s, using 1.5 °/o sucrose, 0. 3 mM ethionine and 0.02 mM p-fluoro-Lphenylalanine. Vegetative colonies were maintained and crosses made on slants of crossing medium 9. This same medium, enriched as described b y FROST 1°, wa s used for production of conidia. Dry weights of mycelia in samples of liquid cultures were obtained by filtration on 1.2-~ Millipore membranes, washing with water, and drying of mycelial pads overnight at IiO °. Uptake experiments were performed as descibed by KAPPY AND METZENBERG 5, the incubation temperature being Io ° for reasons that have been previously discussed. The Warburg manometric technique 11 was used to measure oxygen consumption b y conidia which had been allowed to germinate for 5.5 h and resuspended in fresh medium. Growth experiments were performed on 25-ml suspensions of conidia in siliconized flasks which were shaken rapidly at 25 °. The initial inoculum was made to give an absorbance at 420 m/~ of 0.2. Samples were periodically withdrawn and assayed for protein by the method of LOWRY et al. 12. RESULTS
Isolation of mutants Approx. io 7 conidia of strain Wa were suspended in IOO ml of distilled water and irradiated with a ultraviolet germicidal lamp to approx. 50 % killing. One-ml portions were then plated on Fries medium plus ethionine and p-fluorophenylalanine. 24 Resistant colonies were picked after 3 days growth at 22 °. The resulting cultures were then outcrossed twice to the wild-type strain STA4A, and progeny from each cross were plated on Fries plus ethionine and p-fluorophenylalanine agar and scored Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
142
E . S . JACOBSON, R. L. METZENBERG
for resistance after 36 h at room temperature. Four strains showed a I" I segregation of the resistant trait in the progeny, which suggested that resistance in these strains was attributable to a single gene change.
Genetic linkage of mutants A small number of resistant colonies were isolated from each cross and their mating types determined. From each cross resistant progeny of both mating types were found, indicating that none of the mutations was closely linked to the mating type locus. However, we decided to continue studies on the strain showing the greatest degree of resistance, nap. In order to determine its linkage group it was crossed to Perkins' alcoy stock. (This derived strain contains three translocations which ioin linkage groups I and II, I I I and VI, and IV and V. Near each junction are marker genes that are easily scored: albino, yellow, and cot (colonial temperaturesensitive), respectively. The remaining linkage group, vii, is not marked). Testing of progeny from this cross indicated that the nap gene was situated either in IV of V. A cross to a non-translocation cot strain (linkage group IV) showed random segregation, suggesting that the gene was probably not on this chromosome, but instead on V. This was confirmed by crossing to a strain carrying the inos marker (V), requiring inositol for growth. Of 75 viable ascospores isolated from this cross, only I I , or 15 %, were recombinants (Table I). 95 % confidence limits are from 7 to 23 units in either direction from the inos locus but the nap gene is probably to the left of inos, since it showed no significant linkage to the asp locus. Asp (asparagine) is about 25 units to the right of inos, on chromosome V (ref. 13).
Resistance to noxious substances Growth in the presence of various inhibitors was followed in several ways. In one type of experiment, conidial suspensions were spotted on solid medium and the TABLE I C L A S S I F I C A T I O N OF PROG]~NY F R O M T H E CROSS,
n a p a )< inos A
G e r m i n a t i o n rate : 5 6 %.
inos + inos --
nap + (sensitive)
n a p - - (resistant)
8 (Recombinant) 32 (Parental)
32 (Parental) 3 (Recombinant)
TABLE II GROWTH
OF COLONIES
ON
SOLID
FRIES
MEDIUM
CONTAINING
Inhibitor
None (24 h) o. 3 mM ethionine a n d o.o2 mM p-fluorophenylalanine (24 h) I mM 4 - m e t h y l - D L - t r y p t o p h a n (48 h) 0.5 ° mM a m i n o p t e r i n (48 h)
Biochim. Biophys. Acts, 156 (1968) 14o-147
THE
FOLLOWING
INHIBITORS
Growth (mm) nap
55 7 or
W i l d type
16 8 34 20
29
51
II
o
13 :24
3 4
143
U P T A K E OF N E U T R A L A N D A C I D I C A M I N O A C I D S
growth of the colonies was measured after 24 or 48 h at room temperature. Results in Table II show that the nap gene confers resistance to ethionine-p-fluorophenylalanine, 4-methyltryptophan and aminopterin. It did not protect against actidione, 2-azetidine-carboxylic acid (a proline antagonist), 5-aminouracil or actinomycin D. 55 7 °1, which protected against ethionine and p-fluorophenylalanine, also appeared to give protection against aminopterin and 4-methyltryptophan. These results were confirmed by measurement of dry weights after 18 h growth in liquid culture. These are listed in Table III. A surprising finding was that the wild type was partially inhibited by glycine, and that neutral amino acid permease mutations appeared to abolish this effect. Plates of Fries minimal medium supplemented with increasing amounts of glycine i
I
o
i
1.5
I.E
o
o
o
o E
i.o
o
I.C
o
i
13
o o o
ildtype
tlA 55701"~
~ z~~
z~
212 0 0.5 Q~ (.9
fn"_2 I
i0-7
•
•
~
u
•
o
0.5
I
I
I
i0-5
i0-3
lO-i
GLYGINE CONCENTRATION
[M)
I0"7
I
I
I
lO-S
i0-~
lO-I
GLYCINE C O N C E N T R A T I O N
(M)
Fig, I. R a t e of c h a n g e of r a d i u s of c o l o n i e s g r o w n a t 25 ° o n s o l i d F r i e s m e d i u m s u p p l e m e n t e d w i t h t h e i n d i c a t e d c o n c e n t r a t i o n s of g l y c i n e .
TABLE
III
DRY WEIGHT OF MYCELIUM PER m l OF CULTURE MEDIUM AFTER 18 h GROWTH
Inhibitor
Dry wt. (mg/ml)
Wild type
nap
55 ro7
3-7 1.2
0.4 0. 7
5.3 o. 4
1.37 0.46 0.48
0.42 o.2o o. 12
2.2 o.15 o.09
Expt. I None 0.3 m M e t h i o n i n e a n d 0.02 m M p - t i u o r o p h e n y l a l a n i n e
Expt. I f None 0.25 m g / m l a m i n o p t e r i n i .o m M m e t h y l t r y p t o p h a n
Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
144
E.S.
JACOBSON, R. L. M E T Z E N B E R G
were spotted with conidial suspensions. Fig. I shows a plot of growth rate against glycine concentration. It is apparent that the wild type is inhibited 5o % by I mM glycine, but this effect is not seen in either nap or 55 7 ol.
Peremeability properties Fig. 2 shows that the nap mutation reduced by 25-fold the rate at which germinated conidia concentrate ethionine. This experiment was performed on conidia that had been allowed to germinate by incubating for 5.5 h. Essentially the same results were obtained after 3-5, 7.5 and lO.5 h of incubation. Table IV summarizes I
t
8¢
C
E ,c
/
t / _ 0
~\ 30
.
•
T 60
I 90
TIME (min) Fig. 2. U p t a k e of ~14C]ethionine b y g e r m i n a t e d e o n i d i a of t h e i n d i c a t e d s t r a i n s . TABLE
IX:
ACCUMULATION OF DIFFERENT COMPOUNDS
Substance
Uptake (mFmoles/mg protein) nap
nap__× Ioo %
557oz
557oz × zoo % W a ~Va
5.6 6. 4 6. 3 14.7 3.2 1.6 51.o 290.0 i.o 0.97 -5.4
15 22 IO 9 26 17 300 17o 26 50 80 ioo
Wa Ethionine Methionine Fluorophenylalanine Phenylalanine Aspartate Glutamate Arginine Lysine Proline ~-Aminoisobutyrate Glucose S u l f a t e ( i o rain)
1.4 2.6 I. I 2.8 1.6 1.5 16.o 97.0 2.0 o.37 5.5
4 9 2 2 13 16 94 56 53 18 72 lO4
Biochim. Biophys. Acta, 156 (1968) 1 4 o - 1 4 7
38.4 29.1 65.o 159.o 12.6 9.3 17.o 172.o 3.8 2.0 -5-3
145
UPTAKE OF NEUTRAL AND ACIDIC AMINO ACIDS
the results of twelve such experiments, which compare the amount of accumulation of 12 substances in 30 min (when the uptake curve is still more or less linear). It is evident that only the neutral and acidic amino acids are transported significantly less rapidly in nap than in the wild type. It is possible to suppose that any mutation that resulted in a general impairment of metabolism would manifest itself in many non-specific ways, which could include reduced transport. In order to estimate 'general metabolic rates' we have measured oxygen consumption and protein doubling times during growth in liquid medium. Oxygen consumption is expressed in Table V as the average of three separate experiments; in neither the nap strain nor in 55 7 ol was it dramatically less than that of the wild type, and the same can be said of the growth rates (same table). TABLE V OXYGEN CONSUMPTION AND PROTEIN DOUBLING TIMES nap
nap
x Ioo %
557°z
Wa Oxygen c o n s u p t i o n in #1 per h per m g protein (average of 3 experiments) Protein doubling time (h)
89 2. 9
69 %
557°z × zoo % W a Wa
lO7 4.o
83 %
129 2.6
DISCUSSION
From the data obtained it is apparent that the nap strain derives its resistance from a decreased permeability to neutral and acidic amino acids. Uptake of basic amino acids is not affected, and neither is the uptake of glucose or of sulfate. Since in the mutant strain oxygen consumption and growth rate are not greatly different from normal, it is concluded that the physiological defect is limited to the transport function. These data together with those of others suggest that the products of at least three genetic loci (mtr, 55 7 ol and nap) are required for normal uptake of neutral amino acids from the medium. Both 55 7 ol and nap show greatly decreased uptakes of acidic amino acids, but this has apparently not been measured in the mtr strain. In all three strains the uptake of basic amino acids appears to be unaffected. In nap, as in 55 7 ol, proline transport is reduced by about 50 % ; this is equivocal and probably is not a significant change from the normal. This would be consistent with the finding by DEBUSK AND DEBUSK21 that proline, in contrast to the other neutral amino acids, does not compete with phenylalanine for uptake, and with our own observation that neither mutation confers resistance to azetidine-2-carboxylic acid. KINSEY~° has described an p-fluorophenylalanine-resistant mutant in Neurospora, having an impaired ability to concentrate tryptophan and leucine, but a normal ability to concentrate lysine. This strain, fpr-I, maps in linkage group V and could be allelic with nap. In Sacchararomyces cerevisiae both very general and very specific amino acid transport genes have been found. SURDIN et al. 14 have described a mutant which they call aap; this strain shows a Io-fold reduction of uptake rates of glutamate, aspartate, Biochim. Biophys. Mcta, 156 (1968) 14o-147
I4 6
E. S. JACOBSON, R. L. METZENBERG
threonine and methionine. In addition it is resistant to canavanine and reduces the ability of lysine auxotrophs to grow on added lysine. This locus is thought to be involved in uptake of all amino acids, and m a y be the one that has been studied by SORSOLI, SPENCE AND PARKS1~. The latter group had described an ethionine-resistant strain which showed decreased uptake of randomly labeled algal hydrolysate as well as of labeled ethionine and methionine, and also was resistant to p-fluoromethylalanine 15. On the other hand GRENSON eta/. 16,17 have presented very compelling evidence, both genetic and kinetic, for separate and specific arginine and lysine permeases in Saccharomyces. This appears different from the case in Neurospora, where these basic amino acids reciprocally inhibit each others uptake, presumably because they share a common transport site is. The phenomenon of glycine inhibition has been studied by BEAUD19 in Agro-
bacterium tumefaciens. From the data it can be concluded that there are at least two cistrons which specify machinery common to both acidic and neutral amino acid transport, namely 55701 and nap, and a third, mtr, involved in neutral amino acid transport. Whether the mtr locus is involved in acidic amino acid transport remains to be determined. Neurospora apparently contains two systems for uptake of histidine, one which is subject to competition by neutral amino acids, the other by basic amino acids. WOODWARD, READ AND WOODWARD 22 have obtained mutants for each of these systems. Those for the basic amino acid permease map in linkage group V, in the same general region as does nap. It appears possible, then, that a gene or genes in this part of chromosome V m a y have an important role in determining membrane function; the exact facet seen would then depend on the method used to select mutants. ACKNOWLEDGEMENTS
This work was supported in part by a research grant (GM-o8995-o6) from the U.S. Public Health Service. Support for one of us (E. S. J.) was kindly provided by a Fellowship from tile American Cancer Society, No. PRE-I2, and for the other of us (R. L. M.) by a U.S. Public Health Service Career Development Award (K3-GM19416).
REFERENCES G. LESTER, J. Bacteriol., 91 (1966) 677. D. R. STADLER, Genetics, 54 (1966) 677D. R. STAOLER, Science, 15o (1965) 385 . p. ST. LAWRENCE, B. D. MALING, L. ALTWERGER AND M. RACHMELER, Genetics, 5° (1964) 1383. M. S. NAPPY AND R. L. METZENBERG, Bioehim. Biophys. Acta, lO 7 (1965) 425 . M. S. NAPPY AND R. L. METZENBERG, J. Bacteriol., in t h e press. R. W. BARRATT, Neurospora Newsletter, 2 (1962) 24. G. W. BEADLE AND E. L. TATUM, Am. J. Botany, 32 (1945) 678. M. WESTERGAARD AND H. H. MITCHELL, Am. J. Botany, 34 (1947) 573. C. C. FROST, Neurospora Newsletter, i (1962) i i (2). W. W. UMBREIT, R. H. BURRIS AND J. F. STAUFFER, Manometric Techniques, 3rd ed. Burgess, Minneapolis, Minn., 1959, p. i. 12 O. H. LOWRY, N. J. ROSEBROUGH, A. L. EARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 13 W. STRICKLAND, D. PERKINS AND C. VEACH, Genetics, 44 (1959) 1221. I 2 3 4 5 6 7 8 9 IO it
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UPTAKE OF NEUTRAL AND ACIDIC AMINO ACIDS
147
14 Y. SURDIN, W. SLY, J. SIRE, A. M. BORDES AND H. D~ ROBICHON-SZULMAJSTER, Biochim. Biophys. Acta, lO 7 (1965) 546. 15 W. A. SORSOLI, K. D. SPENCE AND L. W. PARKS, J. Bacteriol., 88 (1964) 20. 16 M. GRENSON, M. MOUSSET, J. M. WIAME AND J. BECHET, Bioehim. Biophys. Acta, 127 (1966) 325 • 17 M. GRENSON, Biochim. Biophys. Acta, 127 (1966) 339. 18 R. H. BAUERLE AND H. R. GARNER, Biochim. Biophys. Acta, 93 (1964) 32o. 19 G. BEAUD, Biochim. Biophys. Acta, 129 (1966) 563 . 2o J. A. KINSEY, Genetics, 56 (1967) 57 °. 21 B. G. DEBUSK AND A. G. DEBUSK, Biochim. Biophys. Acta, lO 4 (1965) 139. 22 C. K. WOODWARD, C. P. READ AND V. W. WOODWARD, Genetics, 56 (1967) 598.
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