Uptake of the Glucose Analogue 2-Deoxyglucose by

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JOURNAL OF BACTERIOLOGY, Nov. 1975, p. 843-848 Copyright C) 1975 American Society for Microbiology Vol. 124, No. 2 Printed in U.S.A. Uptake of the Glucose Analogue 2-Deoxyglucose by Germinating Mitospores of Allomyces macrogynus DANIEL D.

1 JOURNAL OF BACTERIOLOGY, Nov. 1975, p Copyright C) 1975 American Society for Microbiology Vol. 124, No. 2 Printed in U.S.A. Uptake of the Glucose Analogue 2-Deoxyglucose by Germinating Mitospores of Allomyces macrogynus DANIEL D. BURKE Department of Microbiology, University of Illinois, Urbana, Illinois Received for publication 18 August 1975 Mitospores or cysts of Allomyces macrogynus do not take up the glucose analogue 2-deoxyglucose. Uptake of 2-deoxyglucose by germlings begins at 25 min into germination, the start of the rhizoid stage, and increases in rate by approximately 50-fold until 100 min into germination. The rate remains constant from 100 to 200 min, at which time germination is completed and hyphal formation begins. The presence of glucose in the germination medium blocks the uptake of 2-deoxyglucose. Of the other sugars tested, only galactose had any effect on 2-deoxyglucose uptake. Actinomycin D treatment during germination in a glucose-containing medium prevented the appearance of the uptake system, but actinomycin D was not effective after the transfer to a glucose-free medium. Cycloheximide treatment prevented the appearance of the uptake system if it was added at the time of the transfer to the glucose-free medium; it inhibited uptake only partially if the germlings were starved of glucose before its addition. It appears, therefore, that both ribonucleic acid synthesis during germination and protein synthesis after the removal of glucose are required for the uptake of 2-deoxyglucose. The protein synthesis required during the germination of spores of a number of fungi appears to be programmed by stable messenger ribonucleic acid (mrna) present in the spores (1, 3, 4, 6, 16). These include Neurospora (1), Botryodiplodia (16), Blastocladiella (6), and Allomyces (3). Development beyond germination then requires the synthesis of new mrna. Acrylamide gel analysis has shown that a large number of different peptides is synthesized during germination (2, 15; unpublished data), but there is no evidence as to what enzymatic or structural functions are the result of the translation of either the stable mrna in the spores or the mrna transcribed after germination. Mitospores of the phycomycete Allomyces can germinate in the absence of glucose. Glucose, however, is then required as a carbon source to support growth beyond germination (9). We anticipated that the ability to take up glucose might be a cellular function that appears at some time during germination. If this were the case, the uptake system could be a product of either stable mrna of the spore or the mrna synthesized after germination begins. This paper reports on the uptake of a glucose analogue, 2-deoxyglucose (2-DG), during the germination ofallomyces mitospores. We have determined the rate at which the glucose analogue is taken up during germination. We have 843 also examined the requirements for either RNA or protein synthesis in relation to the ability of germlings to accumulate the analogue and in relation to the rate of accumulation of the analogue. MATERIALS AND METHODS Culture conditions. Mitospores ofallomyces macrogynus strain Burma were obtained as previously described (3). To achieve synchronous germination, the mitospores were washed once by pelleting at 200 x g, suspended in 1 ml of cold, dilute salt solution (8), and chilled in an ice bucket for 30 min. The chilled mitospores were then inoculated at a final concentration of 5 x 104 spores/ml into prewarmed (32 C) Machlis B medium (8) containing 500 pg of glutamic acid (MBG) per ml and incubated at 32 C and 125 rpm in a rotary shaker-water bath. Uptake experiments. Germinating spores (germlings) were removed from the growth medium at various times during germination, filtered rapidly on Whatman GSA fiber glass filters, and washed twice with prewarmed MBG minus glucose. The germlings were then resuspended in prewarmed MBG minus glucose. In experiments using actinomycin D (Act D), Act D at a concentration of 10 ;Lg/ml was also present during the filtration and washing step. Cycloheximide, when used, was present at a concentration of 1.0,ug/ml. Uptake of 2-DG was determined by the addition of 3H-labeled 2-DG (0.1 mm) to the germlings at the times indicated for the specific experiment. Samples were removed from the culture at specified times.

2 844 BURKE The samples were filtered on Whatman GSA filters and washed with ice-cold phosphate-buffered saline (140 mm NaCl, 5.0 mm KCl, 0.8 mm MgSO4, 1.8 mm CaCl2, 1 mm NaH2PO4, and 4 mm Na2HCO3 [ph 7.0]). The filters were then air dried in scintillation vials. To each vial, 1.0 ml of 1 M NaOH was added and then the vial was incubated at 65 C for 30 min to solubilize the cell protein and 2-DG. A one-half volume of the NaOH was removed, and the amount of protein present was determined by the Folin reaction (7). To measure the amount of 2-DG uptake, the remainder of the NaOH was counted in Triton-toluene-acetic acid scintillation fluid, and the uptake was normalized to counts per minute per microgram of protein. Zero time determinations were performed routinely to correct for the nonspecific background accumulations of 2-DG. Chemicals. 3H-labeled 2-DG was obtained from New England Nuclear Corp., Boston, Mass. Unlabeled 2-DG was obtained from Sigma Chemical Co., St. Louis, Mo. Act D and cycloheximide were obtained from Calbiochem, Los Angeles, Calif. RESULTS The mitospores of A. macrogynus pass through several distinct morphogenic stages during germination (spore, cyst, and rhizoid stage). By using the cold-shock treatment detailed above, the germination process can be synchronized so that more than 90% of the population is at the same morphogenic stage at selected times (Fig. 1). As a measure of the rate at which germinating spores (all prehyphal stages) take up glucose, the rate of accumulation of the glucose analogue 2-DG was determined. Rather than glucose, 2-DG was used since this analogue has been shown to be transported by the glucose transport system and is phosphorylated but not further metabolized to any significant extent (17). Thus, changes in the rate of substrate metabolism will not affect the measurement of accumulation. The rate of accumulation of 2-DG, as meas- ured by the addition of 3H-labeled 2-DG (0.1 mm) to the glucose-containing medium, was very low. The rates varied from 0.05 to 0.30 nmol of 2-DG/,g of protein per min depending upon the time during germination of the addition of the 3H-labeled 2-DG. To increase the amount of 2-DG accumulated, the experiment was modified so that germlings were transferred to glucose-free medium at specified times after germination in glucose-containing medium (Fig. 2). The rate of 2-DG accumulation in all experiments was then measured over a 10- min period beginning at the time of addition of the 3H-labeled 2-DG. There was a 2-min lag period before uptake began in all cases. The rate was determined as the average rate over the 10-min period beginning at the 3H-labeled 2-DG addition and including the 2-min lag period. There was a much higher rate of 2-DG accumulation in the glucose-free medium than in the glucose-containing medium. The rate increased approximately 50-fold during the germination period. Spores and cysts at 10 min accumulate 2-DG at a low rate (<0.3 nmol of 2- DGIt,g of protein per min). At 25 min, when the population was about 50% cysts and 50% rhizoid, the rate of accumulation was six times that of the spores or 10-min cysts (probably due to the appearance of the rhizoid population), and the rate continued to increase until the 100-min germination, at which point the rate remained constant until the 200-min germination, 200 min being the time at which hyphal formation begins. The specificity of the effect of glucose on the 'I. 12 J. BACTERIOL. 0 FIG. 1. Synchronous germination of mitospores. Germination of mitospores was synchronized by cold shock as detailed in the text. Spores at a final concentration of 5 x 104/ml were innoculated into MBG medium and incubated at 32 C Age of Germlings FIG. 2. Rate of uptake of 2-DG by germlings. Germlings of the age specified were transferred to a glucose-free medium containing 3H-labeled 2-DG (0.10 mm, 0.5,uCi/ml), and their rate of uptake of2- DG was determined over a 10-min period.

3 VOL. 124, 1975 UPTAKE OF 2-DG BY ALLOMYCES MITOSPORES 845 uptake of 2-DG was measured by the transfer of 100-min germlings to the glucose-free medium. The rate of 2-DG uptake was then determined in the presence of added glucose or other sugars (Fig. 3). Uptake of 2-DG was sensitive to added glucose. A glucose concentration one-half that of the germination medium inhibits the accumulation of 2-DG by 95%, whereas even a glucose concentration 5% of that in the germination medium inhibits 2-DG accumulation by approximately 40%. Of the other sugars tested, neither fructose or lactose had any effect, whereas galactose had a small effect at a high concentration. The galactose effect may be due to glucose contamination of commercial galactose. Glucose appears to compete with 2-DG for the transport system. Experiments to be presented later indicate that glucose may also repress the synthesis of some component required for the uptake of 2-DG. Allomyces spores germinated in a glucosecontaining medium in the presence of 10 pg of Act D per ml for periods up to 200 min appeared to develop normally although RNA synthesis was inhibited by over 95%. If the transport system is a product of stable mrna present in the spores, one would predict that its appearance would not be inhibited by the presence of Act D during germination. Spores germinated in the presence of Act D, however, did not accumulate 2-DG to any extent (0.3 nmol of 2- DG/,ug of protein per min) when transferred to a glucose-free medium containing Act D. RNA synthesis, therefore, is required either during germination or after transfer to the glucose-free medium for the appearance of the uptake system. This uptake system itself does not appear to be necessary for germination to occur. A different program of Act D treatment was used to determine whether the required RNA synthesis takes place before or after the transfer to the glucose-free medium. The 100-min germlings, the point at which the maximum uptake rate has been reached, were transferred to the glucose-free medium. Their rate of accumulation of 2-DG was measured immediately after the transfer and at points up to 30 min of glucose starvation. The rate of accumulation increases continually during the period of glucose starvation and doubles at 30 min of starvation (Table 1). This increase is a function of the length of glucose starvation and not the age of the germling (Fig. 3). Apparently, the accumulation system is being synthesized during this entire period. Measurement was then made of the rate of uptake of 2-DG in the presence of Act D after 0 and 30 min of glucose starvation and 30 min of starvation in the presence of Act D Concentration of Added Sugar (mm) FIG. 3. Effect of added sugars on the accumulation of2-dg. Germlings (100 min) were transferred to a glucose-free medium containing 3H-labeled 2-DG (0.10 mm, 0.5 pci/mi). At zero time after transfer, sugars at the specified concentration were added and the accumulation of2-dg was determined over a 10- min period. The concentration ofglucose in MBG is 28 x 10-2 M. Symbols: 0, Glucose; *, galactose; 0, fructose; and *, lactose. TABLE 1. Effect ofact D on 2-DG uptake of100-min germlings during glucose starvation Period in glucos free" medium Time of additionb of Act D Rae (a) 0 d (b) 30 d alength of time in the glucose-free medium before the addition of 2-DG. b Length of time in the glucose-free medium before the addition of Act D (10 g/ml). c Rate (nanomoles of 2-DG/microgram of protein per minute) determined as in the text for a 10-min period beginning at the time indicated in the first column. d No Act D added. (Table 1). Again, as in the case of the glucosecontaining medium, RNA synthesis is inhibited more than 95% in the presence of 10,ug of Act D per ml. In all cases the rate of 2-DG accumulation was essentially the same in the Act D-treated cultures as in the untreated controls. Since Act D treatment during germination blocks the appearance of the uptake system and since the rate of 2-DG accumulation increases in the 30-min glucose-starved, Act D- treated germlings to the same extent that it does in the untreated 30-min starved control, the required RNA synthesis must take place

4 846 BURKE during germination in the glucose-containing medium. RNA synthesis after the transfer to the glucose-free medium is not required for the appearance or for the continued synthesis of the transport system. To determine the point during germination at which the required RNA synthesis takes place, spores were treated with Act D for either the initial 50% or final 50% of their incubation in the glucose-containing medium. The rate of their accumulation of 2-DG was measured after the transfer to the glucosefree medium in the presence of Act D (Table 2). These germlings did accumulate 2-DG, but at a lower rate than untreated germlings of the same age. Their transport rate was similar to that of germlings whose age was the same as the length of the time the test population was free of Act D. Thus, the required RNA synthesis appears to be a continuous process throughout the first 100 min of germination. Since we have shown that this RNA is stable for at least 30 min (in the glucose-free medium), it appears that the increased rate of accumulation during germination is due to a buildup of the RNA required for the system during germination. To determine whether protein synthesis was required for the accumulation of 2-DG, 100-min germlings were transferred to the glucose-free medium in the presence of cycloheximide, and their rate of 2-DG accumulation was determined after 0 and 30 min of glucose starvation (Table 3). In both cases the accumulation of 2- DG was negligible when compared with that of the untreated controls. However, if cycloheximide was added after 30 min of glucose starva- TABLE 2. Effect of Act D treatment during germination of2-dg uptake Age of germling" Act D treatmentb Ratee 25 d _d d a Length of germination in the glucose-containing medium. bperiod of time in the glucose-containing medium during which germlings were treated with 10,ug of Act D per ml. c Nanomoles of 2-DG of protein per minute as detailed in the text. d No Act D treatment. J. BACTERIOL. tion, then the uptake rate was 38% that of the 30-min starved control culture (Table 3). These results indicate that protein synthesis is required for the appearance of the uptake system. After 30 min of starvation, some protein has been synthesized and the system is no longer completely inhibited by cycloheximide. Since the rate is only 38% of the control, this indicates that some turnover of this system occurs in the presence of cycloheximide. If the system were completely stable in cycloheximide, an accumulation rate between that found for the 20-min starvation and that of the 30-min starvation (-75%) would be expected. It seems likely that glucose actually represses the synthesis of the uptake system. If this were not the case, cycloheximide should not completely block the appearance of the system after the transfer from the glucose-containing medium to the glucosefree medium. Uptake after the addition of cycloheximide at 30 min of starvation demonstrates that the system would not have been degraded in the period required for the transfer to the glucose-free medium. DISCUSSION The results reported here demonstrate that during germination there is a dramatic increase in the capacity of Allomyces mitospores to take up 2-DG, a glucose analogue. This uptake requires the synthesis of both RNA and protein. The RNA synthesis occurs in the presence of glucose; however, the required protein synthesis is repressed in the presence of glucose. Thus, this system differs from sugar transport in bacteria in which glucose transport is generally constitutive (10) and the transport of other sugars is inducible (5). TABLE 3. Effect of cycloheximide on 2-DG uptake of 100-min germlings during glucose starvation Period in glucose- Time of additionb free, medium of cycloheximide Rate of uptakee (a) 0 d (b) 30 _d Length of time in the glucose-free medium before the addition of 2-DG. b Length of time in the glucose-free medium before the addition of cycloheximide. I Rate (nanomoles of 2-DG per microgram of protein) determined as in the text for a 10-min period beginning at the time indicated in the first column. d No cycloheximide.

VOL. 124, 1975 Over 95% of the 3H counts per minute extracted after 30 min of accumulation of 3Hlabeled 2-DG was present as 3H-labeled 2-DG or 3H-labeled 2-DGP (unpublished data).

5 VOL. 124, 1975 Over 95% of the 3H counts per minute extracted after 30 min of accumulation of 3Hlabeled 2-DG was present as 3H-labeled 2-DG or 3H-labeled 2-DGP (unpublished data). This indicates that the change in the rate of accumulation could be due to either a change in the transport rate or the rate at which 2-DG is phosphorylated. Our recent experiments indicate that the uptake of 3-0-methylglucose, a non-phosphorylatable glucose analogue (17), exhibits the same characteristics as the uptake of 2-DG. This suggests that we are measuring actual changes in the transport rate and not the phosphorylation of 2-DG. This point, however, will require more detailed study. Experiments measuring the rate at which cell-free extracts prepared at various times during germination will phosphorylate 2-DG are being planned. The absence of a transport system in spores germinated in Act D, coupled with its appearance in untreated germlings whose transport was measured in a glucose-free medium containing Act D, is evidence for an RNA requirement for transport. Since this RNA is transcribed during germination, the transport system is not a product of the stable mrna contained in the mitospore. The inhibition of a function by Act D while germination occurs is indirect evidence for the presence of stable mrna. Two lines of evidence indicate that this mrna does not turn over rapidly. Act D treatment during germination indicated that the mrna made before the treatment is available at least 50 min later (Table 1) and that the mrna made before transfer was not lost after 30 min of incubation with Act D in the glucosefree medium (Table 2). The synthesis of protein for this system is repressed by the presence of glucose, as no transport capacity was in evidence during germination in the glucose-free medium. It apparently has a shorter turnover time than the RNA since some protein was lost during starvation, as measured by the transport rate in the presence of cycloheximide after 30 min of glucose starvation. This is similar to what has been found for tryptophan uptake in Neurospora (18), in which there is continual turnover of the uptake system, and may reflect a more general regulatory mechanism in eucaryotes. A glucose transport system with some similar characteristics has been reported in N. crassa (11, 13). The system in that case is also derepressed by the removal of glucose. In contrast to germinating Allomyces mitospores in which the required RNA synthesis takes place before the transfer to the glucose-free medium, RNA synthesis is apparently required after the UPTAKE OF 2-DG BY ALLOMYCES MITOSPORES 847 transfer to glucose-free medium for the production of the transport system in Neurospora (14). Since the Neurospora experiments were done with mycelium, the possibility exists that at that growth stage there is no stable mrna for transport. Experiments are in progress in our laboratory to investigate this point with Allomyces mycelia. In Neurospora a second transport system has been found that is not repressed by high levels of glucose (12, 13). Although we have no evidence for more than one transport system in Allomyces, it is possible that more than one transport system exists. ACKNOWLEDGMENTS I thank Cathy Ditto for her excellent technical help and M. Weber for his many helpful suggestions. This work was supported by Public Health Service research grant GM from the National Institute of General Medical Sciences. LITERATURE CITED 1. Bhagwat, A. S., and P. R. Mahadevan Conserved mrna from the coridia of Neurospora crassa. Mol. Gen. Genet. 109: Brambl, R. M., and J. L. VanEtten Protein synthesis during fungal spore germination. V. Evidence that the ungerminated conidiospores of Botryodiplodia theobramae contain messenger ribonucleic acid. Arch. Biochem. Biophys. 137: Burke, D. D., T. W. Seale, and B. J. McCarthy Protein and ribonucleic acid synthesis during the diploid life cycle ofallomyces arbuscula. J. Bacteriol. 100: Hollomon, D. W Biochemistry of germination in Pernospora tabacina (Adam) conidia: evidence for the existence of stable messenger RNA. J. Gen. Microbiol. 55: Kepes, A The place of permeases in cellular organization, p In J. F. Hoffman (ed.), The cellular functions of membrane transport. Prentice Hall, Englewood Cliffs, N.J. 6. Lovett, J. S Reactivation of ribonucleic acid and protein synthesis during germination of Blastocladiella zoospores and the role of the ribosomal nuclear cap. J. Bacteriol. 96: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: Machlis, L Growth and nutrition of water molds in the subgenus Euallomyces. II. Optimal composition of the minimal medium. Am. J. Bot. 40: Machlis, L Growth and nutrition of water molds in the subgenus Euallomyces. II. Carbon sources. Am. J. Bot. 40: Roseman, S The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol. 54:138s-180s. 11. Scarborough, G. A Sugar transport in Neurospora crassa. J. Biol. Chem. 245: Scarborough, G. A Sugar transport in Neurospora crassa. II. A second glucose transport system. J. Biol. Chem. 245: Schneider, R. P., and W. R. Wiley Kinetic characteristics of the two glucose transport systems in Neurospora crassa. J. Bacteriol. 106: Schneider, R. P., and W. R. Wiley Regulation of sugar transport in Neurospora crassa. J. Bacteriol.

848 BURKE 106:487-492. 15. Silverman, P. M., M. M. Huh, and L. Sun. 1974. Protein synthesis during zoospore germination in the aquatic phycomycete Blastocladiella emersonii. Dev. Biol. 40:59-70. 16.

6 848 BURKE 106: Silverman, P. M., M. M. Huh, and L. Sun Protein synthesis during zoospore germination in the aquatic phycomycete Blastocladiella emersonii. Dev. Biol. 40: Van Etten, J. L., H. R. Roker, and E. Davies Protein synthesis during fungal spore germination: differential protein synthesis during germination of J. BACTERIOL. Botryodiplodia theobromae spores. J. Bacteriol. 112: Weber, M. J Hexose transport in normal and in Rous Sarcoma virus-transformed cells. J. Biol. Chem. 248: Wiley, W. R., and W. H. Matchett Tryptophan transport in Neurospora crassa. II. Metabolic control. J. Bacteriol. 95:

TRANSPORT OF AMINO ACIDS IN INTACT 3T3 AND SV3T3 CELLS. Binding Activity for Leucine in Membrane Preparations of Ehrlich Ascites Tumor Cells

Journal of Supramolecular Structure 4:441 (401)-447 (407) (1976) TRANSPORT OF AMINO ACIDS IN INTACT 3T3 AND SV3T3 CELLS. Binding Activity for Leucine in Membrane Preparations of Ehrlich Ascites Tumor Cells