Quantitative estimation of channeling from early glycolytic intermediates to CO 2 in intact Escherichia coli Georgia Shearer, Jennifer C. Lee, Jia-an Koo and Daniel H. Kohl Department of Biology, Washington University, St. Louis, MO, USA The idea that intermediates in many metabolic path- ways are ‘channeled’ from one pathway enzyme to the next is widely [1,2], but not universally, accepted. One reason for the controversy is that ‘many of the enzyme complexes are dissociated during isolation owing to dilution effects’ [3]. Srere, in his authoritative 1987 review [4], critically examined the evidence to that date. For more recent reviews, see [1,5]. Contrarians, such as Gutfreund and Chock [6], interpret their kinetic data, compiled from experiments with pure enzymes of the glycolytic pathway in dilute solution, to be compatible with a diffusion model without need to invoke channeling. Atkinson [7] was influential in preparing the ground for the idea of channeling. He pointed out that there is not enough water in the cell to support uniform con- centrations of all pathway intermediates at K M , the approximate concentration traditionally assumed to be necessary to permit pathways to function optimally. Along with other considerations, this led Srere to postulate the existence of ‘metabolons’, transient asso- ciations of pathway enzymes in addition to stable complexes (e.g. cytochrome complexes of the electron transport chain or the covalent linkage of tryptophan synthase subunits). In a metabolon, the presumption is that the proximity of sequential enzymes would cause Keywords glycolysis; metabolic channelling; metabolon; ratio of channeled flux to total flux Correspondence D. H. Kohl, Department Biology, Washington University, St. Louis, MO 63130, USA Fax: +1 314 935 4432 Tel: +1 314 935 5387 E-mail: kohl@biology.wustl.edu Website: http://www.biology.wustl.edu/ (Received 30 December 2004, revised 31 March 2005, accepted 7 April 2005) doi:10.1111/j.1742-4658.2005.04712.x A pathway intermediate is said to be ‘channeled’ when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient inter- action of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole-cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fruc- tose-1,6-bisphosphate, in whole cells of Escherichia coli, was strongly chan- neled all the way to CO 2 , whereas fructose-6-phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO 2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose-6-phosphate was channeled in its transforma- tion to fructose-6-phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined. Abbreviations Fru1,6P 2 , fructose-1,6-bis phosphate; Fru6P, fructose-6-phosphate; F ch , fraction of total flux that is channelled; Glu6P, glucose-6- phosphate; OPPP, oxidative limb of the pentose phosphate pathway; PFK, phosphofructokinase; PGI, phosphoglucoisomerase; TCA, tricarboxyclic acid. 3260 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS the product of the first enzyme to have an advantage in competition for the active site of the second enzyme compared with the same molecular species within the aqueous cytoplasm of the cell. That is, intermediates just made in a pathway are not part of the same pool as are identical molecules within the cell. Intermediates produced within the pathway are ‘channeled’ to the next enzyme. Evidence for channeling Prior investigations of channeling may be divided into two categories: in vitro and in vivo. The former include experiments with cell extracts and purified enzymes. These are inherently unsatisfying. In particular, chan- neling is thought to be the result of protein–protein interactions in the crowded, organized environment of the cell. Although investigators sometimes attempt to simulate the crowding within a cell by, for example, adding polyethylene glycol, the complexity and organ- ization of the system is lost. Masters [8] emphasized the point by noting that ‘many of the conditions com- monly employed … are biologically abnormal and bear little relation to the conditions under which enzymes must act in vivo’. Among tools used for investigating channeling are the following. In vitro experiments (a) Chromatographic techniques. Beeckmans [9] reviewed in great detail the results of experiments util- izing chromatographic techniques. (b) Copurification of sequential pathway enzymes; for example, Law and Plaxton [10]. (c) Coprecipitation of sequential pathway enzymes; for example, Datta et al. [11]. (d) Isotope dilution; for example, Debnam et al. [12]. In vivo experiments (a) Genetic manipulation to disrupt enzyme complexes. Several reports from Srere’s laboratory [3] provide absolutely convincing proof that the interaction of malate dehydrogenase and citrate synthase are essential for the functioning of the tricarboxylic acid (TCA) cycle at its usual rate. (b) Electron microscopy. Micro- graphs showing colocalization of enzymes of the urea cycle across the mitochondrial membrane [13] are con- sistent with the proximity of sequential enzymes that is evoked as a necessary (although not sufficient) condi- tion for channeling. (c) NMR. Incubation of yeast in [4- 13 C]glutamate did not result in the randomization of the label in aspartate formed from it as would be expected if the symmetric intermediates, succinate and ⁄ or fumarate, dissociated from their enzymes and were free to rotate [14]. In addition 19 F NMR studies of citrate synthase 1 tagged with 5-fluorotryptophan showed motional restriction in vivo [15]. (d) The use of stable isotopes. Clegg and Jackson [16] compared the specific activity of 14 C-labelled glycolytic intermediates with that of pyruvate. These studies resulted in much less dilution of the radioactivity in pyruvate than would be expected if intermediates dissociated from their enzymes and entered the cytosol. (In order to facilitate uptake of intermediates these investigators permeabilized the cells with dextran sulfate.) The body of work cited above is strong evidence of channeling in a number of pathways; glycolysis, the TCA cycle, the oxidative limb of the pentose phos- phate pathway (OPPP), the urea cycle. However, these investigations do not provide data that can be used to calculate the fraction of the flux through a pathway that is channeled. The experimental designs used by Negrutskii and Deutscher [17] result in data that could be used to calculate the quantitative importance of channeling in a specific pathway. They used Chi- nese hamster ovary cells in studies of channeling of aminoacyl-tRNA for protein synthesis. The cells were electroporated to facilitate entry of 3 H-labeled amino- acyl-tRNA and 14 C-labeled free amino acids. The quan- tities of [ 14 C]aminoacyl-tRNA (made in the pathway from 14 C-labeled amino acids), [ 3 H]aminoacyl-tRNA, 14 C- and 3 H-labeled protein were measured. The 3 Hin protein was insignificant, showing that the aminoacyl- tRNA made from amino acids and tRNA did not mix with the introduced aminoacyl-tRNA; i.e. there was perfect channeling from free amino acids to protein. In this experiment, it was not necessary to calculate the percentage of the flux that was channeled, because the unchanneled flux was essentially zero. However, had channeling been less than 100%, the data collected would have enabled this calculation. In this study, we describe experiments aimed at cal- culating the fraction of the flux from early glycolytic intermediates to CO 2 in intact Escherichia coli. Cells incubated with [ 14 C]glucose made 14 C-labeled glyco- lytic intermediates. When the incubation mix also included a [ 12 C]intermediate, assuming that this inter- mediate entered the cell, there was a competition between the intermediate just produced in the pathway and the same molecular species in the aqueous cytosol. To the degree the latter was successful, the amount of 14 CO 2 evolved was decreased. Cells were also incuba- ted in [ 12 C]glucose plus [ 14 C]intermediate, [ 14 C]glucose alone and [ 14 C]intermediate alone. Combining these data allowed us to calculate the fraction of the total flux to CO 2 that was channeled. We used an E. coli G. Shearer et al. Quantitation of channeling in intact E. coli FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3261 mutant that was engineered to take up 6-carbon sugar phosphates constitutively. The results indicate a high degree of channeling of fructose 1,6-bisphosphate (Fru1,6P 2 )toCO 2 (99 ± 16% of the flux from Fru1,6P 2 channeled, P ¼ 0.005). This result requires that all of the downstream steps from Fru1,6P 2 to pyruvate and the further oxidation of pyruvate to CO 2 (presumably via mixed acid fermentation) be 100% channeled. We found no significant channeling of Glu6P or Fru6P to CO 2 . However, because Fru6P was not channeled, the signature of any channeling of Glu6P that might exist would be lost. Results The cells grew well on all carbon sources of interest to us, including the 6-carbon sugar phosphates, Glu6P, Fru6P, and Fru1,6P 2 . The doubling times for Glu6P and Fru6P were comparable with the doubling time of the parental strain growing on glucose (Table 1). Doubling times when grown on glycerol and Fru1,6P 2 were considerably longer. Rate of 14 CO 2 evolution and growth rate were poorly correlated. In particular, the rate of 14 CO 2 evo- lution from Fru1,6P 2 was twice that for Glu6P and Fru6P, whereas the doubling time of cells grown on the former was about twice as long as it was for growth on the latter two. The evolution of 14 CO 2 from [U- 14 C]glucose by the mutant strain began with no lag and was linear with time when cell density in the incubation mix was A 600 ¼ 0.8. Thus, at this density the pathways produ- cing 14 CO 2 were in steady state. Above that density, the rate of 14 CO 2 evolution increased with time (data not shown). The amounts of 14 CO 2 evolved in all incubations were highly correlated with the total amounts of 14 C taken up into the cell (three experiments, four treat- ments in each experiment, three or four replicates each treatment ¼ 40 data points; R 2 ¼ 0.97, data not shown). The total amount of 14 C entering the cell was taken to be the sum of the 14 CO 2 evolved plus the amount retained by the cell. Saturation of 14 CO 2 evolution rate by the mutant strain as a function of concentration depended on sub- strate. The rate of 14 CO 2 evolution was saturated when the concentration of glucose labeled with 14 C was 1 mm, although it was not saturated until the concentration reached 5 mm [ 14 C]Glu6P or 10 mm [ 14 C]Fru6P or [ 14 C]Fru1,6P 2 (data not shown). Experiments to determine pathway allocation of CO 2 evolution Using the data shown in Table 2 and calculating as described in Experimental procedures, about 96% of the CO 2 was evolved via oxidation of pyruvate fol- lowed by mixed acid fermentation and the TCA cycle. Therefore, we have ignored the CO 2 evolved by the OPPP when making calculations of the fractional importance of channeled flux in glycolysis. Experiments to investigate channeling of early glycolytic intermediates to CO 2 Figure 1 illustrates our experimental paradigm. In this example, the cells are incubated in [ 14 C]glucose and [ 12 C]-Fru6P (the challenger). This creates a competi- tion for E3 (phosphofructokinase; PFK, EC 2.7.1.10). The measure of channeling is the degree to which the Fru6P just made in the pathway is disproportionately successful in being the substrate for PFK. Invoking the usually proposed mechanism for channeling, the pref- erence for the intermediate just made in the pathway is a consequence of the interaction of PFK and the prior enzyme in the pathway, phosphoglucoisomerase (PGI, EC 5.3.1.9). If all Fru6P molecules just made in the pathway dissociate from PGI and equilibrate with the pool of Fru6P in the aqueous cytoplasm, then there would be no channeling. When there is no channeling, the result of the competition for binding to PFK will be proportional to the number of [ 14 C]Fru6P mole- cules made in the pathway and the [ 12 C]Fru6P in the aqueous cytoplasm. In principle, the challenger can be any glycolytic intermediate or any compound that, on entry into the cell, is converted to a glycolytic inter- mediate (e.g. mannose to mannose-6-phosphate to Fru6P). In addition, results from the inverse experi- ment ([ 12 C]glucose vs. [ 14 C]challenger) are necessary for calculating the fraction of the total flux that is channeled, as discussed later. In this regard, note that, in addition to the putative interaction of two or more Table 1. Growth of E. coli and 14 CO 2 evolution during incubation with [U- 14 C]carbon source, each at 1 mM. C source Strain Doubling time (h) 14 CO 2 evolution during 30 min incubation (37 °C) (nmolÆmin )1 ) Glucose RK 9118 parent 2.62 Glucose RK9117 mutant 2.23 ± 0.32 0.283 ± 0.014 Glycerol RK9117 mutant 3.61 ± 0.42 Glu6P RK9117 mutant 1.77 0.053 ± 0.004 Fru6P RK9117 mutant 2.17 0.042 ± 0.001 Fru1,6P 2 RK9117 mutant 4.05 ± 0.72 0.092 ± 0.001 Quantitation of channeling in intact E. coli G. Shearer et al. 3262 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS enzymes, there are free enzymes whose activity would result in unchanneled flux even if every intermediate just made in the pathway remained within the putative complex. Finally, if channeling is being assessed by measuring the isotopic composition of a downstream compound (such as CO 2 ), the signature of channeling will be lost if any step between the reaction being con- sidered and the downstream intermediate is not chan- neled. Thus, an experiment with our design will result in an evaluation of the minimum channeling in any intervening step. Figure 2 shows the results of experiments in which cells were incubated with [U- 14 C]glucose alone (black bars) and with [U- 14 C]glucose plus unlabeled inter- mediate (white bars). Challenging the glycolytic product of [U- 14 C]glucose with [U- 12 C]Fru1,6P 2 had no effect on the quantity of 14 CO 2 evolved compared with incubation in [U- 14 C]glucose alone (8.8 ± 1.8 vs. 7.9 ± 1.2 nmols). Challenging the [ 14 C]Glu6P made in the pathway from [ 14 C]glucose with [ 12 C]Glu6P taken up by the cell from the incubation mix resulted in only a modest, but significant (P ¼ 0.044), decrease in the counts in CO 2 (8.2 ± 1.0 vs. 5.3 ± 0.6). By contrast, exogenous [ 12 C]Fru6P had a clear impact on the counts in CO 2 (21.2 ± 2.2 vs. 7.9 ± 0.8) originating from [ 14 C]glu- cose. This suggests that Fru1,6P 2 is strongly channeled all the way to CO 2 , that Glu6P is modestly channeled and that Fru6P is channeled to an even lesser degree. But two additional, essential pieces of information are required before even such a qualitative conclusion con- cerning the degree of channeling can be drawn. Also these data by themselves do not allow us to calculate the fraction of the flux that is channeled. The first additional requirement is to show that the exogenous intermediate from the incubation mix entered the cell. Clearly, if the exogenous intermediate did not enter the cell, it could not compete for the active site of the enzyme for which it is substrate. Data relevant to this are shown in Fig. 3. These data establish that all of the exogenous intermediates entered the cells (black bars) and evolved CO 2 , although with varying degrees of success. Cells grown in 2% glycerol evolved 14 CO 2 from Fru1,6P 2 at an even higher rate (32.4 ± 7.7 nmol per 30 min; Fig. 3, solid bar) than did the same cells from glucose (8.8 ± 1.8 nmol per 30 min; Fig. 2, solid bar). In the presence of glucose, cells grown in 2% glycerol evolved almost no CO 2 from Fru6P (data not shown). However when 25 lm Fru6P was added to the growth medium, CO 2 was evolved from Fru6P at a good rate (Fig. 3, solid bar). The implied increase in uptake of Fru6P was unexpected because the cells were engineered to be constitutive for the uptake of 6-carbon sugar phosphates. In addition to establishing the degree to which the exogoenous intermediates enter the cell, the data of Fig. 3 also show the consequence for 14 CO 2 evolution of coincubating [ 14 C]intermediates with [ 12 C]glucose. Whereas the [ 12 C]glucose tended to decrease 14 CO 2 when [ 12 C]glucose was coincubated with a [ 14 C]inter- mediate, the radioactivity in the CO 2 evolved was significant compared to that in the absence of [ 12 C]glucose; 42, 72, and 67% for [ 14 C]Glu6P, [ 14 C]Fru6P and [ 14 C]Fru1,6P 2 , respectively. This is not in conflict with the result that unlabeled Fru1,6P 2 did not dilute the radioactivity in 14 CO 2 when it was coincubated with [ 14 C]glucose (white bar vs. black bar, Fig. 2). Despite the fact that exogenous Fru1,6P 2 entered the cell, it was unsuccessful in competing for the catalytic site of aldolase (EC 4.1.2.13) with the Fru1,6P 2 just made in the pathway originating from [ 14 C]glucose (Fig. 2, members of the third pair are not significantly different). The data in Fig. 3 suggest that some PFK molecules are associated with aldolase and downstream enzymes in a metabolon, while, at the same time, other molecules of aldolase and down- stream glycolytic enzymes are free in the aqueous cytoplasm. These free enzymes are able to convert the exogenous [ 14 C]Fru1,6P 2 to 14 CO 2 in the presence of [ 12 C]glucose. The data in Figs 2 and 3, considered separately, are insufficient to determine the predicted result if Table 2. Fraction of the total flux to 14 CO 2 via the OPPP. Note that only one of the six carbon atoms in [1- 14 C]glucose and [6- 14 C]glucose are labeled, whereas the label in [U- 14 C]glucose is divided among all six atoms. Glc, glucose; % of total flux to 14 CO 2 via the OPPP ¼ 100 · 3.42 ⁄ 79.5 ¼ 4.3%. [1- 14 C]Glucose [6- 14 C]Glucose [1- 14 C]Glucose minus [6- 14 C]glucose (OPPP) [U- 14 C]Glucose [ 14 C]Glucose converted to 14 CO 2 (%) 2.32 0.95 1.37 5.3 Nano equivalents 14 C per nmols [ 14 C]glucose 1 1 1 6 nmol glucose initially present 250 250 250 250 Nano equivalents 14 C initially present 250 250 250 1500 nmol 14 CO 2 produced 5.80 2.38 3.42 79.5 G. Shearer et al. Quantitation of channeling in intact E. coli FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3263 channeling were zero. When the data from both figures are combined, we are able to calculate the expectation if the fraction channeled were zero (F ch ¼ 0). When F ch ¼ 0, the relative success of binding to the enzyme of intermediates just made in the pathway and those in the aqueous cytoplasm will be proportional to their relative numbers. A method for calculating the fraction of the flux that is channeled from each intermediate to CO 2 , using the data of Figs 2 and 3, is developed below. Discussion The first task is to calculate the relative amounts of intermediate made in the pathway vs. the same inter- mediate made from exogenous sources. In this regard, Fig. 2. Effect of unlabeled challenging compound (Glu6P, Fru6P or Fru1,6P 2 ) on the quantity of 14 CO 2 evolved by E. coli cells when incubated with [ 14 C]glucose. Black bars represent the quantity evolved when cells were incubated with [ 14 C]glucose alone. White bars represent the quantity evolved when cells were incubated with [ 14 C]glucose and unlabeled challenging intermediate (Glu6P, Fru6P, or Fru1,6P 2 ). For experiments with Fru6P, cells were grown with a trace of Fru6P (25 l M) in addition to 0.2% (v ⁄ v) glycerol as the carbon source. Fig. 3. Effect of unlabeled glucose on the quantity of 14 CO 2 evolved by E. coli cells when incubated with 14 C-labeled intermedi- ates (Glu6P, Fru6P or Fru1,6P 2 ). Black bars represent the quantity evolved when cells were incubated with 14 C-labeled intermediate alone; white bars represent the quantity evolved when cells were incubated with 14 C-labeled intermediate and unlabeled challenging glucose. For experiments with Fru6P, cells were grown with a trace of Fru6P (25 l M) in addition to 0.2% (v ⁄ v) glycerol as the car- bon source. Fig. 1. Glycolytic pathway in an E. coli cell coincubated with [ 14 C]glucose and a [ 12 C]intermediate (Fru6P in this example). [ 14 C]Glucose is converted to [ 14 C]Glu6P as it enters the cell via the phosphotransferase system (PTS) and thence to [ 14 C]Fru6P by E2 (phosphoglucoisomerase; PGI). In the absence of channeling, [ 14 C]Fru6P will equilibrate with [ 12 C]Fru6P from the exogenous source, competing with [ 14 C]Fru6P for the catalytic site of E3 (phosphofructokinase; PFK), thereby decreasing the amount of 14 CO 2 that would have been evolved had no [ 12 C]Fru6P had been present. To the degree that the 14 C and 12 C intermediates do not equilibrate, the amount of 14 CO 2 evolved will be decreased to a les- ser degree, unless downstream 14 Cand 12 C intermediates equili- brate. Quantitation of channeling in intact E. coli G. Shearer et al. 3264 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS note that the amount of intermediate present at any given time is proportional to the total uptake because the system is in steady state as indicated by the con- stant rate of the evolution of 14 CO 2 from the earliest time point (see Results). Also recall from the Results that the CO 2 evolved from [ 14 C]glucose was strictly proportional to the total uptake of glucose. Thus, we can use CO 2 evolved as a reliable proxy for the total uptake. To facilitate the following discussion, let A ¼ the 14 CO 2 evolved when cells were incubated with [ 14 C]glu- cose alone; B ¼ the 14 CO 2 evolved when cells were incubated with [ 14 C]glucose plus [ 12 C]exogenous inter- mediate; C ¼ the 14 CO 2 evolved when cells were incu- bated with [ 14 C]exogenous intermediate alone; D ¼ the 14 CO 2 evolved when cells were incubated with [ 14 C]exogenous intermediate plus [ 12 C]glucose. The total amount of intermediate is the amount, for example, of Fru6P that has just been made in the pathway plus the exogenous Fru6P that entered the cell. The amount of [ 14 C]Fru6P converted to 14 CO 2 from [ 14 C]glucose as the result of coincubation of [ 14 C]glucose and [ 12 C]intermediate is proportional to ‘B’ as defined above. Likewise the amount of [ 14 C]Fru6P converted to 14 CO 2 from exogenous [ 14 C]Fru6P when coincubated with [ 14 C]Fru6P is pro- portional to ‘D’. Consequently in the absence of channeling (i.e. when the fraction of the flux that is channeled is zero, F ch ¼ 0), the expected dilution of 14 CO 2 originating from 14 C-labeled glucose when challenged by unlabeled intermediate, is equal to B ⁄ (B + D). That is, if B ⁄ A ¼ B ⁄ (B + D), then F ch ¼ 0. When the exogenous intermediate is completely unsuccessful in decreasing the radioactivity of the CO 2 evolved from [ 14 C]glucose, then B=A ¼ 1; and F ch ¼ 1 In principle, the exogenous intermediate (say Fru6P) could be channeled as the result, for instance, of its permease interacting with PFK. By analogy with the above if D ⁄ C ¼ D ⁄ (B + D), then F ch ¼ 0. Likewise, when the intermediate made from glucose does not dilute the radioactivity of CO 2 from a coincu- bation of [ 12 C]glucose and [ 14 C]intermediate, then D=C ¼ 1; and F ch ¼ 1 Values of F ch between 0 and 1 can be calculated by interpolation as illustrated in Fig. 4. An important caveat, if one is to make this interpolation, is that a substantial quantity of the exogenous intermediate must enter the cell during coincubation with glucose. Clearly, the exogenous intermediate cannot compete with the intermediate just made in the pathway for occupancy at the active site of the appropriate enzyme if it does not get into the cell. In our notation, if not much of the intermediate gets into the cell when glu- cose is present, then B » D and B ⁄ (B + D)  1. If the expected value when there is no channeling is close to 1 and the value for 100% channeling is also 1, then there is no ‘room’ to interpolate as required by the procedure outlined in Fig. 4. The data in Figs 2 and 3 permit calculation of B ⁄ (B + D), the ratio by which 14 CO 2 evolved from [ 14 C]glucose would be affected by coincubation with an unlabeled intermediate, if no channeling occurred, compared with B⁄ A, the observed effect (Fig. 5). When the exogenous intermediate was Fru1,6P 2 , there was a large, significant difference between the observed impact on 14 CO 2 evolution from [ 14 C]glucose when coincubated with [ 12 C]Fru1,6P 2 (B ⁄ A) vs. the values that would be expected if there were no channeling Fig. 4. Scheme for calculating the fraction of the total flux that is channeled (F ch ). Fig. 5. Expected fractional effect on 14 CO 2 evolved of coincubating E. coli cells with [ 14 C]glucose and unlabeled intermediate in the absence of channeling [B ⁄ (B + D)] compared to the observed effect (B ⁄ A). Black bars; the expected effect ¼ B ⁄ (B + D). White bars; the observed effect ¼ B ⁄ A. G. Shearer et al. Quantitation of channeling in intact E. coli FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3265 [B ⁄ (B + D)] (Fig. 5). This result is consistent with the Fru1,6P 2 just made in the pathway being channeled. By contrast, for Glu6 P and Fru6P, Fig. 5 shows no significant difference between the observed effect (Fig. 5, Glu6P or Fru6P, white bar) and the value expected if there were no channeling (Fig. 5, Glu6P or Fru6P, black bar). This is a necessary but not suffi- cient indication of the absence of channeling. The data in Fig. 3 rule out the exogenous intermediate not entering the cell as an explanation. There is a second alternative explanation for the apparent absence of channeling of Glu6P. Glu6P just made in the pathway could be strongly channeled to Fru6P, but the signa- ture of that channeling would be lost because Fru6P was apparently not channeled. The data in Table 3 show the method for and the results of calculating the fraction of the total flux that is channeled. The most striking result is that essentially all of the flux from Fru1,6P 2 to CO 2 was channeled (Table 3; F ch ¼ 0.99 ± 0.16). Note that this required almost perfect channeling of each intermediate in the pathway to pyruvate and in the reactions that oxidize pyruvate to CO 2 . This is a surprising result. When the paradigm of Fig. 4 is used to calculate F ch for Glu6P or Fru6P just made in the pathway, the results are F ch ¼ 0.10 ± 0.26 and 0.04 ± 0.01, respectively. That is, the data are consistent with no channeling. However, as noted above, Glu6P could be strongly channeled to Fru6P but the signature would be lost because Fru6P is not channeled. It would be surprising if Glu6P were very strongly channeled in glycolysis because it has other metabolic fates. In addi- tion, Clegg and Jackson’s data [13] are consistent with Glu6P, if it is channeled at all, being channeled to a lesser degree than were the other intermediates tested. It is theoretically possible that the appropriate enzyme might ‘prefer’ the exogenous substrate (after it enters the cell) to the intermediate just made in the pathway. In the context of the usual model, this would require interaction between the permease responsible for the import of the exogenous intermediate and the pathway enzyme responsible for its entry into glycoly- sis. However, the data [D ⁄ (B + D) vs. D⁄ C] provide no evidence for channeling from any exogenous Glu6P or Fru1,6P 2 to CO 2 . Although there was a significant difference between D ⁄ (B + D) vs. D ⁄ C for Fru6P, the fraction channeled was small (0.16 ± 0.01). The evidence presented here for channeling of Fru1,6P 2 to CO 2 is consistent with the existence of a glycolytic complex that holds together long enough for the Fru1,6P 2 that binds to it to be converted through Table 3. Calculation of the fraction of the total flux that is channeled (F ch ). Values are means ± SE. Challenging intermediate Glu6P Fru6P Fru1,6P 2 No. experiments 6 2 5 Carbon source in growth medium 22 m M glycerol 22 mM glycerol +25 l M Fru6P 22 m M glycerol A (nmol 14 CO 2 evolved from incubation with [ 14 C]glucose alone) 8.2 ± 1.0 21.2 ± 2.2 8.8 ± 1.8 B (nmol 14 CO 2 evolved from incubation with [ 14 C]glucose plus unlabeled intermediate) 5.3 ± 0.6 7.9 ± 0.8 7.9 ± 1.2 C (nmol 14 CO 2 evolved from incubation with [ 14 C]intermediate alone) 6.4 ± 1.7 22.0 ± 1.8 32.4 ± 7.7 D (nmol 14 CO 2 evolved from incubation with [14 C]intermediate plus unlabeled glucose) 2.7 ± 1.0 15.8 ± 2.6 21.6 ± 5.9 Evaluation of channeling of intermediate just made in the pathway B ⁄ (B + D) (expected effect of unlabeled challenger on 14 CO 2 evolution from [ 14 C]glucose if no channelling 0.695 ± 0.090 0.333 ± 0.004 0.311 ± 0.050 B ⁄ A (observed effect) 0.707 ± 0.119 0.358 ± 0.001 0.961 ± 0.104 F ch 0.103 ± 0.256 (NS) 0.036 ± 0.012 (NS) 0.994 ± 0.158 (P ¼ 0.005) Evaluation of channeling of exogenous intermediate D ⁄ (B + D) (expected effect of unlabeled challenger on 14 CO 2 evolution from 14 C-labeled intermediate if no channelling 0.305 ± 0.090 0.667 ± 0.008 0.689 ± 0.053 D ⁄ C (observed effect 0.386 ± 0.051 0.719 ± 0.005 0.637 ± 0.047 F ch 0.065 ± 0.089 (NS) 0.155 ± 0.006 (P ¼ 0.017) 0.229 ± 0.145 NS Quantitation of channeling in intact E. coli G. Shearer et al. 3266 FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS the subsequent steps of glycolysis and on to CO 2 . Such a putative complex has elements in common with the complex proposed by Mowbray and Moses [18] on the basis of observations using E. coli extracts. Observa- tions by Clegg and Jackson [16] support the conclusion that channeling of several glycolytic intermediates in pemeabilized mouse fibroblasts occurred, but that this channeling was somewhat leaky. However, in contrast to the results reported here for E. coli, Fru1,6P 2 was not strongly channeled in mouse L-929 cells. Our results show that, under certain conditions, E. coli cells channel an intermediate (Fru1,6P 2 ) from early in the glycolytic pathway all the way to CO 2 . Whe- ther channeling over such a large number of intermedi- ates is sensitive to growth conditions or other details of the experimental protocol remains to be determined. Experimental procedures Materials [U- 14 C]-, [1- 14 C]- and [6- 14 C]glucose, [U- 14 C]Glu6P, [U- 14 C] Fru6P, and [U- 14 C]Fru1,6P 2 , as well as all other biochemi- cals, were obtained from Sigma (St. Louis, MO). Our colla- borator, Robert Kadner (University of Virginia), made an E. coli mutant (RK9117) that was engineered to take up 4-, 5- and 6-carbon sugar phosphates constitutively. Table 4 gives the genotype of RK9117 and the parent from which it was made (RK9118). Bacterial growth Cells were grown at 37° in a rotary shaker in a defined medium [10.5 g K 2 HPO 4 , 4.5 g KH 2 PO 4 , 1 g (NH 4 ) 2 SO 4 , 0.5 g trisodium citrate, 10 mm MgSO 4 ,1mm CaCl 2 ,5mg thiamine and 0.2% (v ⁄ v) glycerol in 1 L] to an absorbance at 600 nm of 0.8. In cultures to be used for channeling experiments with Fru6P, the growth medium was supple- mented with 25 lm Fru6P. This substantially increased the amount of Fru6P taken up by the cells in the presence of glucose. Cells were harvested by centrifugation (10 000 g, 10 min, 8 °C) and washed with growth medium free of carbon source and washed pellets were stored at )80° until needed. Incubation conditions Just before each experiment, cells were resuspended in a vol- ume of growth medium (lacking any carbon source or Mg 2+ or Ca 2+ ) such that the absorbance was  10 at 600 nm. About 20 l L of the concentrated cells were added to 230 lL of incubation medium in order to incubate cells at an absorb- ance at 600 nm of 0.8. Each incubation mixture contained appropriate carbon sources, one labeled with 14 C and, when required by the experimental design, a second unlabeled car- bon source. Incubations were carried out in 25 mL vials that were sealed with a rubber septum fitted with a straight pin. A small strip of filter paper was placed on the pin and wetted with 10 lL 10% (w ⁄ v) NaOH. Incubations were carried out for 30 min at 37 °C in a rotary shaker water bath. Incuba- Table 4. Genotypes of E. coli strains used in this report. Strain Genotype RK9117 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA + B + C91::4 (Con)T + Rk9118 D(argF-lac)U169 araD139 thi gyrA219 relA rpsL150 non polA1 D(ilvBN-uhpABCT ¢)2095 zig621::Tn10 uhpA + B + C91::8 (Neg)T + Table 5. Effect of NaOH and filter paper on CPM and DPM of [U- 14 C]glucose. Each vial contained 9000 DPM [U- 14 C]glucose in 100 lLH 2 O, plus the indicated additions. The greatest difference (9302 vs. 9059) is not statistically significant (P ¼ 0.12). Vial Filter paper NaOH 10 lL Filter paper plus NaOH CPM DPM Mean CPM ± SE Mean DPM ± SE 1 No No No 8350 8911 8488 ± 74 9059 ± 79 2 No No No 8603 9182 3 No No No 8511 9084 4 Yes No No 8542 9116 8554 ± 23 9135 ± 22 5 Yes No No 8521 9110 6 Yes No No 8599 9178 7 No Yes No 8609 9196 8611 ± 21 9198 ± 27 8 No Yes No 8648 9246 9 No Yes No 8575 9152 10 No No Yes 8648 9232 8719 ± 88 9302 ± 94 11 No No Yes 8616 9186 12 No No Yes 8894 9488 G. Shearer et al. Quantitation of channeling in intact E. coli FEBS Journal 272 (2005) 3260–3269 ª 2005 FEBS 3267 tions were terminated by adding 100 lL 70% (v ⁄ v) HClO 4 . The outgassed CO 2 was captured on the base impregnated fil- ter paper. After incubation, the filter paper was removed and eluted with 100 lL water. Three milliliters of scintillation cocktail (Ecolite, ICN Biomedicals, Irvine, CA) were added. Aliquots of the incubation mixture were also counted in order to calculate percent conversion of the added 14 C- source. The rate of CO 2 production was calculated as nmolsÆmin )1 ¼ nmol equivalents of 14 C source · percentage conversion ⁄ (100 · min). The term ‘nmol equivalents of 14 C-source’ is included in order to take into account the fact that six molecules of CO 2 were produced from one molecule of glucose, Glu6P, Fru6P or Fru1,6P 2 via glycolysis followed by mixed acid fermentation. By contrast, only one molecule of CO 2 was produced via the OPPP from one molecule of glucose or Glu6P. Radioactive counting Radioactivity was measured by a Wallac (Model 1410) liquid scintillation counter (PerkinElmer, Wellesley, MA, USA) equipped with quench correction. Because 10 lLof 10% NaOH was present in samples used to measure CO 2 production, we did a control experiment to assess any possible impact of quenching by NaOH. Ten microliters of NaOH (10%) had no significant effect on either the counts per minute (CPM) or the disintegrations per minute (DPM) recorded by the counter (Table 5). Estimation of the relative contribution of the OPPP to the total CO 2 evolved Because we were investigating channeling from early glyco- lytic intermediates to pyruvate and on to CO 2 , any CO 2 produced via the oxidative limb of the OPPP must be cor- rected for or shown to be small enough to neglect. Catabol- ism of glucose via the OPPP results in CO 2 being evolved from the C-1 position of glucose. When glucose is catabo- lized completely to CO 2 in either the TCA cycle or by a branch of mixed acid fermentation, then both the C-1 and the C-6 positions of glucose give rise to CO 2 . Thus the amount of CO 2 produced in the OPPP is CO 2 from [1- 14 C]glucose minus CO 2 from [6- 14 C]glucose. The total amount of CO 2 produced by the OPPP plus that produced by the oxidation of pyruvate was calculated from incuba- tions with [U- 14 C]glucose. The ratio of the above two values is the fraction of CO 2 evolved by the OPPP. Design of experiments to evaluate channelling Incubations were carried out at 37 °C for 30 min as des- cribed above. There were four treatments, each replicated four times, in each experiment: (a) [U- 14 C]glucose (1 mm) alone, (b) [U- 14 C]glucose (1 mm) plus 12 C challenging intermediate (5 mm Glu6P or 5 mm Fru6P or 10 mm Fru1,6P¼), (c) [U- 14 C]intermediate (5 mm Glu6P or 5 mm Fru6P or 10 mm Fru1,6P 2 ) alone, and (d) [U- 14 C]interme- diate (5 mm Glu6P or 5 mm Fru6P or 10 mm Fru1,6P 2 ) plus [ 12 C]glucose (1 mm). Acknowledgements This work was supported by an SGER NSF grant: NSD Grant # MCB-02004900. References 1 Agius L & Sherratt H, eds. (1997) Channelling in Inter- mediary Metabolism. Portland Press, London. 2 Ovadi J & Srere P (2000) Macromolecular compart- mentation and channelling. Int Rev Cytol 192, 255–280. 3 Velot C & Srere P (2000) Reversible transdominant inhibition of a metabolic pathway. 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He pointed out that there is not enough water in