Reversible Transdominant Inhibition of a Metabolic Pathway

Abstract

The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an “assembly mutation,” i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15-amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context. The enzymes of the Krebs tricarboxylic acid cycle in mitochondria are proposed to form a supramolecular complex, in which there is channeling of intermediates between enzyme active sites. While interactions have been demonstrated in vitro between most of the sequential tricarboxylic acid cycle enzymes, no direct evidence has been obtained in vivo for such interactions. We have isolated, in the Saccharomyces cerevisiae gene encoding the tricarboxylic acid cycle enzyme citrate synthase Cit1p, an “assembly mutation,” i.e. a mutation that causes a tricarboxylic acid cycle deficiency without affecting the citrate synthase activity. We have shown that a 15-amino acid peptide from wild type Cit1p encompassing the mutation point inhibits the tricarboxylic acid cycle in a dominant manner, and that the inhibitory phenotype is overcome by a co-overexpression of Mdh1p, the mitochondrial malate dehydrogenase. These data provide the first direct in vivo evidence of interaction between two sequential tricarboxylic acid cycle enzymes, Cit1p and Mdh1p, and indicate that the characterization of assembly mutations by the reversible transdominant inhibition method may be a powerful way to study multienzyme complexes in their physiological context. citrate synthase yeast mitochondrial aconitase yeast mitochondrial CS yeast peroxisomal CS green fluorescent protein malate dehydrogenase yeast mitochondrial MDH wild type open reading frame An important and unresolved general problem in cellular biochemistry concerns the metabolic organization in cells. Considering a metabolic pathway, a basic question is, “Are the enzymes and the metabolites randomly dispersed inside the cell, or is there a cellular organization such that the sequential enzymes of this pathway remain in a close proximity allowing especially the intermediary metabolites to be passed directly from one enzyme of the sequence to the next one?” Whereas no direct evidence has ever supported the view of the cell as a bag of uniformly dispersed enzymes, the general concept of an intracellular organization of enzymes and channeling of intermediates has in many respects received support from a large body of experimental data during the last 20 years (reviewed in Refs. 1.Srere P.A. Annu. Rev. Biochem. 1987; 56: 89-124Crossref PubMed Scopus (811) Google Scholar, 2.Ovádi J. Cell Architecture and Metabolic Channeling. R. G. Landes Co., Austin, TX1995Google Scholar, 3.Srere P.A. Sherry A.D. Malloy C.R. Sumegi B. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1997: 201-217Google Scholar). This type of organization is often referred to as “microcompartmentation” (4.Jones D.P. Microcompartmentation. CRC Press, Boca Raton, FL1988Google Scholar), and the term “metabolon” has been introduced (5.Srere P.A. Trends Biochem. Sci. 1985; 10: 109-110Abstract Full Text PDF Scopus (244) Google Scholar) to describe supramolecular complexes of sequential metabolic enzymes and structural components. Such “integrated” pathways may present important advantages for metabolic integration and regulation, such as protection of labile intermediates, prevention of intermediates from escaping into the bulk solution where they may be sequestered by other enzymes for use in different metabolic pathways, conservation of cellular solvent capacity, reduction of transition times, and enhancement of metabolic fluxes. Experimental data support the existence of metabolons for many metabolic pathways and especially for the Krebs tricarboxylic acid cycle. For instance, specific interactions have been demonstratedin vitro between six of the eight sequential tricarboxylic acid cycle enzymes including citrate synthase (CS)1 and malate dehydrogenase (MDH) (reviewed in Ref. 3.Srere P.A. Sherry A.D. Malloy C.R. Sumegi B. Agius L. Sherratt H.S.A. Channeling in Intermediary Metabolism. Portland Press Ltd., London1997: 201-217Google Scholar). In the last few years, evidence was presented concerning the channeling of oxaloaxetatein vitro between yeast mitochondrial CS (Cit1p) and MDH (Mdh1p) (6.Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar) as well as between the corresponding pig enzymes (7.Morgunov I. Srere P.A. J. Biol. Chem. 1998; 273: 29540-29544Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,8.Shatalin K. Lebreton S. Rault-Leonardon M. Vélot C. Srere P.A. Biochemistry. 1999; 38: 881-889Crossref PubMed Scopus (39) Google Scholar). However, no direct evidence has been obtained in vivo for the interactions between sequential Krebs tricarboxylic acid cycle enzymes, mainly because these interactions are weak and many of the resulting complexes dissociate during isolation due to dilution effects. A possible strategy to demonstrate the in situexistence and the physiological relevance of such complexes is to screen for and characterize mutations that affect specifically these interactions, i.e. mutations that affect the metabolic pathway without affecting the enzymatic activities. Such mutations, which we refer as to “assembly mutations,” have already been isolated in Saccharomyces cerevisiae. For instance, McCammon (9.McCammon M.T. Genetics. 1996; 144: 57-69Crossref PubMed Google Scholar), who screened for mutants unable to grow on acetate, isolated three mutants with defects in the ACO1 gene (encoding mitochondrial aconitase) that display a wild type level of aconitase activity. Here, we describe the serendipitous isolation of an assembly mutation in the CIT1 gene encoding Cit1p and its characterization using an adaptation and an extension of the transdominant genetic analysis initially described by Caponigro et al. (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar). Transdominant genetic experiments involve introduction and overexpression in wild type cells of small molecules and selection for those that inhibit a particular biological process. This is a new and promising approach to identify and characterize protein interactionsin vivo (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar, 11.Norman T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar, 12.Geyer C.R. Colman-Lerner A. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8567-8572Crossref PubMed Scopus (99) Google Scholar). These authors used expression libraries to inhibitory molecules and protein and a selection for those that inhibit the pathway in and protein that cell in the of and from that already to be in the pathway, and other peptide allowing of that may the the transdominant Caponigro et al. (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar) introduced the term by with of mutations in the level of the specific interactions in cells to a In a as the of a gene a dominant mutation I. 1987; PubMed Scopus Google Scholar), i.e. a gene encoding an protein that the of the wild type The of the transdominant inhibition analysis mainly in three the interactions are in their physiological the physiological relevance of the interaction is directly by the resulting from its and the and the characterization of the a direct to the of the interaction In this we use randomly a specific peptide on the of the assembly mutation in to that this mutation affect an of the identify for the Caponigro et al. (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar), et al. T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar), and et al. C.R. Colman-Lerner A. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8567-8572Crossref PubMed Scopus (99) Google Scholar) used the of Fields and S. PubMed Scopus Google Scholar). Here, the was by for that are with the to the transdominant inhibition to a wild type as a of a of the The provide the first direct in of interaction between two sequential Krebs tricarboxylic acid cycle enzymes and and that the reversible transdominant inhibition method may be a powerful to identify weak interactions in cerevisiae and used in this study from the to the was used in the transdominant inhibition The yeast as described by et al. in Genetics. Scholar) with or as the and and and of yeast and with acid or as a and was to with was for using the of et al. A. PubMed Scopus Google Scholar). The used for was and of cells to A. J. PubMed Google Scholar). cells on with of the used in this study are in I. of the in or The encoding the and have been from from The was used to the of green protein or to the used for and and used in this or Genetics. PubMed Google Genetics. PubMed Google Genetics. PubMed Google to the of K. J. Cell Biol. 1998; PubMed Scopus Google C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google its three last and to CIT1 of CIT1 C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google a between and CIT1 of CIT1 of and CIT1 C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google expression of the to of ACO1 that is in to the sequence encoding that is in to a encoding a acid of ACO1 its three last and to CIT1 of ACO1 of ACO1 study in a new The obtained from or enzymatic as by the and and to et al. J. A Scholar). using was used in in those of the that with of the on an in or of the used to of a or or of to was as described C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). on a and to and The was by in with a dilution of a of two in a of and in 20 with a dilution of a to in and in the was with the method for citrate malate and dehydrogenase as described by Srere et al. P.A. L. Chem. Google Scholar), and B. Scopus Google Scholar), and S. L. Scopus Google Scholar), and and L. J. PubMed Google Scholar), using the protein as described by Smith et al. P.K. Biochem. 1985; PubMed Scopus Google Scholar), with as the CIT1 and from S. cerevisiae mitochondrial and peroxisomal of CS in the Krebs tricarboxylic acid cycle and pathway, Cit1p has been shown to to the of the mitochondrial such G. Srere P.A. Biochem. PubMed Scopus Google Scholar). In to the of Cit1p for its to the mitochondrial we have different between Cit1p and was in the of this that the which to the serendipitous isolation of an assembly mutation in the CIT1 was This was from Genetics. PubMed Google Scholar), a the yeast gene as a and to a protein including the first of Cit1p to the of by the of Cit1p The of the of gene and of that including the last and its first and the of the the of mutations in the The resulting where the CIT1 the expression of a gene encoding a protein was The was using which is to the of C. C. Friend S.H. R. 1994; PubMed Scopus Google Scholar). The of the resulting the of These two as well as encoding Cit1p C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar) used to the yeast mutants which grow on as the G. M. L. Srere P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), a phenotype of a Krebs tricarboxylic acid cycle in S. cerevisiae. The resulting for their to grow on The obtained with and those obtained with the are shown The encoding Cit1p as well as that encoding the to the of the to However, the with the encoding was on the of these to grow on acetate, a in was for of as well as for wild type and as and in the the with a and a cell in with the Cit1p or and CS in these three as well as in wild type and shown in the the Cit1p protein mitochondrial CS the wild type This is a due to the that a is present in one cellular of CS in the the the protein mitochondrial CS the other this the for between these the protein a level of mitochondrial CS with that of the which on a of of in the indicate that a of of wild type mitochondrial CS level is to on For instance, we have C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar) that a a mitochondrial form of on with of wild type CS of CS in the the and in the wild type and CS are in of citrate of mitochondrial or of three and from cells in and in and from cells in and in CS are in of citrate of mitochondrial or of three and from cells in and in in a new These indicate that the mutation affecting the gene we have no between the level of mitochondrial CS and the of the corresponding cells to grow on In other we have isolated an form of Cit1p affect the metabolic in which is the Krebs tricarboxylic acid this mutation is an assembly The of the gene from the of three the CIT1 and these three are these in the of one in the protein in a of sequence of acid as in We this mutation The peptide shown in to is in and in pig where a solvent to between two in the this peptide a possible with other other point mutations in the resulting in two acid and these two are in that are in Cit1p, we that these mutations for the assembly mutation phenotype and on the characterization of In to characterize the mutation to this mutation affect an for the metabolic pathway in which Cit1p is we have used a strategy from the transdominant genetic analysis described by Caponigro et al. (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar). The of this strategy was to the of a peptide encompassing the mutation of to as a transdominant of the Krebs tricarboxylic acid i.e. to the of this in to inhibit the on of a wild type The peptide we have to is a 15-amino acid peptide corresponding to the wild type sequence shown in one on the and one on the be inside the cell as with an protein for instance, the protein of Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), from B. I. J. Brent R. 1996; PubMed Scopus Google Scholar) (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar, M.R. Caponigro G. Kamb A. 1998; PubMed Scopus Google Scholar), or a of T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar). In this we as a with In the sequence to a acid peptide because of by that introduced and of the sequence encoding and the in yeast we used the and the sequence of the ACO1 The which is to Biol. 10: PubMed Scopus Google Scholar) and is on a such as acetate, a sequence in by a sequence in that is a of in S. cerevisiae Roberts S.M. 10: PubMed Scopus Google Scholar). The use of a and was to overexpression of the gene encoding the the experimental to a transdominant inhibition The mitochondrial sequence of ACO1 was for and has been in the of other to be for mitochondrial of other as and peroxisomal C. A. and L. to the of the in the of was to the have been and to to and to a acid peptide by a from of these from Genetics. PubMed Google Scholar), a the yeast gene as a The wild type yeast was with or of the three with Genetics. PubMed Google Scholar), a with the yeast for was the of of to specific that for their to The expression and into mitochondria of by analysis of mitochondrial from the resulting The of mitochondrial was by in mitochondrial and the of a mitochondrial The that of dehydrogenase was in the mitochondria The for their to grow on no in was between the the and the However, a in on was in the the that this peptide as a transdominant of the Krebs tricarboxylic acid cycle and that mutation affect a that is for the Krebs tricarboxylic acid cycle. The the the and to the and This may be due to the that the peptide is with to the Krebs tricarboxylic acid cycle. In other this peptide may present with in this In to that the Krebs tricarboxylic acid cycle enzyme are by the overexpression of that the of the corresponding to is due to an of these and dehydrogenase in mitochondrial from the dehydrogenase in the was to demonstrate the of the shown in for one of the enzymes, no was of specific in mitochondrial from the of the peptide from Cit1p to inhibit the on of a wild type in the or of the overexpression of a specific type cells with the in without and and as in the to that selection was on for aconitase and dehydrogenase of the yeast are in of citrate MDH and or aconitase) of mitochondrial or protein of three and from cells in and in and from cells in and in for are in of citrate MDH and or aconitase) of mitochondrial or protein of three and from cells in and in for activity. in a new CS has been shown to in vitro with pig mitochondrial MDH Srere P.A. Biochem. PubMed Scopus Google Scholar) and with pig mitochondrial aconitase Brent A.P. Srere P.A. The of Cell Metabolism. Press, Google Scholar), its two sequential enzymes in the Krebs tricarboxylic acid cycle. we have shown that the yeast mitochondrial CS and MDH enzymes, Cit1p and Mdh1p, in vitro C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). two possible for are and was important to overexpression of Cit1p on of the the for the we a between the protein and the that metabolic pathway the sequence is in we that this into Cit1p in these The yeast was with and or The from and use the ACO1 to expression and The the three last encoding the peroxisomal sequence and is to the mitochondrial of The resulting for their to grow on Cit1p and with the to the on of the While no was with the the the was to grow on that was to and overexpression of Cit1p, or was by and MDH in the mitochondrial of these dehydrogenase in mitochondrial and to demonstrate the of shown in CS was in mitochondrial from the the However, the mitochondrial form of the is to be in protein C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). the of a biological other the and interactions that the process. that a of genetic have been especially in the yeast S. to directly interactions between and to for a protein of (reviewed in Ref. 1999; PubMed Scopus Google Scholar). A new approach that an strategy to the from peptide display of transdominant of a particular or pathway (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar, 11.Norman T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar, 12.Geyer C.R. Colman-Lerner A. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8567-8572Crossref PubMed Scopus (99) Google Scholar). In this we have used an adaptation and an extension of this approach to characterize an assembly mutation in the S. cerevisiae CIT1 gene encoding mitochondrial The of this characterization evidence that this enzyme vivo with mitochondrial of its sequential enzymes in the Krebs tricarboxylic acid and that this interaction the of the corresponding peptide to be a transdominant of the Krebs tricarboxylic acid and the inhibitory phenotype was overcome by an overexpression of is in pig We have a of a between mitochondrial and aconitase by using the of these three enzymes C. M. Srere P.A. Biochemistry. PubMed Scopus Google Scholar). This on the structural and experimental from obtained with the yeast (6.Lindbladh C. Rault M. Hagglund C. Small W.C. Mosbach K. Bülow L. Evans C. Srere P.A. Biochemistry. 1994; 33: 11692-11698Crossref PubMed Scopus (69) Google Scholar), C. G. and A. with and that the the of we the from the and which be in interactions with of the other of Cit1p, which is to a of the in the mutation in pig CS to which was to be in an acid with or of pig mitochondrial MDH C. M. Srere P.A. Biochemistry. PubMed Scopus Google Scholar). the wild type peptide shown in is in which has been shown to be to Cit1p in in for a Krebs tricarboxylic acid and in vitro for interaction with in the of C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar). these a for an assembly mutation affecting an between Cit1p and Mdh1p, as demonstrated by the reversible transdominant inhibition with these we have shown that is in to Cit1p in with to a wild type phenotype of the cells. The of the inhibitory phenotype by a of by overexpression of a protein other those as Cit1p and was with with This is with where the CS proposed to with aconitase is from that corresponding to C. M. Srere P.A. Biochemistry. PubMed Scopus Google Scholar). with in the of Cit1p in a mitochondrial of the CIT1 gene in cells that are unable to grow on G. M. L. Srere P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar) the that the mitochondrial is with a citrate B. L. of Press, Scholar), which the CS to as a for the In introduction in cells of a Cit1p protein in of tricarboxylic acid cycle and on G. Evans Malloy C. Srere P.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), that the protein a structural in cycle that is of its This was supported by that a form of Cit1p was for of of on C. Lebreton S. I. Srere P.A. Biochemistry. 1999; 38: PubMed Scopus Google Scholar), that mitochondrial of Cit1p is for its in the tricarboxylic acid cycle. using a Cit1p, and J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) have shown that this enzyme is in yeast with its in a multienzyme the most used approach to identify and interactions in vivo is the S. PubMed Scopus Google Scholar). the of the directly provide the physiological relevance of the interactions, of the is to gene many with may be to this or the may be to interactions that in have been for analysis of interactions in cellular other the A. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar, A. M. Biol. PubMed Scopus Google Scholar), most of the interactions are of their physiological context. The of the transdominant inhibition method in the that the study of interactions in their cellular the physiological relevance of the interaction directly by the inhibitory phenotype resulting from overexpression of the from of the of this approach is that the use of specifically yeast wild type be used as as the for selection of The of the method used in this study mainly in two the was from a was specifically on a assembly mutation affecting one of of a of this approach initially of such mutations specifically in to the biological of This be by on the of R. PubMed Scopus Google Scholar) instance, mutation was isolated by using or using that the of the J. Biol. Scholar, R. R. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The that is the of this approach concerns the of assembly mutations from the Such a screen that of the interactions of that be as an enzymatic and that is for the biological in For this the approach used in this study may be to a of The of method concerns the of the Caponigro et al. (10.Caponigro G. Abedi M.R. Hurlburt A.P. Maxfield A. Judd W. Kamb A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7508-7513Crossref PubMed Scopus (76) Google Scholar), et al. T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar), and et al. C.R. Colman-Lerner A. Brent R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8567-8572Crossref PubMed Scopus (99) Google Scholar) used the with the as the In to the of this method the of analysis is the of as by et T.C. Smith D.L. Sorger P.K. Drees B.L. O'Rourke S.M. Hughes T.R. Roberts C.J. Friend S.H. Fields S. Murray A.W. Science. 1999; 285: 591-595Crossref PubMed Scopus (173) Google Scholar). for that are with the to overcome the inhibitory the for a are the interaction between the and its is directly the of the biological in which this interaction is The of this concerns the of the metabolic pathway in a specific such as mitochondria or In these this approach may be to of a of to be for the in the of the study described in this the body of evidence for the organization of the enzymes of the tricarboxylic acid cycle in a multienzyme in which there is channeling of cycle intermediates, the concept of a tricarboxylic acid cycle has has been to an from the The weak interactions between the enzymes that are vivo by the protein in the mitochondrial A. J. PubMed Scopus Google Scholar, A.P. J. Full Text PDF PubMed Scopus Google Scholar) are by the dilution that during cell The of the reversible transdominant inhibition as described in this to the tricarboxylic acid cycle to be a powerful to a of tricarboxylic acid cycle enzyme organization the level of the cycle and especially the channeling of for in the R. A. G. L. and K. for L. for a of this especially L. W. B. C. B. and K. for their the of A. S.