As illustrated in Fig. , the most direct control of the genes that define the extended leucine pathway is exerted by the Leu3p-α-isopropylmalate complex, which functions at six of the seven steps shown (ILV2
, and BAT1
), with the caveat that Leu3p control of BAT1
has not yet been directly demonstrated. Superimposed on this leucine-specific regulation is general amino acid control, mediated by Gcn4p. The effect of Gcn4p is twofold. First, it increases the Leu3p level, as inferred from experiments showing an increase in the rate of production of a Leu3p-β-galactosidase fusion protein in a manner typical for the general amino acid control system (118
). This probably has physiological consequences since increased production of Leu3p can lead to increased target gene expression (110
). Second, it acts directly on at least four genes (ILV3, LEU4
, and BAT1-BAT2
) of the extended leucine pathway; the effect on three more genes (ILV2
, and LEU1
) may be indirect, through Leu3p (49
). The Leu3p regulon thus becomes part of a remarkable regulatory network that encompasses at least 539 bona fide targets, among them a total of 26 genes that encode DNA binding transcription factors (76
). By stimulating LEU3
expression, Gcn4p would amplify its impact in cascade-type fashion. Its simultaneous stimulation of LEU3
ensures that both components of the Leu3p-α-isopropylmalate complex will be made at increased rates. It can be imagined that, in a typical sequence of events, starvation for amino acids (or purines or glucose) would elicit an overriding response in a multitude of pathways, through the Gcn4p general amino acid control system. The Leu3p regulon would obey these starvation signals and augment them. In addition, it would respond independently to other signals, e.g. changes in the CoA/acetyl-CoA ratio, the cell's adenylate energy charge, or the leucine pool, that have an effect on the intracellular α-isopropylmalate level. This would give the yeast cell even more flexibility in its adjustment to environmental changes and demands.
Major regulatory mechanisms impacting the extended leucine pathway of S. cerevisiae. For abbreviations of intermediates and the protein products of the genes shown, see the legend to Fig. . See the text for further explanation.
Yet another layer of control that acts on genes of the extended leucine pathway is represented by Tpk1p (Fig. ). This protein is one of three catalytic subunits of yeast protein kinase A. Like Tpk2p and Tpk3p, Tpk1p is released from an inactive complex under conditions that raise the intracellular cyclic AMP level, for example when cells leave stationary phase after gaining access to glucose or other fermentable carbon sources. The functions of the three subunits were recently studied by genome-wide transcriptional profiling, and it was established by comparing a TPK1
mutant strain with the wild type that there was a significant reduction in the expression of BAT1
in the TPK1
mutant in YPD medium (91
). Why should BAT1
be regulated by Tpk1p? It turns out that the enzymes encoded by these two genes play other important roles besides their catalytic functions in the branched-chain amino acid pathways, and it is likely that the stimulatory effect of Tpk1p is aimed at these other functions, which have to do with mitochondrial integrity.
The pathway function of Bat1p is to serve as mitochondrial branched-chain amino acid aminotransferase; Bat2p is an isoenzyme located in the cytosol (58
). Cells deficient in both proteins do not grow on minimal media unless supplied with all three branched-chain amino acids. However, even in the presence of these amino acids, growth remains sluggish. A possible explanation for this behavior comes from the observation that the Bat proteins, in particular Bat1p, perform an essential function in iron homeostasis by being involved in the efficient transfer of Fe-S clusters from the mitochondria, where the clusters are synthesized, to the cytosol, a process that also involves the mitochondrial ABC transporter Atm1p. In fact, the BAT1
gene was isolated as a suppressor of a temperature-sensitive ATM1
mutant, and elevated levels of Bat1p were able to stabilize mutant Atm1p at the nonpermissive temperature. This suggested direct interaction between Bat1p and Atm1p, but other possibilities have not been ruled out (57
) (Fig. ). An important point is that a disturbance in the process of maintaining iron homeostasis can lead to a severe accumulation of iron in the mitochondria with subsequent damage and loss of mitochondrial DNA (mtDNA).
Ilv5p, which normally functions as acetohydroxy acid reductoisomerase, is also involved in enhancing the stability of mtDNA (114
). This feature of Ilv5p was first discovered when ILV5
was identified as a suppressor of the mtDNA instability phenotype of Δabf2
cells grown on rich glucose medium (Abf2p is required for maintenance of mtDNA under these conditions). Suppression was achieved with a relatively small increase in ILV5
copy number or increased expression of the ILV5
gene in a strain with a constitutively active GCN4
allele. The fact that an ILV5
null mutant showed a strong tendency to produce ρ− petite
strains further supported the notion that Ilv5p stabilizes mtDNA. More recently, it was shown that Gcn4p-mediated activation of ILV5
expression results in increased formation of nucleoids (mtDNA-protein complexes considered to be the basic unit of mtDNA segregation) and that such an increase in nucleoid number also increases the transmission of mtDNA to daughter cells (71
). It was proposed that the simultaneous activation of amino acid biosynthesis and nucleoid formation by Gcn4p might enhance the chance of progeny survival.
By increasing the expression of BAT1
, Gcn4p and Tpk1p are likely to achieve similar results with respect to preservation of mtDNA, parsing of mtDNA into nucleoids, and respiratory competence. Apparently, the action of Tpk1p is reinforced by Gcn4p since the TPK1
gene is controlled, at least in part, by Gcn4p (76
). Also, given the possibility that Gcn4p regulation of ILV5
actually works through the Leu3p-α-isopropylmalate complex, the latter might likewise contribute to mtDNA maintenance.