It has been established that valine is synthesized from pyruvate in a series of at least four steps, and that isoleucine is synthesized in a series of four similar steps from pyruvate and a-ketobutyrate. The four enzymes involved are apparently common to both pathways, but this has not been definitely proved in any one case. The enzymes will be described here as the condensing enzyme, which produces aacetolactate and a-acetohydroxybutyrate from pyruvate, and pyruvate + aketobutyrate, respectively; the reductoisomerase, which converts the a-aceto-acids to the dihydroxy acids; the dehydrase which causes a dehydration of the dihydroxy acids with the formation of the a-keto acids; and the transaminase which transaminates between phenylalanine and the respective a-keto acids to form valine and isoleucine.In at least two organisms, Salmonella and Neurospora, it has been shown that two or more of the genes affecting these enzymes are closely linked.1-3 In Neurospora crassa at least two of the enzymes, reductoisomerase and dehydrase, are apparently controlled by two genes which are estimated to be about four map units apart.4 Between them lies a region in which mutations have been detected only within a small segment. These mutations cause an absolute requirement for both isoleucine and valine, but the mutant strains do not show a deficiency for any of the four enzymes. That the blocks in these are not prior to pyruvate and aacetobutyrate has been established by showing that they accumulate either the a-aceto-acids or dihydroxy acids or both. It was postulated that the defect in these mutants, the group II mutants, lies not in their inability to produce the enzymes, but in their inability to organize them properly to be active in vivo.3 The experiments described below were designed to explore this possibility by first determining whether there exists an isoleucine-valine forming system in the particulate fraction of the wild-type mycelial homogenate.Methods and Materials.-The assay procedures for the individual enzymes, the condensing enzyme, reductoisomerase, dehydrase, and transaminase have been described in detail previously.'-Succinic cytochrome c reductase activity was determined by the method of Crane et al.9 Protein was determined by the method of Lowry et al.10The wild-type strain employed here, KJT196Oa, is a segregant from a cross between the Emer-

A group of mutants of Neurospora crassa which require both isoleucine and valine have been described in a series of previous communications from this laboratory (WAGNER, SOMERS and BERGQUIST 1960; WAGNER, KIRITANI and BERGQUIST 1962;and KIRITANI 1962). These mutants were all found to be closely linked between the linkage group V markers Zys-1 (lysine-1, 33933) and sp

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Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.

Data are presented suggesting that intact Neurospora crassa mitochondria contain an enzyme complex incorporating five enzymes necessary for the biosynthesis of isoleucine and valine. The functional integrity and stability of the complex has been shown to be dependent on the metabolic state of the mitochondria. The complex has been solubilized by digitonin and has an approximate molecular weight of 400,000.We have shown previously in this laboratory that isoleucine and valine are synthesized in the mitochondria of Neurospora crassa (1). We have now obtained evidence for the existence of an active enzyme complex that appears to incorporate the isoleucine and valine enzymes, a-acetohydroxy acid synthetase, a-hydroxy, fl-keto acid reductoisomerase, a,#-dihydroxyacid dehydratase, and an aminotransferase, as well as threonine deaminase. This complex is capable of synthesizing valine from pyruvate and isoleucine from threonine and pyruvate. The active complex is not released from isolated mitochondria unless they are first incubated in a medium that contains the necessary substrates for respiration and oxidative phosphorylation. Otherwise only the individual enzymes are solubilized, and these can be separated from one another on Sephadex columns by virtue of the differences in their molecular weights (M. Collins, K. Kiritani, S. Narise, and R. P. Wagner, unpublished results). MATERIALS AND METHODSPreparation of Mitochondria. Flasks containing one liter of Vogel's minimal medium were inoculated with 6-day-old conidia of Neurospora crassa, strain LSDT(1969)A (2) and incubated for 15.5-16 hr at 250 on a rotary shaker. The harvested mycelium was washed with "(" buffer (a buffer designed to preserve the integrity of the mitochondria during isolation as to respiration, oxidative phosphorylation, and synthesis of valine and isoleucine from pyruvate). The "Q" buffer consists of 0.3 M sucrose, 10 mM potassium phosphate at pH 7.4, 10 mM sodium succinate, 0.15% bovine serum albumin, 50 mM sodium pyruvate, 34 mM 2-mercaptoethanol, 2 mM pyridoxal-5'-phosphate, 0.13 mM thiamine pyrophosphate, 47nM FAD, and 59MM MnSO4. The washed mycelium was ground in an ice-cold mortar with twice its wet weight of acid-washed sand. The ground mycelial preparation was diluted with additional "al" buffer and centrifuged twice for 10 min at 1500 X g. The mitochondria were spun down by centrifugation for 1 hr at 12,000 X g. The pellet was taken up in "a" buffer, and the mitochondria were resuspended in a Teflon grinder to give an adjusted protein concentration of 40 mg/ml. Preincubation. The mitochondrial suspension (40 mg/ml) was divided in two portions, placed in a 30°water bath, and stirred for the time intervals indicated in Results. At zero time, to portion A (7 ml) ( Table 1)