We determined the primary structure of a 9.6-kDa subunit of the respiratory chain NADH : ubiquinone reductase (complex 1) from Neurospora crassa mitochondria and found a close relationship between this subunit and the bacterial or chloroplast acyl-carrier protein. The degree of sequence identity amounts to 80% in a region of 19 residues around the serine to which the phosphopantetheine is bound. The N-terminal presequence of the subunit has the characteristic features of a mitochondrial import sequence. We cultivated the auxotroph pan-2 mutant of N . crassa in the presence of [14C]pantothenate and recovered all radioactivity incorporated into mitochondrial protein in the 9.6-kDa subunit of complex I. We cultivated N . crassa in the presence of chloramphenicol to accumulate the nuclear-encoded peripheral arm of complex I. This pre-assembled arm also contains the 9.6-kDa subunit. These results demonstrate that an acyl-carrier protein with pantothenate as prosthetic group is a constituent part of complex I in N . crassa.In a series of reports, Brody and coworkers [I-41 have demonstrated that mitochondria of Neurospora crassa and other microorganisms contain an acyl-carrier protein (ACP) with phosphopantetheine as prosthetic group. This mitochondrial ACP can mediate de novo fatty acid synthesis, independent of the fatty acid synthetase complex present in the cytoplasm [4]. The role of the mitochondrial synthetic pathway, however, remained unclear, and the authors considered the possibility that the pathway exists to satisfy special needs of lipids of the mitochondrion itself [4]. Antigenically similar proteins of similar apparent molecular masses were also found in the mitochondria of yeast, pea leaves and potato tubers, but not in animal mitochondria [3]. In order to isolate the ACP, the authors had to dissolve the N. crassa mitochondria in 6 M guanidine hydrochloride [2].The respiratory chain complex of NADH : ubiquinone oxidoreductase (complex I) in N . crassa mitochondria is an assembly of some 30 different subunits. Most of these are nuclear encoded and imported from the cytoplasm. In N . crassu, at least six (seven in mammals) are encoded and translated in the mitochondrion. Only a few subunits are directly involved in the binding of the substrates NADH and ubiquinone, the internal redox groups FMN and four (EPRdetectable) iron-sulfur clusters. The functions of the many additional subunits which do not appear to take part directly in electron transport remain largely unknown (for a review,

Two related forms of the respiratory-chain complex, NADH : ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin-A-sensitive form, which consists of some 23 different nuclear-and 6 -7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin-A-insensitive form which consists of only = 13 nuclearencoded subunits. The subunits of the small form are either identical or similar to nuclear-encoded subunits of the large form. The iron-sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR-detectable iron-sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron-transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.The mitochondrial respiratory-chain Complex I, NADH : ubiquinone oxidoreductase, links the transfer of two electrons from NADH to ubiquinone with the translocation of four or five protons across the mitochondrial inner membrane (for reviews see [I, 21). The enzyme is most complicated and consists of some 30 different subunits of which seven in human [3, 41 and at least six in the fungus N. crassa [5,61 are encoded by mitochondrial NADH dehydrogenase genes (ndh genes) and translated on mitochondrial ribosomes. The other subunits are nuclear encoded and imported from the cytoplasm. One FMN, four or five EPR-detectable iron-sulfur clusters [7, 81 and at least one bound form of ubiquinone participate in the electron transfer through the enzyme [9-111. Because of this enormous complexity, the NADH : ubiquinone oxidoreductase has remained the least understood among the proton-translocating respiratory-chain complexes of mitochondria. Therefore, attempts have been made to characterize related, putatively less complicated NADH : ubiquinone oxidoreductases in bacteria [I2 -191.Recently Friedrich et al. [20] have found that in chloramphenicol-poisoned N . crassa a smaller form of the NADH:ubiquinone oxidoreductase is made in place of the large Complex I normally present. This small Complex I consists of only = 13 subunits, all of which are nuclear encoded and synthesized in the cytoplasm. Immunoblotting and peptide mapping of the subunits found in the two forms of Complex I suggest that they are either identical or similar toCorrespondence to

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