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A cDNA encoding the 14.8 kDa subunit of complex I from Neurospora crassa was cloned and sequenced. The deduced primary structure of this subunit reveals a predominantly hydrophilic protein containing no obvious membrane-spanning domain. In agreement with this characteristic, we have localized the 14.8 kDa subunit in the peripheral arm of the enzyme. The 14.8 kDa subunit was found to be conserved in mammalian complex I. The conservation of this subunit in such distantly related organisms suggests that the 14.8 kDa subunit is an important component of complex I. We have used an in organello system to study the biosynthetic pathway of this subunit. The 14.8 kDa polypeptide could be efficiently imported into isolated mitochondria. Furthermore, a fraction of the in-vitro-imported subunit was found to assemble in complex I. This is the first time that assembly in complex I of an in-vitro-synthesized subunit is demonstrated.

We have cloned and sequenced a cDNA encoding a 17.8 kDa subunit of the hydrophobic fragment of complex I from Neurospora crassa. The deduced primary structure of this subunit was partially confirmed by automated Edman degradation of the isolated polypeptide. The sequence data obtained indicate that the 17.8 kDa subunit is made as an extended precursor of 20.8 kDa. Resistance of the polypeptide to alkaline extraction from mitochondrial membranes and the existence of a putative membrane-spanning domain suggests that the 17.8 kDa subunit is an intrinsic (bitopic) membrane protein. The in vitro synthesized precursor of the 17.8 kDa subunit can be efficiently imported into isolated mitochondria, where it is cleaved to the mature species by the metal-dependent matrix-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a significant fraction of the imported polypeptide acquires the same membrane topology as the endogenous subunit, indicating that correct assembly in the mitochondrial inner membrane did occur.

The 12.3 kDa subunit of complex I (respiratory-chain NADH dehydrogenase) is a nuclear-coded protein of the hydrophobic fragment of the enzyme. We have isolated and sequenced a full-length cDNA clone coding for this polypeptide. The deduced protein is 104 amino acid residues long with a molecular mass of 12305 Da. This particular subunit of complex I lacks a cleavable mitochondrial targeting sequence. In agreement with its localization within complex I, we have found that this subunit behaves like an intrinsic membrane protein. Nevertheless, the deduced protein is rather hydrophilic, exhibiting no hydrophobic domain long enough to traverse a membrane in an alpha-helical conformation. The 12.3 kDa subunit shows a significant similarity to the hinge protein of complex III, suggesting that these two polypeptides may be involved in identical functions. This complex I subunit is coded for by a single gene. Applying restriction-fragment-length-polymorphism mapping, we located the gene on the right side of the centromere in linkage group I, linked to the lys-4 locus.

The 20.9 kDa subunit of NADH:ubiquinone oxidoreductase (complex I) from Neurospora crassa is a nuclear-coded component of the hydrophobic arm of the enzyme. We have determined the primary structure of this subunit by sequencing a full-length cDNA and a cleavage product of the isolated polypeptide. The deduced protein sequence is 189 amino acid residues long and contains a putative membrane-spanning domain. Striking similarity over a 60 amino-acid-residue domain with the M (matrix) protein of para-influenza virus was found. No other relationship with already known sequences could be detected, leaving the function of this subunit in complex I still undefined. The biogenetic pathway of this polypeptide was studied using a mitochondrial import system in vitro. The 20.9 kDa subunit synthesized in vitro is efficiently imported into isolated mitochondria, where it obtains distinct features of the endogenous subunit. Our results suggest that the 20.9 kDa polypeptide is made on cytosolic ribosomes lacking a cleavable targeting sequence, interacts with the mitochondrial outer membrane (in a process that does not require an energized inner membrane), and is imported into mitochondria at contact sites. The 20.9 kDa subunit is then inserted into the inner membrane acquiring a topology similar to that of the already assembled subunit.