CLONING, IN-VITRO MITOCHONDRIAL IMPORT AND MEMBRANE ASSEMBLY OF THE 17.8 KDA SUBUNIT OF COMPLEX-I FROM NEUROSPORA-CRASSA

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 automate d Edman degradation of the isolated polypeptide. The sequence data obt ained indicate that the 17.8 kDa subunit is made as an extended precur sor 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 (bi topic) 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 matri x-processing peptidase. The in vitro imported mature subunit is found mainly exposed to the mitochondrial intermembrane space. However, a si gnificant fraction of the imported polypeptide acquires the same membr ane topology as the endogenous subunit, indicating that correct assemb ly in the mitochondrial inner membrane did occur.