A small deletion in the second intron of human NDUFV2 (IVS2+5_+8delGTAA) has been shown to cause hypertrophic cardiomyopathy and encephalomyopathy [Bénit, P., Beugnot, R., Chretien, D., Giurgea, I., de Lonlay-Debeney, P., Issartel, J.P., Kerscher, S., Rustin, P., Rötig, A. & Munnich, A. (2003) Human Mutat.21, 582-586]. Skipping of exon 2 results in a partial deletion of the mitochondrial targeting sequence of the precursor for the 24 kDa subunit of respiratory chain complex I. Immunoreactivity of the 24 kDa subunit and complex I activity, both present at 30-50% of normal levels in patient mitochondria, raised the question of how the mutant 24 kDa subunit precursor can be imported and assembled into functional complex I. In the present study, we have remodelled the human NDUFV2 mutation by deleting codons 17-32 from the orthologous NUHM gene of the obligate aerobic yeast Yarrowia lipolytica. The resulting mutant enzyme was indistinguishable from parental complex I with regard to activity, inhibitor sensitivity and EPR signature. Size, isoelectric point and presumably also N-terminal acetylation were altered, indicating that the residual targeting sequence was retained on the mature 24 kDa protein. Complete removal of the NUHM presequence resulted in the absence of complex I activity, strongly arguing against the presence of an internal mitochondrial targeting sequence within the 24 kDa protein.