Processing of artificial peptide-DNA-conjugates by the mitochondrial intermediate peptidase (MIP)

Import of DNA from the cytoplasm into the mitochondrial matrix is an obliga tory step for an in organello site-directed mutagenesis or gene therapy app roach on mitochondrial DNA diseases. In this context, we have developed an artificial DNA translocation vector that is composed of the mitochondrial s ignal peptide of the ornithine transcarbamylase (OTC) and a DNA moiety, Whi le this vector is capable of directing attached passenger molecules to the mitochondrial matrix, the recognition of this artificial molecule by the en dogenous mitochondrial signal peptide processing machinery as well as the c leavage of the peptide plays a pivotal role in the release of the attached DNA. To study the proteolytic processing of the artificial vector, various signal peptide-DNA-conjugates were treated with purified mitochondrial inte rmediate peptidase, When the leader peptide is directly linked to the DNA m oiety without an intervening spacer, MIP processing is prevented. Cleavage of the peptide can be restored, however, when the first ten amino acid resi dues of the mature part of OTC are appended at the carboxy-terminal end of the signal peptide. Our results show that artificial peptide-DNA-conjugates are recognized by the mitochondrial proteolytic machinery, and therefore a n interference of the peptide with the DNA function can be excluded.