Association of the yeast DNA helicase Pif1p with mitochondrial membranes and mitochondrial DNA.

Previously we demonstrated that the mitochondrial form of the yeast Pif1p DNA helicase, which we found to be attached to mitochondrial DNA (mtDNA), is required for the maintenance of mtDNA under genotoxic stress conditions. Here, we demonstrated that mitochondrial Pif1p is exclusively bound to mitochondrial membranes and part of an about 900kDa protein complex. Pif1p might be incorporated into this complex immediately after its translocation from the cytoplasm into the mitochondrial matrix. Pif1p as well as the mitochondrial DNA polymerase Mip1p could not be released from the mitochondrial membranes by digesting mtDNA with restriction enzymes in permeabilized mitochondria. In contrast, restriction enzyme-digested mtDNA fragments that were covered by the histone-like protein Abf2p were efficiently released from the permeabilized mitochondria. We propose that Pif1p as well as Mip1p are not only bound to mtDNA but also to the inner mitochondrial membrane either directly or indirectly via a protein complex. We also found that in the absence of mtDNA the total amount of cellular Pif1p is highly reduced.