In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organ ization and inheritance, we have developed an in organello formaldehyde cro sslinking procedure to identify proteins associated with mtDNA. Using highl y purified mitochondria, we observed a time-dependent crosslinking of prote in to mtDNA as determined by sedimentation through isopycnic cesium chlorid e gradients. We detected approximate to 20 proteins crosslinked to mtDNA an d identified 11, mostly by mass spectrometry. Among them is Abf2p, an abund ant, high-mobility group protein that is known to function in nucleoid morp hology, and in mtDNA transactions. In addition to several other proteins wi th known DNA binding properties or that function in mtDNA maintenance, we i dentified other mtDNA-associated proteins that were not anticipated, such a s the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2 Delta mutation increases the petite-inducing phenotype of an abf2 Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with hi gh specificity for the template strand of a putative origin of mtDNA replic ation. These data identify bifunctional proteins that participate in the st ability of rho(+) mtDNA.