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

Abstract

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected Ϸ20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as 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⌬ mutation increases the petite-inducing phenotype of an abf2⌬ mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of ؉ mtDNA.

Reduction in the E2k Subunit of the α-Ketoglutarate Dehydrogenase Complex Has Effects Independent of Complex Activity

“…Enzymes such as aconitase, isocitrate dehydrogenase and Kgd2p (a subunit of KGDHC in yeast equivalent to E2k in mammal), are found to have two or more different functions (51)(52)(53)(54)(55)(56). For example, Kgd2p in yeast participates in both the tricarboxylic acid cycle and ϩ mtDNA maintenance (56).…”

Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis

“…In several cases it has been observed that the form of mitochondrial genome may differ among closely related species (Bridge et al, 1992;Fukuhara et al, 1993;Martin, 1995;Laflamme & Lee, 2003). Investigations of mtDNA-binding proteins indicate that similar machineries are responsible for the maintenance of both forms (Miyakawa et al, 1995(Miyakawa et al, , 1996Nosek et al, 1999;Kaufman et al, 2000;Tomaska et al, 2001). The occurrence of linearand circular-mapping genomes in different strains of the same species indicates that both types of mitochondrial genome do not differ radically in their life styles (Fukuhara et al, 1993).…”

Heterogeneous Nuclear Ribonucleoprotein K Enhances Insulin-induced Expression of Mitochondrial UCP2 Protein

“…B, mitochondria and cytosolic post-mitochondrial fractions were prepared from HTC-IR cells grown in serum as outlined in A. Organelle markers were used to verify the mitochondria isolation procedure. Equal amounts of protein from the cytosolic post-mitochondrial fraction (Supernatant II) and from the purified mitochondria were resolved by SDS-PAGE and after electrotransfer the PVDF membrane was stained with antibody to the mitochondrial proteins, HSP60 (71), and cytochrome c (39), and to the endoplasmic protein GRP 94 (42). C, total RNA purified from mitochondrial and from cytosolic post-mitochondrial fractions was used in RT.…”