In organello formaldehyde crosslinking of proteins to mtDNA: Identification of bifunctional proteins
- 27 June 2000
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences
- Vol. 97 (14), 7772-7777
- https://doi.org/10.1073/pnas.140063197
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 ≈20 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 kgd2Δ mutation increases the petite-inducing phenotype of an abf2Δ 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 ρ+ mtDNA.Keywords
This publication has 51 references indexed in Scilit:
- Mitochondrial EvolutionScience, 1999
- The Sorting of Mitochondrial DNA and Mitochondrial Proteins in Zygotes: Preferential Transmission of Mitochondrial DNA to the Medial BudThe Journal of cell biology, 1998
- Analysis of Chromatin Structure byin VivoFormaldehyde Cross-LinkingMethods, 1997
- Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: A molecular model for the formation of heterochromatin in yeastCell, 1995
- The saccharomyces PIF1 DNA helicase inhibits telomere elongation and de novo telomere formationCell, 1994
- A close relative of the nuclear, chromosomal high-mobility group protein HMG1 in yeast mitochondria.Proceedings of the National Academy of Sciences, 1991
- Purification of DNA polymerase II stimulatory factor I, a yeast single-stranded DNA-binding protein.Proceedings of the National Academy of Sciences, 1990
- The Mitochondrial Genotype Can Influence Nuclear Gene Expression in YeastScience, 1987
- TheILVSgene ofSaccharomyces cerevisiaeis highly expressedNucleic Acids Research, 1986
- Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures.Proceedings of the National Academy of Sciences, 1985