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 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 approximately 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 kgd2Delta mutation increases the petite-inducing phenotype of an abf2Delta 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 rho(+) mtDNA.
Kaufman
B A
BA
Department of Molecular Biology, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9148, USA.
Newman
S M
SM
Hallberg
R L
RL
Slaughter
C A
CA
Perlman
P S
PS
Butow
R A
RA
D002458:Cell Fractionation; D018834:Chaperonin 60 / Q000235:genetics / Q000302:isolation & purification; D002952:Citric Acid Cycle; D003432:Cross-Linking Reagents; D004261:DNA Replication; D004271:DNA, Fungal; D004272:DNA, Mitochondrial / Q000737:chemistry*; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D005557:Formaldehyde; D005656:Fungal Proteins / Q000302:isolation & purification*; D007655:Ketoglutarate Dehydrogenase Complex / Q000302:isolation & purification; D013058:Mass Spectrometry; D008928:Mitochondria / Q000737:chemistry*; D017354:Point Mutation; D011485:Protein Binding; D018741:Replication Origin; D012441:Saccharomyces cerevisiae
D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D004271:DNA, Fungal; D004272:DNA, Mitochondrial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D005557:Formaldehyde; D007655:Ketoglutarate Dehydrogenase Complex