Crosslinking of Proteins to mtDNA

Mitochondrial DNA (mtDNA) nucleoids have been isolated from several organisms, including rat (liver) (1 ), Physarum polycephalum (2 ), Saccharomy- ces cerevisiae (3 ) and Pichia jadinii (4 ). Most methods for nucleoid isolation have utilized detergent extraction and sucrose gradient centrifugation to separate bulk mitochondrial protein from mtDNA-associated proteins (e.g., ref. 5 ). The disadvantages of these methods include contamination by non-nucleoid proteins and the possibility of losing those nucleoid proteins that are not tightly bound to the complex under the conditions of the fractionation. To circumvent these problems, we have developed an in organello formaldehyde crosslinking method for isolating mtDNA nucleoids that stabilizes protein content and reduces the potential for contamination by irrelevant mitochondrial (and cellular) proteins. Additionally, it has long been suggested that mtDNA is associated with the inner membrane. To begin exploring that notion, we have used ultraviolet (UV) crosslinking as a means of detecting the binding of proteins from membrane-enriched fractions to specific sequences of mtDNA.