PIM1 protease in mitochondria belongs to a conserved family of ATP-dependent proteases, which includes the Escherichia coli Lon protease. Yeast cells lacking PIM1 are largely defective in degrading misfolded proteins in the mitochondrial matrix, are respiratory deficient, and lose integrity of mitochondrial DNA. In order to analyze whether E. coli Lon protease is functionally equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a mitochondrial targeting sequence fused onto the Lon protease. In these cells, the fusion protein was expressed and imported into mitochondria, and the targeting sequence was removed. In the absence of PIM1 protease, the E. coli Lon protease mediated the degradation of misfolded proteins in the matrix space in cooperation with the mitochondrial hsp70 system. These cells maintained the integrity of the mitochondrial genome and the respiratory function at 30 degrees C but not at 37 degrees C. Stabilization of mitochondrial DNA in Deltapim1 cells depended on protein degradation by the E. coli Lon protease, as a proteolytically inactive Lon variant was not capable of substituting for a loss of PIM1 protease. These results demonstrate functional conservation of Lon-like proteases from prokaryotes to eukaryotes and shed new light on the role of Lon-like proteases in mitochondrial biogenesis.