The inheritance of functional mitochondria depends on faithful replication and transmission of mitochondrial DNA (mtDNA). The mitochondrial genome of the yeast Saccharomyces cerevisiae
encodes eight major proteins. Seven mitochondrially encoded proteins are components of respiratory chain complexes, and one is a subunit of mitochondrial ribosomes. All other mitochondrial proteins are encoded in the nucleus, synthesized on cytosolic ribosomes, and imported into mitochondria. An intact mitochondrial genome is necessary for respiratory competence but not for the viability of yeast cells (1
). Several proteins required for inheritance of mtDNA are known in yeast. These include mtDNA polymerase, Mip1p (17
); a single-stranded DNA binding protein, Rim1p (9
); a histone-like protein, Abf2p (43
); mitochondrial RNA polymerase, Rpo41p (19
); a DNA-binding protein of the mitochondrial inner membrane, Yhm2p (5
); and several other proteins of unclear function. The molecular mechanism of mtDNA replication is only poorly understood. It was suggested that mitochondrial RNA polymerase synthesizes an RNA transcript which is then used as a primer for mtDNA polymerase to synthesize a new DNA strand (42
). Alternatively, yeast mtDNA might be primarily replicated by a rolling circle-like mechanism (36
). In addition, recombination and the conversion of recombination intermediates into replication forks have been proposed to be important during the replication of yeast mtDNA (13
Several cases are known where molecular chaperones play a key role in DNA replication processes. The bacterial dnaK
genes, for example, were originally identified and named based on their essential role in the replication of bacteriophage λ DNA, and the purification of the DnaK and DnaJ proteins was facilitated by their activity in DNA synthesis (for a review, see references 49
). Both chaperones perform essential and direct functions in the replication of bacteriophage λ DNA by the activation of a prepriming complex (50
). A direct function of molecular chaperones in DNA replication, however, is known only for bacteriophages (λ, P1, and Mu) or plasmids (F and RK2) (4
). Propagation of the bacterial chromosome is affected by the inactivation either of DnaK or its partner proteins DnaJ or GrpE. In this case, the chaperones appear to play an indirect role by protecting the DnaA initiator protein against aggregation and inactivation (3
). Similarly, eukaryotic homologs of DnaK and DnaJ have been shown to protect calf thymus DNA polymerase
against thermal inactivation in vitro (48
). So far, there is no evidence for a direct role of molecular chaperones in either bacterial or eukaryotic chromosomal DNA replication. It is still an open question whether the direct involvement of molecular chaperones in the synthesis of plasmids and phage DNA is a specific adaptation, or whether it is a more common feature that characterizes also processes of genomic DNA propagation.
Molecular chaperones of the Hsp70 class play an important role in mitochondrial biogenesis (reviewed in references 7
). Mitochondrial Hsp70 (mtHsp70, also termed Ssc1p), a homolog of bacterial DnaK, is an essential constituent of the mitochondrial protein import machinery. Moreover, mtHsp70 is involved in folding and assembly of newly imported and mitochondrially synthesized proteins, in the degradation of misfolded proteins in the matrix, and in the protection of proteins against heat-induced aggregation (22
). Apparently, all functions of mtHsp70 depend on the activity of its cofactor Mge1p, a homolog of bacterial GrpE (24
). Since both mtHsp70 and Mge1p are indispensable for protein import, the encoding genes are essential in yeast. In contrast, Mdj1p, a homolog of bacterial DnaJ, is not essential for mitochondrial protein import (40
). Mdj1p has been shown to cooperate with mtHsp70 during protein folding and degradation and in the protection of proteins against heat stress (39
). Deletion of the MDJ1
gene leads to a temperature-sensitive growth phenotype. It was reported earlier that cells lacking Mdj1p become [rho0
], i.e., they completely lose mtDNA (40
). This observation implies that Mdj1p directly or indirectly is involved in the maintenance of mtDNA.
The key proteins involved in mtDNA replication are encoded in the nucleus and synthesized on cytosolic ribosomes (6
). After translocation across the mitochondrial membranes, they have to acquire their native conformation in the mitochondrial matrix, a process that might require the assistance of chaperones (21
). Furthermore, mitochondrial chaperones might play a role in the inheritance of mtDNA beyond the step of folding of the required proteins.
In the present study, we investigated the role of Mdj1p in the replication of mtDNA. We show that, under restrictive conditions, the activity of mtDNA polymerase is substantially reduced in a conditional mdj1 mutant, resulting in the rapid loss of mtDNA. Furthermore, we find that the activity of mtDNA polymerase does not depend on Mdj1p at low temperature. Under these conditions, the absence of Mdj1p leads to a rapid conversion of functional [rho+] genomes to nonfunctional [rho−] genomes which are then stably maintained. Taken together, our results suggest a dual role of Mdj1p in the maintenance of mtDNA.