Carina Prip-Buus

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

Mdj1p, a novel member of the DnaJ family, is a heat shock protein that is associated with the inner membrane of mitochondria of Saccharomyces cerevisiae. Disruption of the MDJ1 gene resulted in a petite phenotype, loss of mitochondrial DNA, and inviability at 37 degrees C. Import of precursor proteins was not affected by a lack of Mdj1p, but folding of newly imported proteins was markedly impaired. The efficiency of refolding of a tester protein, dihydrofolate reductase, was significantly reduced in mitochondria lacking Mdj1p after incubation at elevated temperature. We conclude that Mdj1p is an important mitochondrial chaperone that participates in the folding of newly imported proteins and in the protection of proteins against heat denaturation and aggregation.

The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria.

Mge1p, a mitochondrial GrpE homologue, has recently been identified in the yeast Saccharomyces cerevisiae and a role for this protein in precursor import has been reported. To dissect the molecular mechanism of Mge1p function, conditional mge1 mutants were constructed. Cells harbouring mutant mge1 accumulated precursor proteins at restrictive temperature. Both kinetics and efficiency of import were reduced in mitochondria isolated from strains possessing mutant mge1. Binding of mitochondrial‐Hsp70 (mt‐Hsp70) to incoming precursor proteins was abolished at restrictive temperature. Nucleotide‐dependent dissociation of mt‐Hsp70 from the import component MIM44 was reduced in mitochondria from mutant mge1 strains. Furthermore, at restrictive temperature an increase of incompletely folded, newly imported protein and enhanced protein aggregation was observed in mitochondria isolated from the mutant strains. We conclude that Mge1p exerts an essential function in import and folding of proteins by controlling the nucleotide‐dependent binding of mt‐Hsp70 to substrate proteins and the association of mt‐Hsp70 with MIM44.

Role of the mitochondrial DnaJ homologue, Mdj1p, in the prevention of heat-induced protein aggregation

The role of the mitochondrial Hsp70 system in the prevention of heat‐induced protein aggregation was studied in isolated mitochondria from Saccharomyces cerevisiae. Firefly luciferase was employed as a thermolabile tester protein. After shift to 40°C a transient increase of mt‐Hsp70/luciferase complex was observed, which required functional Mdj1p and Mge1p, the mitochondrial homologues of DnaJ and GrpE. The kinetics of luciferase aggregation, however, were not influenced by mutations in either mt‐Hsp70 or Mge1p. Only the absence of Mdj1p led to enhanced protein aggregation. Thus, a central role in the transient protection against heat stress is attributed to this mitochondrial DnaJ homologue.

Topological and functional analysis of the rat liver carnitine palmitoyltransferase 1 expressed in Saccharomyces cerevisiae.

The rat liver carnitine palmitoyltransferase 1 (L‐CPT 1) expressed in Saccharomyces cerevisiae was correctly inserted into the outer mitochondrial membrane and shared the same folded conformation as the native enzyme found in rat liver mitochondria. Comparison of the biochemical properties of the yeast‐expressed L‐CPT 1 with those of the native protein revealed the same detergent lability and similar sensitivity to malonyl‐CoA inhibition and affinity for carnitine. Normal Michaelis‐Menten kinetics towards palmitoyl‐CoA were observed when careful experimental conditions were used for the CPT assay. Thus, the expression in S. cerevisiae is a valid model to study the structure‐function relationships of L‐CPT 1.

The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Contains an Internal Mitochondrial Import Signal and Residues Essential for Folding of Its C-terminal Catalytic Domain

We have previously shown that the first 147 N-terminal residues of the rat liver carnitine palmitoyltransferase 1 (CPT1), encompassing its two transmembrane (TM) segments, specify both mitochondrial targeting and anchorage at the outer mitochondrial membrane (OMM). In the present study, we have identified the precise import sequence in this polytopic OMM protein. In vitroimport studies with fusion and deletion CPT1 proteins demonstrated that none of its TM segments behave as a signal anchor sequence. Analysis of the regions flanking the TM segments revealed that residues 123–147, located immediately downstream of TM2, function as a noncleavable, matrix-targeting signal. They specify mitochondrial targeting, whereas the hydrophobic TM segment(s) acts as a stop-transfer sequence that stops and anchors the translocating CPT1 into the OMM. Heterologous expression in Saccharomyces cerevisiae of several deleted CPT1 proteins not only confirms the validity of the “stop-transfer” import model but also indicates that residues 1–82 of CPT1 contain a putative microsomal targeting signal whose cellular significance awaits further investigation. Finally, we identified a highly folded core within the C-terminal domain of CPT1 that is hidden in the entire protein by its cytosolic N-terminal residues. Functional analysis of the deleted CPT1 proteins indicates that this folded C-terminal core, which may belong to the catalytic domain of CPT1, requires TM2 for its correct folding achievement and is in close proximity to residues 1–47.