The import of mitochondrial preproteins requires an electric potential across the inner membrane and the hydrolysis of ATP in the matrix. We assessed the contributions of the two energy sources to the translocation driving force responsible for movement of the polypeptide chain through the translocation channel and the unfolding of preprotein domains. The import-driving activity was directly analyzed by the determination of the protease resistances of saturating amounts of membrane-spanning translocation intermediates. The ability to generate a strong translocation-driving force was solely dependent on the activity of the ATP-dependent import motor complex in the matrix. For a sustained import-driving activity on the preprotein in transit, an unstructured N-terminal segment of more than 70 to 80 amino acid residues was required. The electric potential of the inner membrane was required to maintain the import-driving activity at a high level. The electrophoretic force of the potential exhibited only a limited capacity to unfold preprotein domains. We conclude that the membrane potential increases the probability of a dynamic interaction of the preprotein with the import motor. Polypeptide translocation and unfolding are mainly driven by the inward-directed translocation activity based on the functional cooperation of the import motor components.Although mitochondria can synthesize a small subset of proteins on their own, the vast majority have to be imported after their synthesis in the cytosol (11,23,36,40). The precursor proteins are transported through specific translocase complexes residing in the outer and inner membranes (21, 37). However, the relatively small inner diameter of the translocation channels presents a major constraint for the import process (17, 49). Experimental evidence indicates that preproteins destined for the mitochondrial matrix cross the membranes in an extended conformation (42). Posttranslational import of mitochondrial preproteins therefore requires the unfolding of folded preprotein domains prior to or during the translocation reaction.Two mitochondrial energy sources are responsible for both vectorial movement of the precursor polypeptide in the translocation channel and unfolding of preproteins. The insertion of the amino-terminal segment of the preprotein into the inner membrane is dependent on the electric potential (⌬) across the inner membrane. It is thought that the potential exerts an electrophoretic force on the positively charged N-terminal targeting sequence (29). In addition, the ⌬ has been shown to be involved in the function of the TIM23 protein complex (2). The full translocation of the polypeptide chain requires a second energy source, the hydrolysis of matrix ATP. The enzymatic machinery that couples preprotein translocation and ATP hydrolysis is provided by the import motor complex, which is located at the inner face of the inner membrane in the direct vicinity of the TIM23 translocation channel (36,39,48,55). Its core component is the main mitochondrial chaperone...

Glutaredoxins belong to a family of small proteins with glutathione-dependent disulfide oxidoreductase activity involved in cellular defense against oxidative stress. The product of the yeast GRX2 gene is a protein that is localized both in the cytosol and mitochondria. To throw light onto the mechanism responsible for the dual subcellular distribution of Grx2 we analyzed mutant constructs containing different targeting information. By altering amino acid residues around the two in-frame translation initiation start sites of the GRX2 gene, we could demonstrate that the cytosolic isoform of Grx2 was synthesized from the second AUG, lacking an N-terminal extension. Translation from the first AUG resulted in a long isoform carrying a mitochondrial targeting presequence. The mitochondrial targeting properties of the presequence and the influence of the mature part of Grx2 were analyzed by the characterization of the import kinetics of specific fusion proteins. Import of the mitochondrial isoform is relatively inefficient and results in the accumulation of a substantial amount of unprocessed form in the mitochondrial outer membrane. Substitution of Met 35 , the second translation start site, to Val resulted in an exclusive targeting to the mitochondrial matrix. Our results show that a plethora of Grx2 subcellular localizations could spread its antioxidant functions all over the cell, but one single Ala to Gly mutation converts Grx2 into a typical protein of the mitochondrial matrix.

Use of proteomics and physiological characteristics to elucidate ecotoxic effects of methyl tert-butyl ether in Pseudomonas putida KT2440We monitored rates of growth, ATP-synthesis, respiration, and death to assess the sensitivity of the model organism Pseudomonas putida KT2440 to methyl tert-butyl ether (MTBE), and its degree of toxicity. The physiological data obtained suggested that the energy conservation system was the most sensitive site. However, with the help of proteomic analysis we obtained further information and deeper insight into the molecular mechanisms involved. This analysis indicated that sensitivity involves oxidative stress since alkylhydroperoxide reductase C (AhpC) and two superoxide dismutases (SodM, SodF) were amplified in the presence of MTBE. Thus, proteomics has major advantages in ecotoxicological investigations where the aims include elucidation of the molecular mechanisms as well as characterization of the ecostress and the potency of the stressor(s).

The protease Pim1/LON, a member of the AAA+ family of homo-oligomeric ATP-dependent proteases, is responsible for the degradation of soluble proteins in the mitochondrial matrix. To establish the molecular parameters required for the specific recognition and proteolysis of substrate proteins by Pim1, we analyzed the in organello degradation of imported reporter proteins containing different structural properties. The amino acid composition at the amino-terminal end had no major effect on the proteolysis reaction. However, proteins with an amino-terminal extension of less than 60 amino acids in front of a stably folded reporter domain were completely resistant to proteolysis by Pim1. Substrate proteins with a longer amino-terminal extension showed incomplete proteolysis, resulting in the generation of a defined degradation fragment. We conclude that Pim1-mediated protein degradation is processive and is initiated from an unstructured amino-terminal segment. Resistance to degradation and fragment formation was abolished if the folding state of the reporter domain was destabilized, indicating that Pim1 is not able to unravel folded proteins for proteolysis. We propose that the requirement for an exposed, large, non-native protein segment, in combination with a limited unfolding capability, accounts for the selectivity of the protease Pim1 for damaged or misfolded polypeptides.

The essential mitochondrial Hsp70 (mtHsp70) is required for the import of mitochondrial preproteins into the matrix compartment. The translocation-specific activity of mtHsp70 is coordinated by its interaction with specific partner proteins, forming the import motor complex that provides the energy for unfolding and complete translocation of precursor polypeptide chains. A major biochemical characteristic of Hsp70-type chaperones is their nucleotide-regulated affinity to polypeptide substrates. To study the role of this allosteric regulation in the course of preprotein translocation, we have generated specific mtHsp70 mutations located within or close to the interface between the nucleotide-binding and the substrate-binding domains. Mitochondria isolated from the mtHsp70 mutants displayed severely reduced import efficiencies in vitro. Two of the mutants exhibited strong growth defects in vivo and were significantly impaired in the generation of an inward-directed, ATPdependent import force on precursor proteins in transit. The biochemical properties of these two mutant proteins were consistent with defects in the transfer of conformational signals to the substrate-binding domain, resulting in a prolonged and enhanced interaction with imported substrate proteins. Furthermore, interference with the allosteric mechanism resulted in defects of translocation-specific partner protein interaction. We conclude that even a partial disruption of the interdomain communication in the mtHsp70 chaperone results in an almost complete breakdown of its translocation-driving properties.Chaperones of the 70-kDa family (Hsp70) are ubiquitous proteins that occur in all species (except archaea) and fulfill crucial cellular functions under normal and stress conditions (1). Eukaryotic cells contain several Hsp70 paralogs that are generally involved in all stages of protein biogenesis, ranging from biosynthesis at the ribosomes, intracellular transport, and eventually proteolysis (2-4). Mitochondria of the model organism Saccharomyces cerevisiae contain three types of Hsp70 proteins, encoded by the genes SSC1, SSQ1, and SSC3 (ECM10) (5). Ssc3 is expressed at very low levels under normal growth conditions and its cellular function is so far unknown. Ssq1 has been shown to assist the assembly of iron/sulfur cluster cofactors in mitochondria. The most abundant and functionally important mitochondrial Hsp70 is Ssc1, generally referred to as mtHsp70. It performs most of the typical chaperone functions in terms of protein folding and quality control for polypeptides localized in the mitochondrial matrix compartment (5-7). In addition, Ssc1 is required for the import of all mitochondrial precursor proteins destined for the matrix (8). In yeast cells, deletion of SSC1 results in a lethal phenotype, a property that has been directly attributed to its crucial role in the import process. Mitochondrial preprotein translocation comprises a series of consecutive steps (9): after synthesis at cytosolic ribosomes, the N-terminal targeting signal of a p...