Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70

Mitochondrial heat shock protein 70 (mt-hsp70) functions as a molecular cha perone in mitochondrial biogenesis. The chaperone in co-operation with its co-proteins acts as a translocation motor pulling the mitochondrial precurs or into the matrix. Mt-hsp70s are highly conserved when compared to the bac terial hsp70 homologue, DnaK. Here we have used DnaK as a model to study th e interaction of mitochondrial presequences with mt-hsp70 applying a DnaK-b inding algorithm, computer modeling and biochemical investigations. DnaK-bi nding motifs have been analysed on all available, statistically relevant mi tochondrial presequences found in the OWL database by running the algorithm . A total of 87% of mammalian, 97% of plant, 71% of yeast and 100% of Neuro spora crassa presequences had at least one DnaK binding site. Based on the prediction, five 13-mer presequence peptides have been synthesized and thei r inhibitory effect on the molecular chaperone (DnaK/DnaJ/GrpE) assisted re folding of luciferase has been analysed. The peptide with the highest predi cted binding likelihood showed the strongest inhibitory effect, whereas the peptide with no predicted binding capacity showed no inhibitory effect. A 3D structure of the pea mt-hsp70 has been construct-ed using homology model ing. The binding affinities of the 13-mer presequence peptides and addition al control peptides to DnaK and pea mt-hsp70 have been theoretically estima ted by calculating the buried hydrophobic surface area of the peptides dock ed to DnaK and to the mt-hsp70 structural model. These results suggest that mitochondrial presequences interact with the mt-hsp70 during or after mito chondrial protein import. (C) 1999 Academic Press.