BINDING OF MITOCHONDRIAL PRESEQUENCES TO YEAST CYTOSOLIC HEAT-SHOCK-PROTEIN-70 DEPENDS ON THE AMPHIPHILICITY OF THE PRESEQUENCE

The interactions between a yeast cytosolic hsp70, Ssa1p, and various s ynthetic peptides, including mitochondrial presequences, have been stu died, The interactions were monitored both indirectly, by measuring th e effects of the presequences on the ATPase activity and oligomeric st ate of the enzyme, and directly, by measuring the increased steady-sta te fluorescence polarization of fluorescent derivatives of the presequ ences as they bind to Ssa1p, The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-depend ent manner. The presequences are also able to stimulate the ATPase act ivity of the enzyme at similar concentrations, Quantification of the b inding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. T he most amphiphilic presequences, as measured by retention times on re versed-phase high pressure liquid chromatography or surface activity i n lipid monolayers, had the highest affinity for Ssa1p, The least amph iphilic presequences, which had previously been shown to be ineffectiv e as mitochondrial targeting sequences, had relatively low affinity fo r Ssa1p, The results show that Ssa1p interacts with a broad range of a mino acid sequences and that the strength of these interactions is rel ated to the physical properties of the sequence, That the physical pro perties recognized by Ssa1p are identical to those necessary for the t argeting function of mitochondrial presequences suggests that Ssa1p ma y interact with mitochondrial precursor proteins in the cytosol, The i nteractions may serve a variety of purposes: the maintenance of precur sors in translocation-competent forms, the prevention of improper asso ciation of precursors with non-mitochondrial membranes, and the delive ry of precursors to the mitochondrial surface.