Endo T, Mitsui S, Nakai M, Roise D
Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan.
J Biol Chem. 1996 Feb 23;271(8):4161-7. doi: 10.1074/jbc.271.8.4161.
The interactions between a yeast cytosolic hsp70, Ssa1p, and various synthetic peptides, including mitochondrial presequences, have been studied. The interactions were monitored both indirectly, by measuring the effects of the presequences on the ATPase activity and oligomeric state of the enzyme, and directly, by measuring the increased steady-state fluorescence polarization of fluorescent derivatives of the presequences as they bind to Ssa1p. The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-dependent manner. The presequences are also able to stimulate the ATPase activity of the enzyme at similar concentrations. Quantification of the binding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. The most amphiphilic presequences, as measured by retention times on reversed-phase high pressure liquid chromatography or surface activity in lipid monolayers, had the highest affinity for Ssa1p. The least amphiphilic presequences, which had previously been shown to be ineffective as mitochondrial targeting sequences, had relatively low affinity for Ssa1p. The results show that Ssa1p interacts with a broad range of amino acid sequences and that the strength of these interactions is related to the physical properties of the sequence. That the physical properties recognized by Ssa1p are identical to those necessary for the targeting function of mitochondrial presequences suggests that Ssa1p may interact with mitochondrial precursor proteins in the cytosol. The interactions may serve a variety of purposes: the maintenance of precursors in translocation-competent forms, the prevention of improper association of precursors with non-mitochondrial membranes, and the delivery of precursors to the mitochondrial surface.
人们已经研究了酵母胞质热休克蛋白70（Ssa1p）与各种合成肽（包括线粒体前导序列）之间的相互作用。这些相互作用通过两种方式进行监测：间接监测，通过测量前导序列对该酶的ATP酶活性和寡聚状态的影响；直接监测，通过测量前导序列的荧光衍生物与Ssa1p结合时稳态荧光偏振的增加。前导序列均能够以浓度依赖的方式将Ssa1p从寡聚形式转变为单体形式。前导序列在相似浓度下也能够刺激该酶的ATP酶活性。通过荧光偏振对结合进行定量分析表明，对Ssa1p的亲和力与前导序列的物理性质直接相关。通过反相高压液相色谱上的保留时间或脂质单层中的表面活性测量，亲水性最强的前导序列对Ssa1p的亲和力最高。亲水性最弱的前导序列，先前已证明其作为线粒体靶向序列无效，对Ssa1p的亲和力相对较低。结果表明，Ssa1p与广泛的氨基酸序列相互作用，并且这些相互作用的强度与序列的物理性质相关。Ssa1p识别的物理性质与线粒体前导序列靶向功能所需的性质相同，这表明Ssa1p可能在细胞质中与线粒体前体蛋白相互作用。这些相互作用可能有多种目的：将前体维持在具有转运能力的形式，防止前体与非线粒体膜的不当结合，以及将前体递送至线粒体表面。
