The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species
The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species
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DOI：
10.1046/j.1432-1327.2000.01053.x
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发表时间：
2000-02-01
期刊：
影响因子：
--
通讯作者：
Kindl, H
中科院分区：
文献类型：
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作者：
Diefenbach, J;Kindl, H
DnaJ蛋白位于真核细胞的各个区室中。如前所述，过氧化物酶体和乙醛酸酶体具有位于胞质表面上的膜锚定形式的DnaJ蛋白。通过检查DnaJ蛋白和Hsp 70家族的各种潜在伴侣之间的亲和力，获得了关于膜结合的共伴侣如何与胞质可溶性伴侣相互作用的提示：从黄瓜子叶中分离并表征了编码胞质Hsp 70同种型的两个基因。此外，制备了编码归因于胞质、质体和内质网腔的Hsp 70形式的cDNA。构建His标记的DnaJ蛋白和谷胱甘肽S-转移酶-Hsp 70融合蛋白。使用这些工具，证明了DnaJ蛋白的可溶性His标记形式仅结合Hsp 70的胞质同种型1。通过表征DnaJ蛋白的乙醛酸结合形式与Hsp 70的各种亚型之间的相互作用，进一步分析了这种相互作用。仅在Hsp 70的胞质同种型1的情况下观察到与glyoxysomal表面的特异性结合。这种相互作用严格依赖于ADP的存在。乙醛酸酶体不结合其他胞质或质体亚型或：BiP相关形式的Hsp 70。分析胞浆Hsp 70的酶性质，我们表明，当测定亚型1时，DnaJ的ATP酶调节活性最高。总的来说，这些数据表明：配偶体的DnaJ蛋白锚定在glyoxysomal膜是热休克蛋白70的胞质异构体1。除了位于乙醛酸酶体表面的分子伴侣外，在乙醛酸酶体基质中还检测到两种Hsp 70亚型和一种可溶形式的DnaJ蛋白。
DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face, Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family: Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form: of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the: BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the:partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.