Parsell D A, Kowal A S, Lindquist S
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
J Biol Chem. 1994 Feb 11;269(6):4480-7.
Heat-shock proteins (hsps) function in a variety of ways to help cells and organisms cope with environmental changes. One class of hsps, the Hsp100 proteins, is especially important for tolerance to a variety of extremely stressful conditions (e.g. high temperatures or high concentrations of ethanol). To begin to characterize the mechanism of action of Hsp100 proteins, we have initiated an in vitro analysis of the Saccharomyces cerevisiae Hsp104 protein. Here, we report the purification and initial structural characterization of the wild-type protein and three variants carrying mutations in the two ATP-binding site consensus elements. As demonstrated by both gel filtration chromatography and by cross-linking studies with glutaraldehyde, Hsp104 forms a homohexameric particle. By electron microscopy, these particles are ring-shaped and reminiscent of proteins in the Hsp60 and TF55/TCP families. In contrast to these other proteins, Hsp104 forms single rings, each containing only six subunits. More strikingly, the assembly and maintenance of Hsp104 particles are dependent upon the presence of adenine nucleotides. Oligomerization appears to primarily depend upon the second of the two ATP-binding sites in the protein.
热休克蛋白（hsps）通过多种方式发挥作用，帮助细胞和生物体应对环境变化。其中一类热休克蛋白，即Hsp100蛋白，对于耐受多种极端应激条件（如高温或高浓度乙醇）尤为重要。为了开始阐明Hsp100蛋白的作用机制，我们启动了对酿酒酵母Hsp104蛋白的体外分析。在此，我们报告了野生型蛋白以及在两个ATP结合位点共有元件中携带突变的三个变体的纯化及初步结构表征。凝胶过滤色谱法和戊二醛交联研究均表明，Hsp104形成同六聚体颗粒。通过电子显微镜观察，这些颗粒呈环状，让人联想到Hsp60和TF55/TCP家族中的蛋白质。与这些其他蛋白质不同的是，Hsp104形成单环，每个环仅包含六个亚基。更引人注目的是，Hsp104颗粒的组装和维持依赖于腺嘌呤核苷酸的存在。寡聚化似乎主要取决于该蛋白两个ATP结合位点中的第二个。
