Narayanan Saravanakumar, Bösl Benjamin, Walter Stefan, Reif Bernd
Institut für Organische Chemie und Biochemie and Lehrstuhl für Biotechnologie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
Proc Natl Acad Sci U S A. 2003 Aug 5;100(16):9286-91. doi: 10.1073/pnas.1233535100. Epub 2003 Jul 22.
The [PSI+] determinant of Saccharomyces cerevisiae, consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [PSI+]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup355-26, results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup355-26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup355-26, whereas the intermediate and monomeric "release" forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup355-26. No interaction can be detected between Hsp104 and higher oligomeric states (>/=8) of Sup355-26. Taking into account the fact that Hsp104 is required for maintenance of [PSI+], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.
酿酒酵母的[PSI+]决定簇由胞质翻译终止因子Sup35组成，是一种朊病毒型遗传元件，可诱导可遗传的构象变化，并将Sup35蛋白转化为淀粉样纤维。分子伴侣Hsp104是维持[PSI+]自我复制所必需的。我们在体外观察到，向Sup35的NM结构域的朊病毒决定区域Sup355-26中添加催化量的Hsp104，会导致寡聚体Sup35解离为单体形式。在此过程中可以检测到Sup355-26的几种中间体。通过饱和转移差异和扩散排序核磁共振光谱实验监测发现，Hsp104与六聚体/四聚体Sup355-26之间存在强相互作用，而中间体和单体“释放”形式与Hsp104的亲和力降低。相互作用主要由Sup355-26中Gln、Asn和Tyr残基的侧链介导。未检测到Hsp104与Sup355-26的更高寡聚状态（≥8）之间存在相互作用。考虑到Hsp104是维持[PSI+]所必需的这一事实，我们认为Sup35的低寡聚体重量物种对酵母中朊病毒的传播很重要。
