The elongation of yeast prion fibers involves separable steps of association and conversion
The elongation of yeast prion fibers involves separable steps of association and conversion
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DOI：
10.1073/pnas.0308754101
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发表时间：
2004-02-24
影响因子：
11.1
通讯作者：
Lindquist, SL
中科院分区：
文献类型：
--
作者：
Scheibel, T;Bloom, J;Lindquist, SL
翻译终止因子 Sup35p 构象的自我延续变化是朊病毒 [PSI+] 的基础，朊病毒是酿酒酵母的一种基于蛋白质的遗传元件。在与体内转化密切相关的过程中，纯化的 Sup35p (NM) 可溶性朊病毒决定区通过有核构象转化转化为淀粉样蛋白纤维。首先，寡聚物转化为细胞核，然后这些细胞核促进可溶性蛋白质聚合成淀粉样蛋白纤维。为了阐明聚合步骤的性质，我们在 NM 的不同位置创建了单半胱氨酸取代突变体，为各种探针提供独特的附着位点。在体内，突变体在 [psi(-)] 和 [PSI+] 状态下都表现得像野生型蛋白质。在体外，它们以野生型动力学组装并形成具有相同形态的纤维。当用荧光探针标记时，两种突变体 NMT158C 和 NME167C 表现出与淀粉样蛋白组装一致的荧光变化。这些突变体为纤维伸长的动力学和转化的滞后阶段提供了灵敏的测量。突变体 NMK184C 中的半胱氨酸在组装后仍然暴露。当用生物素标记并与链霉亲和素珠结合时，它用于在转化过程中捕获放射性标记的可溶性 NM。该过程确定了纤维伸长过程中对洗涤剂敏感的中间体的存在。因此，有核构象转化的第二阶段，即纤维伸长，本身至少包含两个步骤：可溶性蛋白质与预形成的纤维缔合，形成组装中间体，然后构象转化为淀粉样蛋白。
A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of nucleated conformational conversion. First, oligomeric species convert to nuclei, and these nuclei then promote polymerization of soluble protein into amyloid fibers. To elucidate the nature of the polymerization step, we created single-cysteine substitution mutants at different positions in NM to provide unique attachment sites for various probes. In vivo, the mutants behaved like wild-type protein in both the [psi(-)] and [PSI+] states. In vitro, they assembled with wild-type kinetics and formed fibers with the same morphologies. When labeled with fluorescent probes, two mutants, NMT158C and NME167C, exhibited a change in fluorescence coincident with amyloid assembly. These mutants provided a sensitive measure for the kinetics of fiber elongation, and the lag phase in conversion. The cysteine in the mutant NMK184C remained exposed after assembly. When labeled with biotin and bound to streptavidin beads, it was used to capture radiolabeled soluble NM in the process of conversion. This process established the existence of a detergent-susceptible intermediate in fiber elongation. Thus, the second stage of nucleated conformational conversion, fiber elongation, itself contains at least two steps: the association of soluble protein with preformed fibers to form an assembly intermediate, followed by conformational conversion into amyloid.