酵母是用于研究蛋白质聚集的方便模型,因为已知其繁殖淀粉样蛋白病毒。[PSI+]是释放因子eRF3(Sup35)的朊病毒形式。聚合的Sup35导致翻译终止的缺陷,这导致携带过早终止密码子的菌株的无义抑制。Sup35的N-末端和中间(M)结构域对于维持细胞中的[PSI+]同时保持朊病毒株的性质是必要和充分的。出于这个原因,与荧光蛋白融合的Sup35NM通常用于[PSI+]检测和调查。然而,我们发现在这样的嵌合构建体中,并非所有的荧光蛋白都可以可靠地检测Sup35聚集体。特别是,Sup35NM-mCherry的瞬时过量产生导致[PSI+]细胞中的扩散荧光模式,同时没有观察到朊病毒的损失,也没有观察到对Sup35NM朊病毒特性的影响。这种效应在各种不相关的菌株背景和朊病毒变体中再现。相比之下,Sup35NM与另一种红色荧光蛋白融合,TagRFP-T,允许检测[PSI+]聚集体。蛋白质裂解物的分析表明Sup35NM-mCherry在细胞中被主动降解。这种降解不是由液泡蛋白酶和与Sup35加工有关的泛素-蛋白酶体系统引起的。尽管这种蛋白水解的强度高于Sup35NM-GFP,与Sup35NM-TagRFP-T的情况大致相同。因此,有可能,与TagRFP-T相比,Sup35NM-mCherry的降解产物仍然保留其荧光特性,同时失去装饰预先存在的Sup35聚集体的能力。尽管细胞中存在朊病毒聚集体,但这导致扩散荧光。因此,标记荧光蛋白应谨慎使用,这样的蛋白水解可能会增加检测携带朊病毒的细胞时的假阴性率。
Yeast is a convenient model for studying protein aggregation as it is known to propagate amyloid prions. [PSI+] is the prion form of the release factor eRF3 (
Sup35). Aggregated
Sup35 causes defects in termination of translation, which results in nonsense suppression in strains carrying premature stop codons. N-terminal and middle (M) domains of
Sup35 are necessary and sufficient for maintaining [PSI+] in cells while preserving the prion strain\'s properties. For this reason, Sup35NM fused to fluorescent proteins is often used for [PSI+] detection and investigation. However, we found that in such chimeric constructs, not all fluorescent proteins allow the reliable detection of
Sup35 aggregates. Particularly, transient overproduction of Sup35NM-mCherry resulted in a diffuse fluorescent pattern in the [PSI+] cells, while no loss of prions and no effect on the Sup35NM prion properties could be observed. This effect was reproduced in various unrelated strain backgrounds and prion variants. In contrast, Sup35NM fused to another red fluorescent protein, TagRFP-T, allowed the detection of [PSI+] aggregates. Analysis of protein lysates showed that Sup35NM-mCherry is actively degraded in the cell. This degradation was not caused by vacuolar proteases and the ubiquitin-proteasomal system implicated in the
Sup35 processing. Even though the intensity of this proteolysis was higher than that of Sup35NM-GFP, it was roughly the same as in the case of Sup35NM-TagRFP-T. Thus, it is possible that, in contrast to TagRFP-T, degradation products of Sup35NM-mCherry still preserve their fluorescent properties while losing the ability to decorate pre-existing Sup35 aggregates. This results in diffuse fluorescence despite the presence of the prion aggregates in the cell. Thus, tagging with fluorescent proteins should be used with caution, as such proteolysis may increase the rate of false-negative results when detecting prion-bearing cells.