Introduction
The prion-folding states orchestrated by chaperone systems in protein homeostasis are closely related to prion diseases, such as transmissible spongiform encephalopathy (TSE) [1-3]. Previous studies have revealed that prion transmission is a universal phenomenon, which shows intriguingly similar self-assembly behavior and heredity stability fromE. coli and yeast to mammals [4-6].However, cells in different organisms exhibit distinct responses and regulatory mechanisms for prion assembly [7]. Investigations on the heterologous expression of a prion protein in a foreign host should allow one to gain insight into the prion self-assembly mechanism.
S. cerevisiae Sup35 protein is an ideal model for examining prion self-assembly and genetic stability from parental cells to filial cells. In its normal monomer state, Sup35 serves as a translation termination release factor, dictated by the C-terminal [8,9]. In contrast, the N-terminal, comprising relatively conserved amino acids, serves as the core functional domain for amyloid formation [10]. Sup35-NM, which was constructed using the N-terminal and middle (M) domain, is typically chosen for in vitro self-assembly studies. Transforming in vitro -prepared amyloid fibrils into non-prion state yeast cells can effectively induce prion state phenotypes, and these phenotypes possess genetic stability from one generation to the next [11,12].Meanwhile, Sup35 overexpression in yeast causes the prion state phenotype [PSI +] [13], which limits the investigation of prion transmission in the in vivo state by homologous overexpression. HSP104 has been suggested as a contributing factor in the development of prion states in [PSI +] [14,15], but the process by which exhaustive genetic and self-assembly stability is maintained in yeast cells has remained unclear.
Many conclusions about the in vitro prion self-assembly mechanism have been drawn from prokaryotic purified proteins [16-18]. Examinations of Sup35 heterologous expression have primarily focused on two aspects. For one, GPI-anchored Sup35 in mammalian cells can form aggregates, which are different from the fibrils assembled in vitro [19,20]. This suggests that heterologous expression is feasible and can contribute to the understanding of the prion self-assembly mechanism. In addition, studies examining Sup35 and Sup35-NM self-assembly have been conducted in E. coli . Sup35-NM fusion expression with green fluorescent protein in E. colishowed bright foci and amyloid-like structures [21]. Several lines of evidence have shown that overexpressed Sup35 could be purified in soluble states in E. coli [22,23]. In contrast, Sup35 from yeast formed high molecular weight aggregates. These findings suggest that Sup35 may exist in the cytoplasm of E. coli cells as monomers. The cellular components responsible for maintaining the monomer state for Sup35 in heterologous E. coli are not fully understood. Furthermore, whether Sup35 monomer constitutes the sole surviving state in E. coli remains unclear. Answering these questions will provide a solid foundation for achieving prion self-assembly in a controlled fashion and can offer deeper insight into prion in vivo heredity and infectivity.