Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.

许多可以组装成称为淀粉样蛋白的高级结构的蛋白质也可以通过液-液相分离浓缩成细胞质内含物。在这里，我们研究了人类高尔基体相关植物发病机制相关蛋白 1 (GAPR-1)、富含半胱氨酸的分泌蛋白抗原 5 和发病机制相关蛋白 1 蛋白 (CAP) 蛋白超家族的淀粉样蛋白，组装到细胞溶质中。酿酒酵母中的内含物. GAPR-1-GFP 的过度表达导致酵母细胞质中形成 GAPR-1 寡聚体和荧光包涵体。这些胞质内含物是动态和可逆的细胞器，在过度表达期间逐渐增加，在启动子关闭后逐渐减少。然而，内含物的形成是一个受调节的过程，受蛋白质表达水平以外的因素影响。我们将 GAPR-1 的 N-肉豆蔻酰化确定为包裹体形成早期的重要决定因素。此外，保守的金属结合位点（His54 和 His103）的突变增强了包涵体的形成，表明这些残基防止了不受控制的蛋白质螯合。与此一致，我们发现添加 Zn ²⁺金属离子增强了夹杂物的形成。此外，Zn ²⁺减少 GAPR-1 蛋白质降解，这表明 GAPR-1 在夹杂物中的稳定性。我们提出，淀粉样蛋白生成特性和 GAPR-1 可逆螯合到包裹体中的特性在 GAPR-1 和其他 CAP 家族成员的生物学功能中发挥作用。