Deposition of amyloid aggregates and terminally misfolded or damaged proteins at the cellular protein quality control site IPOD in yeast.

淀粉样蛋白的聚集与神经退行性疾病有关。越来越多的证据表明，较大的可见聚集沉积物通常是细胞保护性的，而不是细胞毒性的。因此，重要的是要确定淀粉样蛋白聚集体以及其他类型的聚集或受损蛋白质在细胞中被处理以抵消其有害特性的机制。我们使用酵母作为模型来研究淀粉样蛋白聚集体和被称为不溶性蛋白存款（IPOD）的末端聚集或受损蛋白的沉积位点。IPOD直接位于噬菌体组装位点（PAS）附近，在PAS中细胞启动自噬体和细胞质-液泡靶向（CVT）囊泡的形成。以前，对IPOD的底物募集机制、其分子组成或沉积在该位点的底物的全谱知之甚少。在前一个资助期，我们发现，淀粉样蛋白聚集体通过囊泡招募机制沿着肌动蛋白电缆招募到IPOD，并确定了该机制的核心组成部分。然而，包括将淀粉样蛋白聚集体与其连接的因子在内的机制的详细分子组成仍有待阐明。有趣的是，我们观察到这种机制与PAS的结构组分和底物的靶向机制共享。我们建议扩展我们以前的流式细胞术为基础的筛选方法和联合收割机结合它与候选人为基础的方法和一种新的基于FACS的方法分离IPOD从细胞裂解物，以确定IPOD的结构组成，并研究该沉积位点的招聘机制的全面分子组成，以及额外的内源性底物沉积在这里。接下来，我们将确定不同类别底物的共同性和差异。IPOD的这种特性应该允许我们操纵它，然后询问在IPOD处沉积不同的末端聚集或受损蛋白质和淀粉样蛋白聚集体的生理重要性。最后，我们将揭示沉积在IPOD上的不同基板的命运。

Aggregation of amyloidogenic proteins is associated with neurodegenerative diseases. Mounting evidence suggests that larger visible aggregate depositions are often cell protective rather than cytotoxic. It is therefore important to identify the mechanism(s) by which amyloid aggregates as well as other types of aggregated or damaged proteins, are handled in the cell to counteract their detrimental properties. We use yeast as a model to study the deposition site for amyloid aggregates and terminally aggregated or damaged proteins termed Insoluble PrOtein Deposit (IPOD). The IPOD is located directly adjacent to the Phagophore Assembly site (PAS) where the cell initiates the formation of autophagosomes and Cytoplasm-to-vacuole Targeting (CVT) vesicles. Previously, not much was known about the mechanism of substrate recruitment to the IPOD, its molecular composition or the full spectrum of substrates deposited at this site. In the previous funding period, we revealed that amyloid aggregates are recruited to the IPOD via a vesicular recruitment machinery along actin cables and identified core components of this machinery. However, the detailed molecular composition of the machinery including the factor(s) that link the amyloid aggregates to it remains to be elucidated. Intriguingly, we observed that this machinery is shared with the targeting machinery for structural components and substrates to the PAS. We propose to extend our previous flow cytometry-based screening approaches and combine it with candidate-based approaches and a novel FACS-based method for isolation of IPODs from cell lysates to identify structural components of the IPOD and to study the comprehensive molecular composition of the recruitment machinery for this deposition site, as well as additional endogenous substrates deposited here. Next, we will determine communalities and differences for the recruitment of the different classes of substrates. This characterization of the IPOD should allow us to manipulate it and then ask about the physiological importance of depositing different terminally aggregated or damaged proteins and amyloid aggregates at the IPOD. Finally, we will reveal what the fate of the different substrates deposited at the IPOD is.