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，较大的可见聚集体沉积通常是细胞保护而不是细胞毒性。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(S)。我们使用酵母作为模型来研究淀粉样聚集体和终末聚集或损伤的蛋白质的沉积位置，称为不溶蛋白沉积(IPOD)。IPod位于噬菌体组装位点(PAS)附近，细胞在这里启动自噬小体和细胞质到空泡靶向(CVT)囊泡的形成。此前，人们对iPod的底物募集机制、其分子组成或沉积在该位置的底物的全谱知之甚少。在之前的融资期间，我们揭示了淀粉样蛋白聚集体是通过肌动蛋白电缆上的囊泡募集机制招募到iPod上的，并确定了该机制的核心组件。然而，这一机制的详细分子组成，包括将淀粉样蛋白聚集体与其联系起来的因子(S)，仍有待阐明。耐人寻味的是，我们观察到这种机械与PAS的结构部件和基材的定位机械是相同的。我们建议扩展我们以前基于流式细胞仪的筛选方法，并将其与基于候选者的方法和基于FACS的新方法相结合，从细胞裂解物中分离iPod，以确定iPod的结构成分，并研究该沉积位置的招募机制的全面分子组成，以及在此沉积的额外内源底物。下一步，我们将确定不同类别基质招募的共同性和差异性。IPod的这种特征应该允许我们操纵它，然后询问在iPod中沉积不同的末端聚集或受损的蛋白质和淀粉样聚集体的生理重要性。最后，我们将揭示沉积在iPod上的不同基片的命运是什么。

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.