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

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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.