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Recognition and Targeting of Prion Aggregates to the Insoluble Protein Deposit (IPOD) in Saccharomyces cerevisiae

Kumar, Rajesh

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Abstract

Protein quality control is a well-organized cellular process in which potentially toxic misfolded proteins are either refolded back to their native state, degraded or deposited into special deposition sites. Sequestration of misfolded protein species into specific deposition sites occurs in all kingdoms of life and serves as a second line of defense when refolding or degradation machineries that normally deal with these misfolded proteins are overwhelmed. In Saccharomyces cerevisae, three major subcellular sequestration sites have been described for deposition of different protein aggregates: INQ (intranuclear quality control compartment)/JUNQ (juxtanuclear quality control compartment), IPOD (insoluble protein deposit) and CytoQ. Amorphously aggregating proteins are targeted either to the INQ/JUNQ by the nuclear sorting factor Btn2, or they are targeted to a peripheral deposition site termed as Cyto Q with the aid of the cytosolic small heat shock protein Hsp42.

Amyloidogenic aggregates including yeast prions are predominantly sequestered at the IPOD, a perivacuolar deposition site. The perivacuolar IPOD is located in close proximity to the PAS (Phagophore Assembly Site) where the cells initiate formation of autophagosomes and CVT (Cytoplasm-to-Vacuole Targeting) vesicles. The cellular machinery, however, by which amyloid aggregates are recognized and deposited at the IPOD is still unknown.

Using a fishing approach with immobilized prion fibers formed by the prion domain of Sup35 (PrD), I identified components of actin cable-based and SNARE-mediated vesicular transport machinery to bind to PrD fibers. Using an auxin-based depletion system, I show that proper recruitment of the model prion amyloid PrD-GFP to the IPOD and the CVT substrate preApe1 to the PAS is disrupted upon depletion of essential components of the actin-based transport machinery, Myo2, Cmd1 and Tpm1/2, as well as Sec18-mediated SNARE function. Interestingly, the IPOD substrate PrD-GFP and the two PAS markers preApe1 and Atg8 accumulate reversibly in the cytosol in these mutants. Using fluorescence microscopy, I observed that PrD-GFP aggregates are associated with Atg9 transport vesicles similar to preApe1 and are targeted to the IPOD through these vesicles along actin cables. In addition, these PrD-GFP aggregates are shown to interact with Myo2 in vivo upon disruption of Sec18 SNARE function.

In a next approach, I investigated the possible fate of PrD-GFP aggregates deposited at the IPOD, that is located adjacent to the site in the cell where autophagosomes/CVT vesicles are formed. I demonstrate that PrD-GFP aggregates are not turned over in bulk via autophagy, but can be degraded by proteasomal and other unknown cellular degradation pathway(s) only after their slow and progressive extraction by the disaggregase Hsp104 from the IPOD. Thus accumulation of PrDGFP amyloids at the IPOD might serve a temporary storage function when downstream cellular degradation systems are overwhelmed. Based on the above findings, a model was proposed where PrD-GFP and preApe1 use an Atg9-vesicular transport machinery and Myo2 as a linking factor to be deposited at their recruitment sites IPOD and PAS, respectively, along tropomyosin-coated actin cables.

Item Type: Dissertation
Supervisor: Bukau, Prof. Dr. Bernd
Place of Publication: Heidelberg
Date of thesis defense: 16 December 2016
Date Deposited: 02 Jan 2017 14:22
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
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