Deposition of amyloid aggregates and terminally misfolded or damaged proteins at the cellular protein quality control site IPOD in yeast.
Cell Biology
Final Report Abstract
The appearance of protein aggregates is a hallmark of several pathologies including many neurodegenerative diseases. Mounting evidence suggests that the sequestration of misfolded proteins into specialized aggregate deposition sites has a cell protective function. We studied the yeast Insoluble PrOtein Deposit (IPOD) as a model system for ordered deposition of amyloid aggregates. In particular, we focused on three major aims: 1) Revealing the molecular composition of the recruitment machinery for amyloid aggregates to the IPOD; 2) Identification and characterization of additional substrates targeted to the IPOD including oxidatively damaged proteins; 3) Determination of the fate of proteins deposited at the IPOD. While me made good progress in characterizing the recruitment machinery for amyloid aggregates to the IPOD and in studying the fate of amyloid aggregates deposited there, our studies on additional substrate classes deposited at the IPOD were limited due to technical difficulties to generate robust model systems for the other potential substrate classes. Nonetheless, we were able to further support our hypothesis that oxidatively damaged proteins are also deposited at the IPOD. To study our first aim, we used various different experimental approaches. Basis of these approaches were GFP fusions of different model substrates known to aggregate and to be deposited at the IPOD. This allowed us to visualize the IPOD as a single fluorescent focus located adjacent to vacuole. Impairment of the faithful recruitment to the IPOD, e.g. by genetic manipulations of the cells, became visible as multiple small fluorescent foci of our model substrates. Aggregation patterns of our model substrates were analyzed by fluorescence microscopy as well as by a newly developed method employing flow cytometry. A high throughput screen combined with flow cytometry using the yeast deletion library as well as a pull-up assay with recombinant amyloid fibers to identify amyloid binding proteins followed my mass spectrometry identified several proteins that were crucial for IPOD targeting of amyloid aggregates. These proteins were related to actin-based transport processes as well as vesicular transport pathways. Co-localization studies with candidate proteins as well as time lapse microscopy further confirmed their involvement in IPOD targeting of amyloid aggregates. These findings led to the hypothesis that amyloid aggregates are associated with vesicular structures of yet unknown nature that traverse along the actin cytoskeleton to the IPOD. The IPOD is known to be located adjacent to the Phagophore Assembly Site (PAS). Therefore, with regard to our third aim, we tested whether the deposition of amyloid aggregates at the PAS is related to autophagic turn-over of aggregates. Using different cellular assays, we could not find any hint for autophagic turnover of the used amyloid aggregate substrates. Instead, we observed that amyloid aggregates deposited at the IPOD are subjected to slow liberation by the molecular chaperone Hsp104, followed by turn-over that was partially inhibited by inhibitors of the proteasome.
Publications
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(See online at https://doi.org/10.1080/19336896.2017.1293226) - The Insoluble Protein Deposit (IPOD) in Yeast. Front Mol Neurosci. 2018 Jul 12;11:237
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(See online at https://doi.org/10.3389/fnmol.2018.00237)
