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Abstract

Cells have evolved elaborate protein quality control systems (PQS), which include molecular chaperones and proteolytic machineries. However, when the occurrence of misfolded proteins exceeds the PQS’s capacity, they accumulate and can form aggregates. More and more evidence suggests that the accumulation of misfolded protein species into specific spatially separated deposition sites is a cytoprotective response of the cell. The yeast S. cerevisiae has at least three different such protein quality control sites: the JUxtaNuclear Quality control (JUNQ)/IntraNuclear Quality control site (INQ) and the Cyto-Q harbours unstructured, amorphously misfolded proteins, while the perivacuolar Insoluble PrOtein Deposit (IPOD) has been initially described as a deposition site for amyloid aggregates. However, more recently it has been suggested that the IPOD may also harbour other types of substrates, such as oxidatively damaged proteins and inactive/damaged proteasomes or subunits thereof. Interestingly, many of these potential substrate classes can form high molecular weight aggregates or represent large protein complexes, respectively. Because the IPOD lies directly adjacent to the phagophore assembly site at the vacuole, it was hypothesized that the perivacuolar IPOD may represent a sorting center for aggregates and larger protein complexes destined for autophagic turnover.

This study focuses on the enrichment of IPODs visualized with the model substrate PrD-GFP under different conditions, including oxidative stress, to characterize other IPOD substrates through an unbiased mass spectrometry approach. This strategy identified several proteins that co-enriched with the IPOD, mainly after oxidative stress. Among these was Pyruvate decarboxylase 1 (Pdc1), a protein susceptible to carbonylation which has been previously hypothesized to be present at the IPOD after oxidative insult.

For a Pdc1-mCh fusion protein, it was observed that the number of cells which formed Pdc1-mCh foci was increased after different forms of oxidative stress such as H2O2 or menadione treatment. The majority of these stress-induced foci colocalized with PrD-GFP marked IPODs. Other proteins found enriched at the IPOD after oxidative stress include Enolase 2 (Eno2) and Glyceraldehyde-3-phosphate dehydrogenase isozyme 3 (Tdh3). Along these lines, by staining for carbonylated proteins it was found that the overall levels of carbonylated proteins co-enriching with IPODs were much higher after application of oxidative stress. This supports the hypothesis that aggregates of oxidatively damaged proteins are another substrate group for the IPOD.

Furthermore, it has been shown that aberrant stress granules transiently associate with the aggresome on their way to autophagic degradation in mammalian cells. I hypothesized that the IPOD may play a similar role to the aggresome in yeast in this regard and indeed, in a dCuz1 background that hinders proteasomal degradation of stress granules and makes them more persistent, a proportion of aberrant arsenite-induced stress granules marked by Pab1mCh colocalized with the IPOD after arsenite stress.