α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast

Abstract

Parkinson’s disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.

Inhibition of 26S proteasome activity by α‐synuclein is mediated by the proteasomal chaperone Rpn14/PAAF1

“…Downregulation of the base proteasome subunit genes RPT2 , RPT4 , and RPT6 significantly enhances α‐syn toxicity. Furthermore, elevated levels of α‐syn increase the pool of ubiquitinated substrates upon downregulation of RPT2 (Popova, Galka, et al., 2021 ). Therefore, we next examined whether the impact of α‐syn is affected by different levels of Rpn14 in growth assays (Figure 3 ).…”

“…The interplay of Rpn14 and α‐syn was further studied under proteolytic stress conditions. α‐Synuclein significantly alters ubiquitin homeostasis (Popova, Galka, et al., 2021 ). It was examined whether the impact of α‐syn is affected by different protein levels of Rpn14.…”

“…Rpt2 is one of the AAA+ ATPase subunits of the 19S regulatory particle (RP), which is essential for substrate unfolding and translocation (Sakata et al., 2021 ). An α‐syn subpopulation is localized in proximity of Rpt2 (Popova, Galka, et al., 2021 ). α‐Synuclein may directly or indirectly interact with the 19S RP, which could interfere with the assembly with the 20S core particle to 26S proteasome.…”

“…Fluorescence measurements were performed after subtraction of the background fluorescence. Flow cytometry was performed as previously described (Popova, Galka, et al., 2021 ).…”

“…It enhances α‐syn degradation by the 26S proteasome as well as by autophagy (Shahpasandzadeh et al., 2014 ; Tenreiro et al., 2014 ). Quantitative cellular proteomics revealed that α‐syn expression significantly changes the yeast proteome, leading to a decrease in the abundance of multiple 26S proteasome subunits (Popova, Galka, et al., 2021 ). This effect correlates with α‐syn turnover including phosphorylation of α‐syn at S129.…”