Mapping quality control systems for mislocalized proteins
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Abstract
The functional state of a cell is ultimately determined by the state of its proteome. Maintaining a balanced proteome requires not only correct protein synthesis and folding, but also proper trafficking of proteins to different organelles and assembly of multi-protein complexes with precise stoichiometry. Proteins that fail to reach their native compartments or assemble into native complexes are defined as mislocalized proteins. Protein mislocalization is a constitutive problem caused by intrinsic inefficiencies in cellular processes and increases with aging. Aberrant accumulation of mislocalized proteins brings harmful effects on normal cellular functions and is associated with a wide range of diseases, such as neurodegenerative diseases and some types of cancer. Therefore, eukaryotic cells have evolved protein quality control systems to prevent the abnormal accumulation of mislocalized proteins. Consequently, due to their low abundance, detecting mislocalized proteins that are subject to quality control can be challenging under non-perturbed conditions.
Here we sought to address this issue in the budding yeast Saccharomyces cerevisiae. By modeling protein mislocalization through overexpression of individual proteins combined with the tandem fluorescent protein timer assay, we identified about 15% of the proteome that is attenuated specifically upon overexpression. This set of attenuated proteins is strongly enriched for subunits of protein complexes. Extensive validation experiments proved the reproducibility of the high-throughput screening results. Moreover, cell culture conditions and the large size of the C-terminal tFT tag did not widely affect the protein attenuation phenotypes.
Protein abundance can be reduced by either deceleration of protein synthesis or acceleration of protein degradation. Taking advantage of the tFT readout, whereby the redFP/greenFP fluorescence ratio reports on protein degradation kinetics, we found that at least 43.5% of attenuated proteins are also destabilized. Further experiments on several randomly chosen destabilized proteins showed that they are mainly targeted for degradation by the ubiquitin-proteasome system. In order to know which E3 ligases mediate their degradation, we tested how protein abundance is affected by mutations in key E3s involved in protein quality control. Our results show that 20% of attenuated and destabilized proteins accumulate in the ubr1Δ, san1Δ or tom1Δ mutants, suggesting these E3s have important roles in elimination of mislocalized proteins.
This work provides a systematic survey of mislocalized proteins that are subject to quality control and suggests Ubr1, San1, and Tom1 as key quality control factors responsible for removal of mislocalized proteins. Our work paves the way for further dissecting features and mechanisms involved in the recognition of mislocalized proteins by quality control machinery.