Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.

Quality control of mislocalized and orphan proteins.

A healthy and functional proteome is essential to cell physiology. However, this is constantly being challenged as most steps of protein metabolism are error-prone and changes in the physico-chemical environment can affect protein structure and function, thereby disrupting proteome homeostasis. Among a variety of potential mistakes, proteins can be targeted to incorrect compartments or subunits of protein complexes may fail to assemble properly with their partners, resulting in the formation of mislocalized and orphan proteins, respectively. Quality control systems are in place to handle these aberrant proteins, and to minimize their detrimental impact on cellular functions. Here, we discuss recent findings on quality control mechanisms handling mislocalized and orphan proteins. We highlight common principles involved in their recognition and summarize how accumulation of these aberrant molecules is associated with aging and disease.

Post-transcriptional negative feedback regulation of proteostasis through the Dis3 ribonuclease and its disruption by polyQ-expanded Huntingtin.

When proteostasis is disrupted by stresses such as heat shock, the heat stress response will be stimulated, leading to up-regulation of molecular chaperones by transcriptional activation and mRNA stabilization for restoring proteostasis. Although the mechanisms for their transcriptional activation have been clearly defined, how chaperone mRNAs are stabilized remains largely unknown. Starting by exploring the coupling between the apparently unrelated RNA degradation and protein quality control (PQC) systems, we show that the Dis3 ribonuclease, catalytic subunit of the RNA exosome required for RNA degradation, suppresses PQC activity in unstressed cells by degrading mRNAs encoding the Hsp70 cofactors Sis1, Ydj1 and Fes1, as well as some other chaperones or PQC factors, thereby limiting their protein expression. Dis3 is stabilized through its binding to Sis1 and the Hsp70s Ssa1/2. Upon heat stress, loss of Sis1 and Ssa1/2 availability triggers Dis3 ubiquitination and degradation, leading to stabilization of those chaperone mRNAs originally targeted by Dis3. We further demonstrate that polyQ-expanded huntingtin delays Dis3 degradation during heat stress and thus hinders chaperone mRNA stabilization. Our findings not only reveal a post-transcriptional negative feedback loop for maintaining proteostasis, but also uncover a mechanism that contributes to the impaired heat stress response in Huntington's disease.

Cotranscriptional recruitment of yeast TRAMP complex to intronic sequences promotes optimal pre-mRNA splicing.

Most unwanted RNA transcripts in the nucleus of eukaryotic cells, such as splicing-defective pre-mRNAs and spliced-out introns, are rapidly degraded by the nuclear exosome. In budding yeast, a number of these unwanted RNA transcripts, including spliced-out introns, are first recognized by the nuclear exosome cofactor Trf4/5p-Air1/2p-Mtr4p polyadenylation (TRAMP) complex before subsequent nuclear-exosome-mediated degradation. However, it remains unclear when spliced-out introns are recognized by TRAMP, and whether TRAMP may have any potential roles in pre-mRNA splicing. Here, we demonstrated that TRAMP is cotranscriptionally recruited to nascent RNA transcripts, with particular enrichment at intronic sequences. Deletion of TRAMP components led to further accumulation of unspliced pre-mRNAs even in a yeast strain defective in nuclear exosome activity, suggesting a novel stimulatory role of TRAMP in splicing. We also uncovered new genetic and physical interactions between TRAMP and several splicing factors, and further showed that TRAMP is required for optimal recruitment of the splicing factor Msl5p. Our study provided the first evidence that TRAMP facilitates pre-mRNA splicing, and we interpreted this as a fail-safe mechanism to ensure the cotranscriptional recruitment of TRAMP before or during splicing to prepare for the subsequent targeting of spliced-out introns to rapid degradation by the nuclear exosome.

Building yeast libraries to dissect terminal degrons with fluorescent timers.

Selective degradation of unnecessary or abnormal proteins by the ubiquitin-proteasome system is an essential part of proteostasis. Ubiquitin ligases recognize substrates of selective protein degradation and modify them with polyubiquitin chains, which mark them for proteasomal degradation. Substrate recognition by ubiquitin ligases often involves degradation signals or degrons, which are typically short linear motifs found in intrinsically disordered regions, e.g., at protein termini. However, specificity in selective protein degradation is generally not well understood, as for most ubiquitin ligases no degrons have been identified thus far. To address this limitation, high-throughput mutagenesis approaches, such as multiplexed protein stability (MPS) profiling, have been developed, enabling systematic surveys of degrons in vivo or allowing to define degron motifs recognized by different ubiquitin ligases. In MPS profiling, thousands of short peptides can be assessed in parallel for their ability to trigger degradation of a fluorescent timer reporter. Here, we describe common types of libraries used to identify and dissect degrons located at protein termini using MPS profiling in budding yeast, and provide protocols for their construction.

Multiplexed protein stability (MPS) profiling of terminal degrons using fluorescent timer libraries in Saccharomyces cerevisiae.

N-terminal protein sequences and their proteolytic processing and modifications influence the stability and turnover of proteins by creating potential degrons for cellular proteolytic pathways. Understanding the impact of genetic perturbations of components affecting the processing of protein N-termini and thereby their stability, requires methods compatible with proteome-wide studies of many N-termini simultaneously. Tandem fluorescent timers (tFT) allow the in vivo measurement of protein turnover completely independent of protein abundance and can be deployed for proteome-wide studies. Here we present a protocol for Multiplexed Protein Stability (MPS) profiling of tFT-libraries encoding large numbers of different protein N-termini fused to tFT in the yeast Saccharomyces cerevisiae. This protocol includes fluorescence cell sorting based profiling of these libraries using a pooling approach. Analysis of the sorted pools is done by using multiplexed deep sequencing, in order to generate a stability index for each N-terminally peptide fused to the tFT reporter, and to evaluate half-life changes across all species represented in the library.

Orphan quality control by an SCF ubiquitin ligase directed to pervasive C-degrons.

Selective protein degradation typically involves substrate recognition via short linear motifs known as degrons. Various degrons can be found at protein termini from bacteria to mammals. While N-degrons have been extensively studied, our understanding of C-degrons is still limited. Towards a comprehensive understanding of eukaryotic C-degron pathways, here we perform an unbiased survey of C-degrons in budding yeast. We identify over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C­terminome. Combining machine learning, high-throughput mutagenesis and genetic screens reveals that the SCF ubiquitin ligase targets ~40% of degrons using a single F-box substrate receptor Das1. Although sequence-specific, Das1 is highly promiscuous, recognizing a variety of C-degron motifs. By screening for full-length substrates, we implicate SCFDas1 in degradation of orphan protein complex subunits. Altogether, this work highlights the variety of C-degron pathways in eukaryotes and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.

Yeast arginine methyltransferase Hmt1p regulates transcription elongation and termination by methylating Npl3p.

The heterogeneous nuclear ribonucleoprotein Npl3p of budding yeast is a substrate of arginine methyltransferase Hmt1p, but the role of Hmt1p in regulating Npl3p's functions in transcription antitermination and elongation were unknown. We found that mutants lacking Hmt1p methyltransferase activity exhibit reduced recruitment of Npl3p, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-Delta56 terminator. Remarkably, hmt1Delta cells also exhibit diminished recruitment of elongation factor Tho2p and a reduced rate of transcription elongation in vivo. Importantly, the defects in Npl3p and Tho2p recruitment, antitermination and elongation in hmt1Delta cells all were mitigated by substitutions in Npl3p RGG repeats that functionally mimic arginine methylation by Hmt1p. Thus, Hmt1p promotes elongation and suppresses termination at cryptic terminators by methylating RGG repeats in Npl3p. As Hmt1p stimulates dissociation of Tho2p from an Npl3p-mRNP complex, it could act to recycle these elongation and antitermination factors back to sites of ongoing transcription.

Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD+-dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV.

Loss of APD1 in yeast confers hydroxyurea sensitivity suppressed by Yap1p transcription factor.

Ferredoxins are iron-sulfur proteins that play important roles in electron transport and redox homeostasis. Yeast Apd1p is a novel member of the family of thioredoxin-like ferredoxins. In this study, we characterized the hydroxyurea (HU)-hypersensitive phenotype of apd1Δ cells. HU is an inhibitor of DNA synthesis, a cellular stressor and an anticancer agent. Although the loss of APD1 did not influence cell proliferation or cell cycle progression, it resulted in HU sensitivity. This sensitivity was reverted in the presence of antioxidant N-acetyl-cysteine, implicating a role for intracellular redox. Mutation of the iron-binding motifs in Apd1p abrogated its ability to rescue HU sensitivity in apd1Δ cells. The iron-binding activity of Apd1p was verified by a color assay. By mass spectrometry two irons were found to be incorporated into one Apd1p protein molecule. Surprisingly, ribonucleotide reductase genes were not induced in apd1Δ cells and the HU sensitivity was unaffected when dNTP production was boosted. A suppressor screen was performed and the expression of stress-regulated transcription factor Yap1p was found to effectively rescue the HU sensitivity in apd1Δ cells. Taken together, our work identified Apd1p as a new ferredoxin which serves critical roles in cellular defense against HU.