High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers

Fung, Jia Jun; Blöcher-Juárez, Karla; Khmelinskii, Anton

doi:10.1007/978-1-0716-1732-8_6

Cited by 6 publications

(3 citation statements)

References 46 publications

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“…To better define the abundance and localization of Nsp1 we endogenously tagged Nsp1 with a fast-folding and bright mNeonGreen fluorescent protein 65 . Nsp1 indeed shows a punctate rim staining at the NE that is typical for nucleoporins, but also a cytosolic pool is observed (Figure 1C, Figure S2AB).…”

Section: Nsp1 Localizes In Three Distinct Pools and Decreases With Agingmentioning

confidence: 99%

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Otto

Bergsma

Dekker

et al. 2023

Preprint

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Transport through the NPC relies on intrinsically disordered FG-Nups forming a selective barrier. Away from the NPC, FG-Nups readily form condensates and aggregates, and we address how this behavior is surveilled in cells. FG-Nups, including Nsp1, together with nuclear transport receptor Kap95, form a native cytosolic condensate in yeast. In aged cells this condensate disappears as cytosolic Nsp1 levels decline. Biochemical assays and modeling show that Nsp1 is a modulator of FG-Nup liquid-liquid phase separation, promoting a liquid-like state. Nsp1s presence in the cytosol and condensates is critical, as a reduction of cytosolic levels in young cells induces NPC assembly and transport defects and a general decline in protein quality control, all quantitatively mimicking aging phenotypes. Excitingly, these phenotypes can be rescued by cytosolic Nsp1. We conclude that Nsp1 is a phase state regulator that surveils FG-Nups and impacts general protein homeostasis.

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Section: Nsp1 Localizes In Three Distinct Pools and Decreases With Agingmentioning

confidence: 99%

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Otto

Bergsma

Dekker

et al. 2023

Preprint

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“…It is possible that in some cases partial degradation results in low protein levels that are below the detection limit of our assay but are sufficient for viability. In addition, it is likely that the fraction of strains with the complete degradation phenotype is overestimated in our analysis as colony fluorescence usually scales with colony size, which would result in underestimated fluorescence levels for strains with fitness defects (Fung et al, 2022). Indeed, OsTIR1 + strains with impaired fitness typically exhibited fluorescence levels below background autofluorescence (Fig.…”

Section: Resultsmentioning

confidence: 99%

Genome-wide conditional degron libraries for functional genomics

Gameiro,

Juárez-Núñez,

Fung

et al. 2024

Preprint

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The yeast knockout library, comprised of strains carrying precise deletions of individual open reading frames (ORFs), has been a vital resource to understand gene function. However, accumulation of suppressor mutations in knockout strains can lead to erroneous functional annotations. Moreover, the library is limited to non-essential ORFs, and must be complemented with hypomorphic alleles of essential ORFs for genome-wide studies. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for conditional degradation of AID-tagged proteins. First, we determined that N-terminal tagging is at least twice more likely to inadvertently impair protein function across the proteome. We thus constructed two genome-wide libraries with over 5600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Almost 90% of AID-tagged proteins were completely or partially degraded in the presence of auxin, with protein abundance and tag accessibility as limiting factors. Genome-wide screens for DNA damage response factors with the AID libraries revealed a role for the glucose signaling factorGSF2in resistance to hydroxyurea, highlighting how these resources extend the toolbox for functional genomics in yeast.

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“…To assess this hypothesis, we analyzed the role of Das1 in turnover of yeast proteins that contain Das1 Cdegrons. We expressed each ORF N-terminally tagged with an mNeonGreen-mCherry timer 46,47 at endogenous levels or overexpressed from a strong constitutive promoter, which could lead to production of unnecessary (e.g., orphan subunits of protein complexes) or abnormal molecules (e.g., misfolded or mislocalized) 3,4,48,49 . When expressed at endogenous levels, none of the tested potential Das1 substrates exhibited Das1-dependent turnover (Fig.…”

Section: Endogenous Substrates and Functions Of Scf Das1mentioning

confidence: 99%

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

Kong

Shankar

Ruehle

et al. 2023

Preprint

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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, we performed an unbiased survey of C-degrons in budding yeast. We identified over 5000 potential C-degrons by stability profiling of random peptide libraries and of the yeast C‐terminome. Combining machine learning, high-throughput mutagenesis and genetic screens revealed 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 endogenous substrates, we implicate SCFDas1in degradation of orphan protein complex subunits. Altogether, this work provides a comprehensive view of a eukaryotic C-degronome and uncovers how an SCF/C-degron pathway of broad specificity contributes to proteostasis.

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High-Throughput Analysis of Protein Turnover with Tandem Fluorescent Protein Timers

Cited by 6 publications

References 46 publications

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Nucleoporin Nsp1 surveils the phase state of FG-Nups

Genome-wide conditional degron libraries for functional genomics

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

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