Building yeast libraries to dissect terminal degrons with fluorescent timers

Kong, Ka-Yiu Edwin; Reinbold, Christian; Knop, Michael; Khmelinskii, Anton

doi:10.1016/bs.mie.2023.02.012

Cited by 2 publications

(2 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Highthroughput N-terminal tagging was performed with the SWAp-Tag approach using the N-SWAT library 45,63 and the donor plasmids pKEK431, pKEK432, pKEK433 and pKEK434 (Table S8). Unless stated otherwise, yeast strains were grown at 30℃ in synthetic complete (SC) medium lacking histidine to select for constructs based on the C-degron reporter plasmid pNS002 64 and with 2% (w/v) glucose as carbon source.…”

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

“…The C-degron reporter was constructed with a tFT designed specifically for studying C-degrons, in which the slower maturing mCherry is proximal and the faster maturing sfGFP is distal to the degron, such that incomplete proteasomal degradation of sfGFP does not affect the tFT readout 46,64 . tFT-peptide libraries were constructed by homologous recombination in yeast using degenerate oligonucleotides or oligonucleotide pools 26,64 . Degenerate oligonucleotides were designed with 42 nucleotide overhangs, homologous to the insertion site in the vector pNS002, flanking the variable region.…”

Section: Multiplexed Protein Stability Profilingmentioning

confidence: 99%

See 1 more Smart Citation

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

Kong

Shankar

Ruehle

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Yeast Strains and Growth Conditionsmentioning

confidence: 99%

Section: Multiplexed Protein Stability Profilingmentioning

confidence: 99%

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

Kong

Shankar

Ruehle

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

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

Kong,

Shankar,

Rühle

et al. 2023

Nat Commun

View full text Add to dashboard Cite

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 C‑terminome. 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.

show abstract

Building yeast libraries to dissect terminal degrons with fluorescent timers

Cited by 2 publications

References 26 publications

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

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

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

Contact Info

Product

Resources

About