iAID: an improved auxin-inducible degron system for the construction of a ‘tight’ conditional mutant in the budding yeast<i>Saccharomyces cerevisiae</i>

Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hideki

doi:10.1002/yea.3080

Cited by 47 publications

(65 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, as previously reported for budding yeast (Tanaka, Miyazawa-Onami, Lida, & Araki, 2015), the presence of the NLS had little effect on depletion rate of a nuclear protein (Prp22). We speculate that TIR1 protein when N-terminally fused to an NLS may have reduced stability, but that this may result in similar amounts of nuclear localised TIR1 protein in the presence or absence of an NLS, such that the target protein depletion rates are similar.…”

Section: Discussionsupporting

confidence: 76%

A fast and tuneable auxin‐inducible degron for depletion of target proteins in budding yeast

Mendoza‐Ochoa

Barrass

Terlouw

et al. 2018

Yeast

View full text Add to dashboard Cite

The auxin‐inducible degron (AID) is a useful technique to rapidly deplete proteins of interest in nonplant eukaryotes. Depletion is achieved by addition of the plant hormone auxin to the cell culture, which allows the auxin‐binding receptor, TIR1, to target the AID‐tagged protein for degradation by the proteasome. Fast depletion of the target protein requires good expression of TIR1 protein, but as we show here, high levels of TIR1 may cause uncontrolled depletion of the target protein in the absence of auxin. To enable conditional expression of TIR1 to a high level when required, we regulated the expression of TIR1 using the β‐estradiol expression system. This is a fast‐acting gene induction system that does not cause secondary effects on yeast cell metabolism. We demonstrate that combining the AID and β‐estradiol systems results in a tightly controlled and fast auxin‐induced depletion of nuclear target proteins. Moreover, we show that depletion rate can be tuned by modulating the duration of β‐estradiol preincubation. We conclude that TIR1 protein is a rate‐limiting factor for target protein depletion in yeast, and we provide new tools that allow tightly controlled, tuneable, and efficient depletion of essential proteins whereas minimising secondary effects.

show abstract

Section: Discussionsupporting

confidence: 76%

A fast and tuneable auxin‐inducible degron for depletion of target proteins in budding yeast

Mendoza‐Ochoa

Barrass

Terlouw

et al. 2018

Yeast

View full text Add to dashboard Cite

show abstract

“…The TET-shp1-aid allele was constructed using a modified version of the auxin-inducible degron system (Nishimura et al., 2009, Tanaka et al., 2015). First, the tetracycline-repressible promotor was introduced at the 5′ end of the SHP1 gene in diploid cells expressing the rice Tir1 E3 ubiquitin ligase.…”

Section: Methodsmentioning

confidence: 99%

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

et al. 2017

View full text Add to dashboard Cite

SummaryDisassembly of the Cdc45-MCM-GINS (CMG) DNA helicase is the key regulated step during DNA replication termination in eukaryotes, involving ubiquitylation of the Mcm7 helicase subunit, leading to a disassembly process that requires the Cdc48 “segregase”. Here, we employ a screen to identify partners of budding yeast Cdc48 that are important for disassembly of ubiquitylated CMG helicase at the end of chromosome replication. We demonstrate that the ubiquitin-binding Ufd1-Npl4 complex recruits Cdc48 to ubiquitylated CMG. Ubiquitylation of CMG in yeast cell extracts is dependent upon lysine 29 of Mcm7, which is the only detectable site of ubiquitylation both in vitro and in vivo (though in vivo other sites can be modified when K29 is mutated). Mutation of K29 abrogates in vitro recruitment of Ufd1-Npl4-Cdc48 to the CMG helicase, supporting a model whereby Ufd1-Npl4 recruits Cdc48 to ubiquitylated CMG at the end of chromosome replication, thereby driving the disassembly reaction.

show abstract

“…For construction of the YPT6 ARL1 conditional double-knockout strain ( ypt6 Δ arl1 AID ), the TIR1 gene from Oryza sativa was integrated into the SSN6 locus of the ypt6 Δ strain by cutting the plasmid pST1868 67 with BbvC I and performing homologous recombination. To tag Arl1 with AID, the fragment of 3xmini AID-5xFLAG along with the KanMX gene was amplified from the plasmid pST1933 67 with the primers containing gene flanking regions of the C-terminal region of ARL1 .…”

Section: Methodsmentioning

confidence: 99%

“…To tag Arl1 with AID, the fragment of 3xmini AID-5xFLAG along with the KanMX gene was amplified from the plasmid pST1933 67 with the primers containing gene flanking regions of the C-terminal region of ARL1 . Homologous recombination was performed to integrate the fragment into the C terminus of ARL1 .…”

Section: Methodsmentioning

confidence: 99%

Autophagy inSaccharomyces cerevisiaerequires the monomeric GTP-binding proteins, Arl1 and Ypt6

Yang

Rosenwald

2016

Autophagy

View full text Add to dashboard Cite

Macroautophagy/autophagy is a cellular degradation process that sequesters organelles or proteins into a double-membrane structure called the phagophore; this transient compartment matures into an autophagosome, which then fuses with the lysosome or vacuole to allow hydrolysis of the cargo. Factors that control membrane traffic are also essential for each step of autophagy. Here we demonstrate that 2 monomeric GTP-binding proteins in Saccharomyces cerevisiae, Arl1 and Ypt6, which belong to the Arf/Arl/Sar protein family and the Rab family, respectively, and control endosome-trans-Golgi traffic, are also necessary for starvation-induced autophagy under high temperature stress. Using established autophagy-specific assays we found that cells lacking either ARL1 or YPT6, which exhibit synthetic lethality with one another, were unable to undergo autophagy at an elevated temperature, although autophagy proceeds normally at normal growth temperature; specifically, strains lacking one or the other of these genes are unable to construct the autophagosome because these 2 proteins are required for proper traffic of Atg9 to the phagophore assembly site (PAS) at the restrictive temperature. Using degron technology to construct an inducible arl1Δ ypt6Δ double mutant, we demonstrated that cells lacking both genes show defects in starvation-inducted autophagy at the permissive temperature. We also found Arl1 and Ypt6 participate in autophagy by targeting the Golgi-associated retrograde protein (GARP) complex to the PAS to regulate the anterograde trafficking of Atg9. Our data show that these 2 membrane traffic regulators have novel roles in autophagy.

show abstract

iAID: an improved auxin-inducible degron system for the construction of a ‘tight’ conditional mutant in the budding yeastSaccharomyces cerevisiae

Cited by 47 publications

References 27 publications

A fast and tuneable auxin‐inducible degron for depletion of target proteins in budding yeast

A fast and tuneable auxin‐inducible degron for depletion of target proteins in budding yeast

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

Autophagy inSaccharomyces cerevisiaerequires the monomeric GTP-binding proteins, Arl1 and Ypt6

Contact Info

Product

Resources

About