An auxin-based degron system for the rapid depletion of proteins in nonplant cells

Nishimura, Kohei; Fukagawa, Tatsuo; Takisawa, Haruhiko; Kakimoto, Tatsuo; Kanemaki, Masato T.

doi:10.1038/nmeth.1401

Cited by 1,536 publications

(1,679 citation statements)

References 35 publications

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“…Northern blot analyses of two well‐characterized NNS‐targets, snR47 and snR13, in a Sen1‐AID (auxin‐induced degron, Nishimura et al , 2009) strain carrying a plasmid (pFL39) expressing either the wt or a ΔLP version of SEN1 . A strain harboring an empty vector was included as a positive control for termination defects.…”

Section: Resultsmentioning

confidence: 99%

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

et al. 2017

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The superfamily 1B (SF1B) helicase Sen1 is an essential protein that plays a key role in the termination of non‐coding transcription in yeast. Here, we identified the ~90 kDa helicase core of Saccharomyces cerevisiae Sen1 as sufficient for transcription termination in vitro and determined the corresponding structure at 1.8 Å resolution. In addition to the catalytic and auxiliary subdomains characteristic of the SF1B family, Sen1 has a distinct and evolutionarily conserved structural feature that “braces” the helicase core. Comparative structural analyses indicate that the “brace” is essential in shaping a favorable conformation for RNA binding and unwinding. We also show that subdomain 1C (the “prong”) is an essential element for 5′‐3′ unwinding and for Sen1‐mediated transcription termination in vitro. Finally, yeast Sen1 mutant proteins mimicking the disease forms of the human orthologue, senataxin, show lower capacity of RNA unwinding and impairment of transcription termination in vitro. The combined biochemical and structural data thus provide a molecular model for the specificity of Sen1 in transcription termination and more generally for the unwinding mechanism of 5′‐3′ helicases.

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Section: Resultsmentioning

confidence: 99%

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

et al. 2017

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“…To test whether this process contributed to the cohesin landscape on URA3, cohesin loading was blocked conditionally by depleting Scc2, a subunit of the cohesin loading complex. The protein was fused to an auxin-inducible degron (AID) in strains expressing the SCF OsTir1 , an alternative F box protein required for SCF-mediated degradation (41). Addition of the synthetic auxin analog 1-naphthaleneacetic acid (NAA) blocked colony formation of a strain expressing both Scc2-AID and OsTir1 and caused clearance of the Scc2-AID protein in 60 min (Fig.…”

Section: Resultsmentioning

confidence: 99%

Binding, sliding, and function of cohesin during transcriptional activation

Borrie

Campor

Joshi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The ring-shaped cohesin complex orchestrates long-range DNA interactions to mediate sister chromatid cohesion and other aspects of chromosome structure and function. In the yeast Saccharomyces cerevisiae, the complex binds discrete sites along chromosomes, including positions within and around genes. Transcriptional activity redistributes the complex to the 3′ ends of convergently oriented gene pairs. Despite the wealth of information about where cohesin binds, little is known about cohesion at individual chromosomal binding sites and how transcription affects cohesion when cohesin complexes redistribute. In this study, we generated extrachromosomal DNA circles to study cohesion in response to transcriptional induction of a model gene, URA3. Functional cohesin complexes loaded onto the locus via a poly(dA:dT) tract in the gene promoter and mediated cohesion before induction. Upon transcription, the fate of these complexes depended on whether the DNA was circular or not. When gene activation occurred before DNA circularization, cohesion was lost. When activation occurred after DNA circularization, cohesion persisted. The presence of a convergently oriented gene also prevented transcription-driven loss of functional cohesin complexes, at least in M phase-arrested cells. The results are consistent with cohesin binding chromatin in a topological embrace and with transcription mobilizing functional complexes by sliding them along DNA.T he protein complex known as cohesin organizes eukaryotic genomes into structures that segregate faithfully between dividing cells. The complex was first discovered for its role in mediating sister chromatid cohesion, but it is now known to participate in numerous aspects of chromosome biology (1, 2). Cohesin is clinically relevant because mutations in subunits of the complex or in factors that load and activate the complex lead to developmental disorders such as Cornelia de Lange syndrome, Roberts syndrome, and Warsaw breakage syndrome (3, 4).Cohesin is a ring-shaped complex composed of four subunits named Smc1, Smc3, Scc3, and Mcd1/Scc1 in budding yeast. The complex is thought to embrace DNA topologically with chromatin fibers passing through a central open channel (5-7). A substantial body of work supports a model in which cohesion arises from single cohesin complexes that embrace both sister chromatids [the double embrace model (6)]. Whether the central channel is large enough to accommodate both chromatids has recently been brought into question (8). In addition, the behavior of cohesin has not always been consistent with a double embrace by single complexes (for examples, see refs. 9-11). These situations usually involve a perturbation that causes loss of cohesion but not a loss of cohesin binding from chromatin. Such results force consideration of alternative models in which cohesin complexes embrace only one chromatid topologically and interact with other cohesin complexes or chromatin features to mediate cohesion (9,11,12).Cohesin binds densely in centromeric regions of chromos...

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“…pFA6a-natMX6-PADH-3HA was purchased from Euroscarf (plasmid # P30346) [36]. pNHK53 (YIp, ADH1::OsTIR1-9Myc1) (TIR1) was obtained from the National BioResource Project, Japan (depositor: M. Kanemaki) [37]. …”

Section: Methodsmentioning

confidence: 99%

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

et al. 2018

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Organelle distribution is regulated over the course of the cell cycle to ensure that each of the cells produced at the completion of division inherits a full complement of organelles. In yeast, the protein Num1 functions in the positioning and inheritance of two essential organelles, mitochondria and the nucleus. Specifically, Num1 anchors mitochondria as well as dynein to the cell cortex, and this anchoring activity is required for proper mitochondrial distribution and dynein-mediated nuclear inheritance. The assembly of Num1 into clusters at the plasma membrane is critical for both of its anchoring functions. We have previously shown that mitochondria drive the assembly of Num1 clusters and that these mitochondria-assembled Num1 clusters serve as cortical attachment sites for dynein. Here we further examine the role for mitochondria in dynein anchoring. Using a GFP-αGFP nanobody targeting system, we synthetically clustered Num1 on eisosomes to bypass the requirement for mitochondria in Num1 cluster formation. Utilizing this system, we found that mitochondria positively impact the ability of synthetically clustered Num1 to anchor dynein and support dynein function even when mitochondria are no longer required for cluster formation. Thus, the role of mitochondria in regulating dynein function extends beyond simply concentrating Num1; mitochondria likely promote an arrangement of Num1 within a cluster that is competent for dynein anchoring. This functional dependency between mitochondrial and nuclear positioning pathways likely serves as a mechanism to order and integrate major cellular organization systems over the course of the cell cycle.

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An auxin-based degron system for the rapid depletion of proteins in nonplant cells

Cited by 1,536 publications

References 35 publications

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

Sen1 has unique structural features grafted on the architecture of the Upf1‐like helicase family

Binding, sliding, and function of cohesin during transcriptional activation

The role of mitochondria in anchoring dynein to the cell cortex extends beyond clustering the anchor protein

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