Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.

2.50
Hdl Handle:
http://hdl.handle.net/10541/84408
Title:
Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.
Authors:
Sanchez-Diaz, Alberto; Kanemaki, Masato; Marchesi, Vanessa; Labib, Karim
Abstract:
One effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.
Affiliation:
Cancer Research U.K., Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Citation:
Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron. 2004, 2004 (223):PL8 Sci. STKE
Journal:
Science's STKE
Issue Date:
9-Mar-2004
URI:
http://hdl.handle.net/10541/84408
DOI:
10.1126/stke.2232004pl8
PubMed ID:
15010550
Language:
en
ISSN:
1525-8882
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorSanchez-Diaz, Alberto-
dc.contributor.authorKanemaki, Masato-
dc.contributor.authorMarchesi, Vanessa-
dc.contributor.authorLabib, Karim-
dc.date.accessioned2009-10-19T15:36:38Z-
dc.date.available2009-10-19T15:36:38Z-
dc.date.issued2004-03-09-
dc.identifier.citationRapid depletion of budding yeast proteins by fusion to a heat-inducible degron. 2004, 2004 (223):PL8 Sci. STKEen
dc.identifier.issn1525-8882-
dc.identifier.pmid15010550-
dc.identifier.doi10.1126/stke.2232004pl8-
dc.identifier.urihttp://hdl.handle.net/10541/84408-
dc.description.abstractOne effective way to study the biological function of a protein in vivo is to inactivate it and see what happens to the cell. For proteins that are dispensable for cell viability, the corresponding gene can simply be deleted from its chromosomal locus. The study of essential proteins is more challenging, however, because the function of the protein must be inactivated conditionally. Here, we describe a method that allows the target protein to be depleted rapidly and conditionally, so that the immediate effects on the cell can be examined. The chromosomal locus of a budding yeast gene is modified so that a "heat-inducible degron cassette" is added to the N terminus of the encoded protein, causing it to be degraded by a specific ubiquitin-mediated pathway when cells are shifted from 24 degrees to 37 degrees C. Degradation requires recognition of the degron cassette by the evolutionarily conserved Ubr1 protein, which is associated with a ubiquitin-conjugating enzyme. To promote rapid and conditional depletion of the target protein, we use a yeast strain in which expression of the UBR1 gene can be either repressed or strongly induced. Degron strains are constructed by a simple "one-step" approach using the polymerase chain reaction.en
dc.language.isoenen
dc.subject.meshBase Sequence-
dc.subject.meshCell Survival-
dc.subject.meshChromosome Mapping-
dc.subject.meshChromosomes, Fungal-
dc.subject.meshDNA, Fungal-
dc.subject.meshGene Expression Regulation, Fungal-
dc.subject.meshGenes, Essential-
dc.subject.meshGenes, Fungal-
dc.subject.meshHaploidy-
dc.subject.meshHot Temperature-
dc.subject.meshMolecular Sequence Data-
dc.subject.meshMutagenesis, Insertional-
dc.subject.meshNucleic Acid Amplification Techniques-
dc.subject.meshOligonucleotides-
dc.subject.meshPolymerase Chain Reaction-
dc.subject.meshProtein Processing, Post-Translational-
dc.subject.meshRecombinant Fusion Proteins-
dc.subject.meshSaccharomyces cerevisiae-
dc.subject.meshSaccharomyces cerevisiae Proteins-
dc.subject.meshTemperature-
dc.subject.meshTransformation, Genetic-
dc.titleRapid depletion of budding yeast proteins by fusion to a heat-inducible degron.en
dc.contributor.departmentCancer Research U.K., Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.en
dc.identifier.journalScience's STKEen

Related articles on PubMed

All Items in Christie are protected by copyright, with all rights reserved, unless otherwise indicated.