Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.
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.
Sanchez-Diaz
Alberto
A
Cancer Research U.K., Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK.
Kanemaki
Masato
M
Marchesi
Vanessa
V
Labib
Karim
K
D001483:Base Sequence; D002470:Cell Survival / Q000235:genetics; D002874:Chromosome Mapping / Q000379:methods; D015825:Chromosomes, Fungal / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000235:genetics*; D020043:Genes, Essential / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D006238:Haploidy; D006358:Hot Temperature; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014170:Transformation, Genetic / Q000235:genetics
D004271:DNA, Fungal; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins