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

Alberto Sanchez-Diaz, Masato Kanemaki, Vanessa Marchesi, Karim Labib (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

54 Citations (Scopus)

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.

Original languageEnglish
Article numberPL8
Number of pages20
JournalScience's STKE : signal transduction knowledge environment
Volume2004
Issue number223
DOIs
Publication statusPublished - 9 Mar 2004

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