TY - JOUR
T1 - Rapid depletion of budding yeast proteins by fusion to a heat-inducible degron.
AU - Sanchez-Diaz, Alberto
AU - Kanemaki, Masato
AU - Marchesi, Vanessa
AU - Labib, Karim
PY - 2004/3/9
Y1 - 2004/3/9
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=12144288039&partnerID=8YFLogxK
U2 - 10.1126/stke.2232004pl8
DO - 10.1126/stke.2232004pl8
M3 - Article
C2 - 15010550
AN - SCOPUS:12144288039
SN - 1525-8882
VL - 2004
JO - Science's STKE : signal transduction knowledge environment
JF - Science's STKE : signal transduction knowledge environment
IS - 223
M1 - PL8
ER -