Figure 3.

Grr1p and Cdc4p are ubiquitinated in vivo and degraded by the proteasome. (A) Grr1p is ubiquitinated in vivo. (Left) Immunoblot with 9E10 antibodies of extracts prepared from cells expressing Grr1p-myc (JMG101; lane b) or for control untagged Grr1p (W303; lane a) demonstrating a ladder of distinct slower-migrating species separated by approximately 10 kDa, characteristic for ubiquitinated forms. The arrowhead indicates the position of unmodified Grr1p-myc; the bracket marks ubiquitinated species of Grr1p. (Right) Extracts from Grr1p-myc cells (lanes e–g) or untagged control cells (lanes c and d) overexpressing wild-type ubiquitin (Ub; lane e) or dominant-negative Ub3R (lanes d, f, and g) from a Cu²⁺-inducible promoter were immunoblotted with 9E10 (Upper) or anti-ubiquitin antibodies (Lower). Reduced levels of Ub3R are detected in cells that were not treated with Cu²⁺ (lane f). Note that expression of Ub3R interferes with the formation of multi-ubiquitin species (Lower) and results in accumulation of mono-ubiquitinated Grr1p (Upper). (B) Mono-ubiquitinated Grr1p-myc and Cdc4p-myc are stabilized in vivo. The half-life of Grr1p-myc (Upper pair) and Cdc4p-myc (Lower pair) were determined in cells overexpressing either wild-type ubiquitin (upper panel of each pair) or Ub3R (lower panel of each pair). Note that overexpression of Ub3R stabilizes Grr1p-myc and Cdc4p-myc. (C) (Upper) Grr1p-myc and Cdc4p-myc are stabilized in the presence of the proteasome inhibitor MG132. The half-life of Grr1p-myc (JMG67; Left) or Cdc4p-myc (JMG66; Right) was determined with CX in the presence of MG132 (right lanes) or the solvent DMSO (−; left lanes). Note the accumulation of partially degraded forms of Grr1p-myc and Cdc4p-myc in the presence of MG132 (bar). (Lower) The half-life of Grr1p-myc was determined as described in wild-type (JMG53; left lanes) or cim5–1 cells (JMG54; right lanes) which are defective for proteasome function. An isogenic cim5–1 strain (YMP241) expressing untagged Grr1p is included for control (right lane). Note the accumulation of partially degraded forms of Grr1p-myc in cim5–1 cells (bar).