Figure 2.—

get3 mutants suppress npl4-1 phenotypes. (A) Δget3 rescues ERAD-mediated CPY* degradation in npl4-1 cells. Wild-type (NPL4), npl4-1, and npl4-1 Δget3 mutant cells were grown to log phase and protein synthesis was halted by treatment with cyclohexamide. Samples were collected 30, 60, and 120 min after cyclohexamide treatment, separated by SDS–PAGE, and subjected to Western blot analysis with anti-CPY antibodies. (B) get3^tn restores OLE1 transcription in npl4-1 cells. Northern analysis was performed on total RNA isolated from wild type (WT), npl4-1, or npl4-1 with the suppressing mutations spt23^tn or get3^tn, using OLE1- and ACT1-specific DNA probes. Cells were either continuously grown at 25° or shifted to 37° for 2 hr (labeled as 25° and 37°, respectively) prior to RNA purification. The ratio of OLE1/ACT1 signal for each sample is given (at the bottom) relative to that for the WT 25° sample.