Figure 4. Overexpression of Msp1... | Rockefeller University Press

Figure 4.

$Overexpression of Msp1 restores the degradation inhibition of Pex15Δ30 in the GET mutants. (A) Cells expressing Get3-AID*-9xMyc from its own promoter and 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter were grown in SCD at 30°C, in SCGal for 2 h at 30°C, and further incubated for 30 min after addition of 1 mM IAA. Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (bottom). Values are mean ± SD from three independent experiments. (B)get3Δ cells expressing Get3-HA or its ATPase-inactive mutant Get3(D57N)-HA under the control of its own promoter were grown in SCD at 30°C and then in SCGal for 3 h at 30°C to induce 3xFLAG-mNG-Pex15Δ30, with or without Msp1 overexpression (Msp1 O/E) from the ADH1 promoter. Cell extracts were prepared at the indicated times at 30°C after addition of 100 µg/ml CHX, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30 in get3Δ cells, right after the addition of CHX (chase time = 0), relative to that in get3Δ cells with Get3-HA, but without Msp1 overexpression, is shown (center). Values are mean ± SD from three independent experiments. (C)doa10Δ cells used in Fig. 1 were grown in SCD at 30°C, and then 3xFLAG-mNG-Pex15Δ30 was induced by addition of 5 mM cyanamide for 3 h at 30°C. The cells were washed with fresh SCD medium to shut off 3xFLAG-mNG-Pex15Δ30 expression and incubated for 2 h at 30°C. The cells were treated with or without 1 μM β-estradiol for 30 min at 30°C and were harvested. Total cell lysates (T) were separated into the supernatant/cytosol fraction (S) and pellet/membrane fraction (P) by centrifugation (1 h, 100,000 g), and 3xFLAG-mNG-Pex15Δ30 was immunoprecipitated from the supernatant fractions with anti-FLAG magnetic beads (IP: FLAG; see Coimmunoprecipitation in Materials and methods). Amounts of 3xFLAG-mNG-Pex15Δ30 in fraction S relative to the total amounts in fractions S and P are indicated below the uppermost gel. Proteins in the flow-through (F), washed (W), and eluted (E) fractions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The arrowheads indicate the proteins of interest. The asterisk in immunoblotting using anti-Pgk1 antibodies indicates an IgG heavy chain of anti-FLAG antibodies. (D)get3Δ cells expressing 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter, with or without overexpression of Msp1 (Msp1O/E) under the control of the ADH1 promoter, were grown in SCD at 30°C, and then in SCGal for 3 h at 30°C. Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 are plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30, right after the addition of CHX (chase time = 0), relative to that in WT cells without Msp1 overexpression, is shown (center). Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData F4.$

Overexpression of Msp1 restores the degradation inhibition of Pex15Δ30 in the GET mutants. (A) Cells expressing Get3-AID*-9xMyc from its own promoter and 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter were grown in SCD at 30°C, in SCGal for 2 h at 30°C, and further incubated for 30 min after addition of 1 mM IAA. Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (bottom). Values are mean ± SD from three independent experiments. (B)get3Δ cells expressing Get3-HA or its ATPase-inactive mutant Get3(D57N)-HA under the control of its own promoter were grown in SCD at 30°C and then in SCGal for 3 h at 30°C to induce 3xFLAG-mNG-Pex15Δ30, with or without Msp1 overexpression (Msp1 O/E) from the ADH1 promoter. Cell extracts were prepared at the indicated times at 30°C after addition of 100 µg/ml CHX, and proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies (left). Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 were plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30 in get3Δ cells, right after the addition of CHX (chase time = 0), relative to that in get3Δ cells with Get3-HA, but without Msp1 overexpression, is shown (center). Values are mean ± SD from three independent experiments. (C)doa10Δ cells used in Fig. 1 were grown in SCD at 30°C, and then 3xFLAG-mNG-Pex15Δ30 was induced by addition of 5 mM cyanamide for 3 h at 30°C. The cells were washed with fresh SCD medium to shut off 3xFLAG-mNG-Pex15Δ30 expression and incubated for 2 h at 30°C. The cells were treated with or without 1 μM β-estradiol for 30 min at 30°C and were harvested. Total cell lysates (T) were separated into the supernatant/cytosol fraction (S) and pellet/membrane fraction (P) by centrifugation (1 h, 100,000 g), and 3xFLAG-mNG-Pex15Δ30 was immunoprecipitated from the supernatant fractions with anti-FLAG magnetic beads (IP: FLAG; see Coimmunoprecipitation in Materials and methods). Amounts of 3xFLAG-mNG-Pex15Δ30 in fraction S relative to the total amounts in fractions S and P are indicated below the uppermost gel. Proteins in the flow-through (F), washed (W), and eluted (E) fractions were analyzed by SDS-PAGE and immunoblotted with the indicated antibodies. The arrowheads indicate the proteins of interest. The asterisk in immunoblotting using anti-Pgk1 antibodies indicates an IgG heavy chain of anti-FLAG antibodies. (D)get3Δ cells expressing 3xFLAG-mNG-Pex15Δ30 from the GAL1 promoter, with or without overexpression of Msp1 (Msp1O/E) under the control of the ADH1 promoter, were grown in SCD at 30°C, and then in SCGal for 3 h at 30°C. Normalized relative amounts of 3xFLAG-mNG-Pex15Δ30 are plotted against chase time (right), and the amount of 3xFLAG-mNG-Pex15Δ30, right after the addition of CHX (chase time = 0), relative to that in WT cells without Msp1 overexpression, is shown (center). Values are mean ± SD from three independent experiments. Source data are available for this figure: SourceData F4.

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