Figure 1. Met32 degradation by SCF^Met30 is required for cell proliferation.

(A) Regulation of Met4 transcription complex components. Cells expressing Met31^3HA, Met32^3HA, Met28^3HA, or Cbf1^3HA from their endogenous loci were grown in minimal medium supplemented with 1mM methionine (+Met), shifted to medium without methionine (−Met) for 30′ to activate Met4, before methionine was added back to the cultures for an additional 15′ (+Met) to inactivate Met4. Cell lysates were analyzed by immunoblotting using anti-HA antibodies (Met28, Met32, Met31, Cbf1), and anti-Met4 antibodies to detect Met4 activation as indicated by loss of the ubiquitylated forms and appearance of phosphorylated species. The proteasome subunit Rpt1 was detected as a loading control.

(B) Met32 degradation depends on the F-box protein Met30. Met32 stability was analyzed by promoter shut-off experiments (“gal-shut-off“) and immunoblotting using anti-HA antibodies. R: uninduced sample grown in raffinose medium.

(C) Degradation of Met32 is essential for cell proliferation. Five fold serial dilutions of yeast strains as indicated were spotted onto YEP-dextrose (no Met32 expression) and YEP-galactose plates (Met32 expression). Plates were incubated at 30° C.

(D) Stabilization of Met32 induces a G1 cell cycle arrest. Yeast cells as indicated were grown in YEP-dextrose or YEP-galactose for 4hrs. Cell cycle distribution was analyzed by flow cytometry.