Back to article: The ubiquitin-conjugating enzyme, Ubc1, indirectly regulates SNF1 kinase activity via Forkhead-dependent transcription

FIGURE 5: Snf1 protein and glucose-responsive SUC2 expression levels are reestab-lished by Fkh1 or Fkh2 overexpression in the ubc1Δ strain.

(A) α-factor arrest release of the WT yeast strain harboring an endogenous Snf1-GFP tag, with detection of Snf1-GFP and endogenous Clb2. Equal cell numbers were taken after release from G1 phase at the time points indicated, and Western analysis of the cell lysates as shown.

(B) Corresponding flow cytometry of samples in (A) at the indicated time points, with unreplicated (1n) and replicated (2n) DNA abundance indicated.

(C) Western analysis of steady-state Snf1-GFP protein levels in WT and ubc1Δ strains with or without 2μ plasmid expression of Fkh1 (pFkh1) and Fkh2 (pFkh2) in 2% glucose.

(D) RT-PCR (26 cycles) of SNF1 and rRNA expression in the ubc1Δ strain with and without independent 2μ plasmid expression of Fkh1 (pFkh1) and Fkh2 (pFkh2), with comparison to isogenic WT.

(E) RT-PCR (26 cycles) of SUC2 expression under repressive (H: 2%) and acti-vating (L: 0.05%) glucose levels with strains and plasmids as described in (D).

(F) RT-PCR of genes encoding SNF1 kinase γ subunit (SNF4), β subunit (GAL83), SUC2 repressor, MIG1, and the forkheads (FKH1, FKH2), comparing the ubc1Δ and WT strains. Asy: asynchronous. G1: α-factor arrest in G1.

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