Fig 5.
Pho85 activity increases the half-life of Cln3 protein. (A) Different strains with a genomic tagged version of CLN3 were sampled and then analyzed for CLN3 mRNA levels (by RT-PCR) or Cln3-Myc levels (by immunoblotting). Wild-type cells were grown exponentially in synthetic complete medium with (wt) or without (wt −PO42−) phosphate, pho85Δ strain cells were grown in the same complete medium with phosphate, and GAL-PHO85 cells were grown for 5 h in synthetic complete medium with galactose as a carbon source. (B) YAM67 strain cells were incubated with either 1-Na PP1 (a specific pho85-as inhibitor) or drug vehicle (DMSO). Forty minutes later (time zero), cycloheximide (CHX) was added to the cultures (final concentration, 10 μg/ml). At the indicated times, samples were taken and analyzed for Cln3-Myc levels by immunoblotting. Data ± standard deviations from four independent experiments are shown. t1/2, half-life. (C) Downregulation of autophagy does not restore the diminished Cln3 levels of pho85Δ mutants. Strains of the indicated genotypes were grown exponentially in phosphate-rich medium, and Cln3-Myc was analyzed by immunoblot assay using specific antibodies. (D) Absence of the PEST region restores Cln3 levels in pho85Δ cells. Wild-type or pho85Δ cells carrying a plasmid with a cln3-1 allele without the PEST region (41) were grown exponentially in YPD. Cln3-HA levels were measured by immunoblotting. (E) Absence of UBC4 restores Cln3 levels in a pho85Δ strain. Strains of the indicated genotypes carrying a genomic Myc-tagged version of Cln3 were grown exponentially in phosphate-rich medium. Cells were harvested, and Cln3 levels were evaluated by immunoblotting.