Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4 is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4 is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCFCDC4 activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

The yeast transcription factor Gcn4 is regulated by amino acid starvation at the levels of both protein synthesis and stability. Gcn4 degradation depends on the ubiquitination complex SCF CDC4 and requires phosphorylation by the cyclin-dependent kinase Pho85. Here, we show that Pcl5 is the Pho85 cyclin specifically required for Gcn4 degradation. PCL5 is itself induced by Gcn4 at the level of transcription. However, even when PCL5 is constitutively overexpressed, Pho85-associated Gcn4 phosphorylation activity is reduced in starved cells and Gcn4 degradation is decreased. Under these conditions, the Pcl5 protein disappears because of rapid constitutive turnover. We suggest that, by virtue of its constitutive metabolic instability, Pcl5 may be a sensor of cellular protein biosynthetic capacity. The fact that PCL5 is transcriptionally induced in the presence of Gcn4 suggests that it is part of a homeostatic mechanism that reduces Gcn4 levels upon recovery from starvation.

The ubiquitin‐conjugating enzyme Cdc34, in conjunction with the SCF ubiquitin ligase complexes, catalyzes the ubiquitination of protein substrates involved in various cellular pathways, including the cell cycle and amino acid synthesis. A synthetic lethality screen was carried out in S. cerevisiae with the hypomorphic allelele cdc34‐2 to uncover new genetic interactions with this component of the ubiquitin system. Three temperature‐sensitive mutants that are synthetically lethal with cdc34‐2 at its permissive temperature were characterized. They represent new alleles of the F‐box protein gene CDC4, of the ubiquitin‐activating enzyme Uba1, and an allele of SUP70/CDC65, encoding the unique tRNAGln(CUG). The uba1‐118 mutant displayed extremely enlarged cells and defects in cell cycle regulation. The N‐end rule substrate Arg‐lacZ and the UFD pathway substrate Pro‐LacZ were stabilized in the uba1‐118 strain. However, no defects were detected in either in vitro activity of Uba1‐118, or in degradation of several natural substrates of the ubiquitin system at the non‐permissive temperature, suggesting that the uba1‐118 allele differentially affects degradation of specific substrates. The sup70‐65 allele, which is synthetically lethal with cdc34‐2, consists of a basepair‐disrupting mutation at the top of the anticodon stem of tRNAGln(CUG). This allele was suggested to disrupt a regulatory role of tRNAGln(CUG) in nitrogen metabolism and morphogenesis at permissive temperature. At restrictive temperature, sup70‐65 cells arrest predominantly in G1, possibly reflecting the requirement for protein synthesis in cell‐cycle START control. The synthetic interaction of cdc34‐2 and sup70‐65 suggests that common targets are affected by both Cdc34 activity and tRNAGln(CUG).