Synthetic Genetic Interaction Between the Ubiquitin‐Conjugating Enzyme Cdc34 and a tRNA Mutant

Abstract

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).