Functional subdomains of yeast elongation factor 3. Localization of ribosome-binding domain.

Elongation factor 3 (EF-3) is an essential requirement of the fungi for translational elongation. EF-3 is an ATPase, and the hydrolytic activity is stimulated 2 orders of magnitude by yeast ribosomes. Limited trypsinolysis of EF-3 results in the cleavage of a single peptide bond between residues 774 (Arg) and 775 (Gln), generating polypeptides of approximate molecular mass 90 and 30 kDa. The 90-kDa fragment is relatively resistant to proteolysis and retains ribosome-independent ATPase activity. The 30-kDa fragment is further proteolyzed into smaller fragments and retains the specificity for binding to yeast ribosomes. Both the intact EF-3 and the 30-kDa fragment are protected from proteolysis by yeast ribosomes. EF-3 is NH2 terminally blocked, and so is the 90-kDa fragment. The COOH terminally derived 30-kDa fragment contains glutamine (residue 775) at the NH2-terminal end. A construct was designed representing the COOH-terminal domain of EF-3 (30-kDa fragment), subcloned, and expressed as a glutathione S-transferase fusion in yeast. The glutathione S-transferase-30-kDa peptide remains stringently associated with ribosomes. Isolated fusion peptide rebinds to yeast ribosomes with high affinity. Based on these results, we propose that at least one of the ribosome-binding sites of EF-3 resides at the COOH-terminal end of the protein.