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.
Kambampati
R
R
Medical College of Wisconsin, Department of Biochemistry, Milwaukee, Wisconsin 53226, USA.
Chakraburtty
K
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010441:Peptide Chain Elongation, Translational; D010445:Peptide Elongation Factors / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014357:Trypsin
D005656:Fungal Proteins; D010445:Peptide Elongation Factors; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C111584:YEF3 protein, S cerevisiae; D014357:Trypsin; D000251:Adenosine Triphosphatases