Elongation factor 2 (EF-2), the protein which promotes the translocation step during protein synthesis in the cytoplasm of eukaryotic cells, contains an unusual amino acid, designated X by Robinson, Hendriksen, and Maxwell (J. Biol. Chem. 249, 5088, 1974). Amino acid X appears to be essential to the function of EF-2 because its covalent modification by diphtheria toxin inactivates the protein. Although the toxin will apparently inactivate all eukaryotic EF-2s, through this modification, it does not appear to modify any other protein. This suggests that all EF-2s contain a functionally essential and unique structural feature which is specifically recognized by diphtheria toxin. The toxin inactivates EF-2 by transferring the -ADP-ribose of NAD+ to the unusual amino acid X. Amino acid X almost certainly arises by posttranslational modification of a precursor of EF-2 because it is not one of the 20 common amino acids. Interestingly, it also appears to differ from the previously recognized amino acids known to arise by posttranslational modification of proteins. We have developed a large scale method for the purification of amino acid X from yeast EF-2. The initial goal of this proposal is to utilize the material we have prepared by this method to determine the structure of amino acid X and the mode of ADP ribose attachment. For this purpose we will employ primarily nuclear magnetic resonance spectroscopy and mass spectroscopy. Immediately following the determination of the structure of amino acid X we propose to initiate studies designed to determine: 1) The distribution of amino acid X in other proteins of various biological materials; 2) The mechnism of biosynthesis of amino acid X; 3) The role of amino acid X in the function of EF-2; 4) The nature of the diphtheria toxin recognition site on EF-2.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026832-08
Application #
3274275
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1979-07-01
Project End
1988-04-30
Budget Start
1986-07-01
Budget End
1988-04-30
Support Year
8
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Veldman, S; Rao, S; Bodley, J W (1994) Differential transcription of the two Saccharomyces cerevisiae genes encoding elongation factor 2. Gene 148:143-7
Phan, L D; Perentesis, J P; Bodley, J W (1993) Saccharomyces cerevisiae elongation factor 2. Mutagenesis of the histidine precursor of diphthamide yields a functional protein that is resistant to diphtheria toxin. J Biol Chem 268:8665-8
Perentesis, J P; Phan, L D; Gleason, W B et al. (1992) Saccharomyces cerevisiae elongation factor 2. Genetic cloning, characterization of expression, and G-domain modeling. J Biol Chem 267:1190-7
Donovan, M G; Veldman, S A; Bodley, J W (1992) A screening procedure for the intracellular expression of native proteins by Saccharomyces cerevisiae: discrimination of diphtheria toxin-resistant mutants. Yeast 8:629-33
Perentesis, J P; Miller, S P; Bodley, J W (1992) Protein toxin inhibitors of protein synthesis. Biofactors 3:173-84
Donovan, M G; Bodley, J W (1991) Saccharomyces cerevisiae elongation factor 2 is phosphorylated by an endogenous kinase. FEBS Lett 291:303-6
Miller, S P; Bodley, J W (1991) Alpha-sarcin cleavage of ribosomal RNA is inhibited by the binding of elongation factor G or thiostrepton to the ribosome. Nucleic Acids Res 19:1657-60
Miller, S P; Bodley, J W (1988) Alpha-sarcin cleaves ribosomal RNA at the alpha-sarcin site in the absence of ribosomal proteins. Biochem Biophys Res Commun 154:404-10
Chen, J Y; Bodley, J W (1988) Biosynthesis of diphthamide in Saccharomyces cerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase. J Biol Chem 263:11692-6
Miller, S P; Bodley, J W (1988) The ribosomes of Aspergillus giganteus are sensitive to the cytotoxic action of alpha-sarcin. FEBS Lett 229:388-90

Showing the most recent 10 out of 15 publications