Elongation factor 2 mutants deficient in diphthamide formation show temperature-sensitive cell growth

Area of Science:

• Molecular Biology
• Protein Biochemistry
• Genetics

Background:

• Eukaryotic protein synthesis elongation factor 2 (EF-2) possesses a unique modified histidine residue called diphthamide.
• Diphthamide is known as a target for ADP-ribosylation by bacterial toxins, but its biological role is not well understood.

Purpose of the Study:

• To elucidate the function of diphthamide in EF-2.
• To investigate the impact of altering the diphthamide modification site on EF-2 activity and cellular growth.

Main Methods:

• Expressed mutant EF-2 genes in yeast cells, substituting His-699 (the diphthamide precursor) with 19 different amino acids.
• Assessed EF-2 activity, cellular growth inhibition, and susceptibility to diphtheria toxin-mediated ADP-ribosylation for each mutant.

Main Results:

• Mutants were classified into three groups based on EF-2 activity and cellular effects.
• Group 1 mutants (Arg, Lys substitutions) were inactive and inhibited growth. Group 2 mutants (Gly, Pro, Ser, Asp) were inactive but did not inhibit growth.
• Group 3 mutants (13 other substitutions) were functional but not ribosylated, indicating lack of diphthamide formation. These mutants exhibited slower growth and temperature sensitivity.

Conclusions:

• EF-2 can be active without diphthamide at normal temperatures.
• Diphthamide modification likely enhances EF-2's heat resistance, suggesting a role in cellular thermotolerance.