Diphthamide, a unique post-translationally modified amino acid
that is found only in EF-2, is conserved in archaebacteria and
eukaryotes. Although diphthamide is the molecular target for diphtheria
toxin, its biological significance has remained elusive. Biosynthesis of
diphthamide requires the function of at least five proteins (Dph1-5).
Here we report the cloning and characterization of the remaining three
DPH genes. Dph1, Dph2, Dph4, and Dph5 appear to be dedicated to
diphthamide biosynthesis, whereas the pleiotropic defects of dph3
mutants suggest that Dph3 may have a more general role in cellular
metabolism. In contrast, strains deleted for DPH1 , DPH2 ,
DPH4 , or DPH5 are indistinguishable from wild type when
tested under a multitude of growth conditions suggesting that
diphthamide is not required for general EF-2 function. However,
dph mutants are characterized by two stationary phase phenotypes
that implicate a role in nutrient sensing and response. Specifically,
dph mutants lose viability more rapidly than isogenic wild type
strains upon extended culture and also exhibit an increased lag upon
return to growth. Additionally, dph mutants are hypersensitive to
treatment with rapamycin, a drug that blocks the nutrient-dependent
activation of translation by Tor. These phenotypes are recapitulated by
mutations in the diphthamide modification site of EF-2. Together these
data implicate diphthamide in the integration of protein translation
with nutrient availability.
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