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Molecular and Cellular Biology, November 2004, p. 9487-9497, Vol. 24, No. 21
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.21.9487-9497.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of the Proteins Required for Biosynthesis of Diphthamide, the Target of Bacterial ADP-Ribosylating Toxins on Translation Elongation Factor 2{dagger}

Shihui Liu,1,{ddagger} G. Todd Milne,2,{ddagger},§ Jeffrey G. Kuremsky,1 Gerald R. Fink,2,3* and Stephen H. Leppla1*

Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland,1 Whitehead Institute for Biomedical Research,2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts3

Received 11 June 2004/ Returned for modification 19 July 2004/ Accepted 9 August 2004

Diphthamide, a posttranslational modification of translation elongation factor 2 that is conserved in all eukaryotes and archaebacteria and is the target of diphtheria toxin, is formed in yeast by the actions of five proteins, Dph1 to -5, and a still unidentified amidating enzyme. Dph2 and Dph5 were previously identified. Here, we report the identification of the remaining three yeast proteins (Dph1, -3, and -4) and show that all five Dph proteins have either functional (Dph1, -2, -3, and -5) or sequence (Dph4) homologs in mammals. We propose a unified nomenclature for these proteins (e.g., HsDph1 to -5 for the human proteins) and their genes based on the yeast nomenclature. We show that Dph1 and Dph2 are homologous in sequence but functionally independent. The human tumor suppressor gene OVCA1, previously identified as homologous to yeast DPH2, is shown to actually be HsDPH1. We show that HsDPH3 is the previously described human diphtheria toxin and Pseudomonas exotoxin A sensitivity required gene 1 and that DPH4 encodes a CSL zinc finger-containing DnaJ-like protein. Other features of these genes are also discussed. The physiological function of diphthamide and the basis of its ubiquity remain a mystery, but evidence is presented that Dph1 to -3 function in vivo as a protein complex in multiple cellular processes.

* Corresponding author. Mailing address for Stephen H. Leppla: Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892. Phone: (301) 594-2865. Fax: (301) 480-0326. E-mail: sleppla{at}niaid.nih.gov. Mailing address for Gerald R. Fink: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142. Phone: (617) 258-5215. Fax: (617) 258-6425. E-mail: gfink{at}wi.mit.edu.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} S.L. and G.T.M. contributed equally to this work.

§ Present address: Microbia, Inc., Cambridge, MA 02141.

Molecular and Cellular Biology, November 2004, p. 9487-9497, Vol. 24, No. 21
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.21.9487-9497.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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