Retroviral insertional mutagenesis identifies a small protein required for synthesis of diphthamide, the target of bacterial ADP-ribosylating toxins.
Retroviral insertional mutagenesis was used to produce a mutant Chinese hamster ovary cell line that is completely resistant to several different bacterial ADP-ribosylating toxins. The gene responsible for toxin resistance, termed diphtheria toxin (DT) and Pseudomonas exotoxin A (ETA) sensitivity required gene 1 (DESR1), encodes two small protein isoforms of 82 and 57 residues. DESR1 is evolutionally conserved and ubiquitously expressed. Only the longer isoform is functional because the mutant cell line can be complemented by transfection with the long but not the short isoform. We demonstrate that DESR1 is required for the first step in the posttranslational modification of elongation factor-2 at His(715) that yields diphthamide, the target site for ADP ribosylation by DT and ETA. KTI11, the analog of DESR1 in yeast, which was originally identified as a gene regulating the sensitivity of yeast to zymocin, is also required for diphthamide biosynthesis, implicating DESR1/KTI11 in multiple biological processes.
Liu
Shihui
S
Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Leppla
Stephen H
SH
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D000818:Animals; D001424:Bacterial Infections / Q000276:immunology*; D001427:Bacterial Toxins / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D005057:Eukaryotic Cells / Q000378:metabolism / Q000382:microbiology; D006639:Histidine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism*; D007113:Immunity, Innate / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009154:Mutation / Q000235:genetics; D020652:Peptide Elongation Factor 2 / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000302:isolation & purification; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins / Q000235:genetics / Q000302:isolation & purification*; D012190:Retroviridae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D001427:Bacterial Toxins; C459979:KTI11 protein, S cerevisiae; D020652:Peptide Elongation Factor 2; D020033:Protein Isoforms; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D006639:Histidine; C027527:diphthamide; D020727:ADP-Ribosylation Factors