A synonymous mutation in Yersinia enterocolitica yopE affects the function of the YopE type III secretion signal.
Yersinia spp. inject virulence proteins called Yops into the cytosol of target eukaryotic cells in an effort to evade phagocytic killing via a dedicated protein-sorting pathway termed type III secretion. Previous studies have proposed that, unlike other protein translocation mechanisms, Yops are not recognized as substrates for secretion via a solely proteinaceous signal. Rather, at least some of this information may be encoded within yop mRNA. Herein, we report that the first seven codons of yopE, when fused to the reporter protein neomycin phosphotransferase (Npt), are sufficient for the secretion of YopE1-7-Npt when type III secretion is induced in vitro. Systematic mutagenesis of yopE codons 1 to 7 reveals that, like yopQ, codons 2, 3, 5, and 7 are sensitive to mutagenesis, thereby defining the first empirical similarity between the secretion signals of two type III secreted substrates. Like that of yopQ, the secretion signal of yopE exhibits a bipartite nature. This is manifested by the ability of codons 8 to 15 to suppress point mutations in the minimal secretion signal that change the amino acid specificities of particular codons or that induce alterations in the reading frame. Further, we have identified a single nucleotide position in codon 3 that, when mutated, conserves the predicted amino acid sequence of the YopE1-7-Npt but abrogates secretion of the reporter protein. When introduced into the context of the full-length yopE gene, the single-nucleotide mutation reduces the type III injection of YopE into HeLa cells, even though the predicted amino acid sequence remains the same. Thus, yopE mRNA appears to encode a property that mediates the type III injection of YopE.
Ramamurthi
Kumaran S
KS
Department of Microbiology, University of Chicago, 920 East 58th St., Chicago, IL 60637, USA.
Schneewind
Olaf
O
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D003062:Codon; D004847:Epithelial Cells / Q000382:microbiology; D016368:Frameshift Mutation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport; D012329:RNA, Bacterial / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015008:Yersinia enterocolitica / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity
D001425:Bacterial Outer Membrane Proteins; D003062:Codon; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C063239:yopE protein, Yersinia; C067570:YopQ protein, Yersinia enterocolitica; D019868:Kanamycin Kinase