Identification of a novel selD homolog from Eukaryotes, Bacteria, and Archaea: Is there an autoregulatory mechanism in selenocysteine metabolism?

Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme’s putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.

Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach.

Pathogenic yersiniae secrete a set of antihost proteins, called Yops, by a type III secretion mechanism. Upon infection of cultured epithelial cells, extracellular Yersinia pseudotuberculosis and Yersinia enterocolitica translocate cytotoxin YopE across the host cell plasma membrane. Several lines of evidence suggest that tyrosine phosphatase YopH follows the same pathway. We analyzed internalization of YopE and YopH into murine PU5-1.8 macrophages by using recombinant Y. enterocolitica producing truncated YopE and YopH proteins fused to a calmodulin-dependent adenylate cyclase. The YopE-cyclase and YopH-cyclase hybrids were readily secreted by Y. enterocolitica. The N-terminal domain required for secretion was not longer than 15 residues of YopE and 17 residues of YopH. Internalization into eukaryotic cells, revealed by cAMP production, only required the N-terminal 50 amino acid residues of YopE and the N-terminal 71 amino acid residues of YopH. YopE and YopH are thus modular proteins composed of a secretion domain, a translocation domain, and an effector domain. Translocation of YopE and YopH across host cell's membranes was also dependent on the secretion of YopB and YopD by the same bacterium. The cyclase fusion approach could be readily extended to study the fate of other proteins secreted by invasive bacterial pathogens.