Functional replacement of the essential ESS1 in yeast by the plant parvulin DlPar13.

A functionally Pin1-like peptidyl-prolyl cis/trans isomerase (PPIase(1)) was isolated from proembryogenic masses (PEMs) of Digitalis lanata according to its enzymatic activity. Partial sequence analysis of the purified enzyme (DlPar13) revealed sequence homology to members of the parvulin family of PPIases. Similar to human Pin1 and yeast Ess1, it exhibits catalytic activity toward substrates containing (Thr(P)/Ser(P))-Pro peptide bonds and comparable inhibition kinetics with juglone. Unlike Pin1-type enzymes it lacks the phosphoserine or phosphothreonine binding WW domain. Western blotting with anti-DlPar13 serum recognized the endogenous form in nucleic and cytosolic fractions of the plant cells. Since the PIN1 homologue ESS1 is an essential gene, complementation experiments in yeast were performed. When overexpressed in Saccharomyces cerevisiae DlPar13 is almost as effective as hPin1 in rescuing the temperature-sensitive phenotype caused by a mutation in ESS1. In contrast, the human parvulin hPar14 is not able to rescue the lethal phenotype of this yeast strain at nonpermissive temperatures. These results suggest a function for DlPar13 rather similar to parvulins of the Pin1-type.

Unusual sites of arginine methylation in Poly(A)-binding protein II and in vitro methylation by protein arginine methyltransferases PRMT1 and PRMT3.

Arginine methylation is a post-translational modification found mostly in RNA-binding proteins. Poly(A)-binding protein II from calf thymus was shown by mass spectrometry and sequencing to contain NG, NG-dimethylarginine at 13 positions in its amino acid sequence. Two additional arginine residues were partially methylated. Almost all of the modified residues were found in Arg-Xaa-Arg clusters in the C terminus of the protein. These motifs are distinct from Arg-Gly-Gly motifs that have been previously described as sites and specificity determinants for asymmetric arginine dimethylation. Poly(A)-binding protein II and deletion mutants expressed in Escherichia coli were in vitro substrates for two mammalian protein arginine methyltransferases, PRMT1 and PRMT3, with S-adenosyl-L-methionine as the methyl group donor. Both PRMT1 and PRMT3 specifically methylated arginines in the C-terminal domain corresponding to the naturally modified sites.

Purification and biochemical characterization of SELB, a translation factor involved in selenoprotein synthesis.

The product of the selB gene from Escherichia coli is required for co-translational insertion of selenocysteine into protein. To make the SELB protein accessible to biochemical analysis, the protein was purified from cells that overexpressed the selB gene from a phage T7 promoter plasmid. It was calculated that the overproduced SELB protein was purified 20-fold. The N-terminal amino acid sequence of the purified protein was determined, and it confirmed that the initiation codon of selB mRNA translation overlaps the stop codon of the preceding selA gene by 4 bases. Structural similarity between SELB and elongation factors was demonstrated by limited proteolysis of SELB by trypsin. The cleavage sites within SELB were identified by N-terminal sequencing of the two proteolytic products. The position in the SELB protein of the major cleavage site was homologous to a tryptic cleavage site which is characteristic for elongation factors. Immunological analysis showed that the levels of SELB are equivalent in aerobically and anaerobically grown cells; the amount of the protein was estimated to be approximately 1100 copies/E. coli cell. Upon fractionation of cell extracts, SELB was found to be partially associated with the ribosomes. The results therefore indicate that SELB is the first known elongation factor-like protein that has specificity for a particular charged tRNA.