Schizosaccharomyces pombe contains separate CC- and A-adding tRNA nucleotidyltransferases.

A specific cytidine-cytidine-adenosine (CCA) sequence is required at the 3'-terminus of all functional tRNAs. This sequence is added during tRNA maturation or repair by tRNA nucleotidyltransferase enzymes. While most eukaryotes have a single enzyme responsible for CCA addition, some bacteria have separate CC- and A-adding activities. The fungus, Schizosaccharomyces pombe, has two genes (cca1 and cca2) that are thought, based on predicted amino acid sequences, to encode tRNA nucleotidyltransferases. Here, we show that both genes together are required to complement a Saccharomyces cerevisiae strain bearing a null mutation in the single gene encoding its tRNA nucleotidyltransferase. Using enzyme assays we show further that the purified S. pombe cca1 gene product specifically adds two cytidine residues to a tRNA substrate lacking this sequence while the cca2 gene product specifically adds the terminal adenosine residue thereby completing the CCA sequence. These data indicate that S. pombe represents the first eukaryote known to have separate CC- and A-adding activities for tRNA maturation and repair. In addition, we propose that a novel structural change in a tRNA nucleotidyltransferase is responsible for defining a CC-adding enzyme.

Mutations in TRNT1 cause congenital sideroblastic anemia with immunodeficiency, fevers, and developmental delay (SIFD).

Mutations in genes encoding proteins that are involved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturation of both nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations result in partial loss of function of TRNT1 and lead to metabolic defects in both the mitochondria and cytosol, which can account for the phenotypic pleiotropy.

The ability of an arginine to tryptophan substitution in Saccharomyces cerevisiae tRNA nucleotidyltransferase to alleviate a temperature-sensitive phenotype suggests a role for motif C in active site organization.

We report that the temperature-sensitive (ts) phenotype in Saccharomyces cerevisiae associated with a variant tRNA nucleotidyltransferase containing an amino acid substitution at position 189 results from a reduced ability to incorporate AMP and CMP into tRNAs. We show that this defect can be compensated for by a second-site suppressor converting residue arginine 64 to tryptophan. The R64W substitution does not alter the structure or thermal stability of the enzyme dramatically but restores catalytic activity in vitro and suppresses the ts phenotype in vivo. R64 is found in motif A known to be involved in catalysis and nucleotide triphosphate binding while E189 lies within motif C previously thought only to connect the head and neck domains of the protein. Although mutagenesis experiments indicate that residues R64 and E189 do not interact directly, our data suggest a critical role for residue E189 in enzyme structure and function. Both R64 and E189 may contribute to the organization of the catalytic domain of the enzyme. These results, along with overexpression and deletion analyses, show that the ts phenotype of cca1-E189F does not arise from thermal instability of the variant tRNA nucleotidyltransferase but instead from the inability of a partially active enzyme to support growth only at higher temperatures.

The folding capacity of the mature domain of the dual-targeted plant tRNA nucleotidyltransferase influences organelle selection.

tRNA-NTs (tRNA nucleotidyltransferases) are required for the maturation or repair of tRNAs by ensuring that they have an intact cytidine-cytidine-adenosine sequence at their 3'-termini. Therefore this enzymatic activity is found in all cellular compartments, namely the nucleus, cytoplasm, plastids and mitochondria, in which tRNA synthesis or translation occurs. A single gene codes for tRNA-NT in plants, suggesting a complex targeting mechanism. Consistent with this, distinct signals have been proposed for plastidic, mitochondrial and nuclear targeting. Our previous research has shown that in addition to N-terminal targeting information, the mature domain of the protein itself modifies targeting to mitochondria and plastids. This suggests the existence of an as yet unknown determinate for the distribution of dual-targeted proteins between these two organelles. In the present study, we explore the enzymatic and physicochemical properties of tRNA-NT variants to correlate the properties of the enzyme with the intracellular distribution of the protein. We show that alteration of tRNA-NT stability influences its intracellular distribution due to variations in organelle import capacities. Hence the fate of the protein is determined not only by the transit peptide sequence, but also by the physicochemical properties of the mature protein.

Human parotid secretory protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain.

Parotid secretory protein (PSP) (C20orf70) is a salivary protein of unknown function. The protein belongs to the palate, lung, and nasal epithelium clone (PLUNC) family of mucosal secretory proteins that are predicted to be structurally similar to lipid-binding and host-defense proteins including bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. However, the PLUNC proteins exhibit significant sequence variation and different biological functions have been proposed for different family members. This study tested the functional implications of the proposed similarity of PSP to the acute phase protein lipopolysaccharide-binding protein (LBP). PSP was identified in human saliva and was soluble in 70% ethanol, as shown for other PLUNC proteins. PSP binds lipopolysaccharide and can be eluted by non-ionic detergent, but not by urea or high salt. A synthetic PSP peptide, GL13NH2, which corresponds to a lipopolysaccharide-inhibiting peptide from LBP, inhibited the binding of lipopolysaccharide to both PSP and lipopolysaccharide-binding protein. Peptides from other regions of PSP and the control peptide polymyxin B showed no effect on the binding of PSP to lipopolysaccharide. GL13NH2 also inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor from macrophages. The other PSP peptides had no effect in this assay. PSP peptides had no or only minor effect on macrophage cell viability. These results indicate that PSP is a lipopolysaccharide-binding protein that is functionally related to LBP, as suggested by their predicted structural similarities.

Characterization of a temperature-sensitive mutation that impairs the function of yeast tRNA nucleotidyltransferase.

ATP(CTP) : tRNA nucleotidyltransferase catalyses the posttranscriptional addition of cytidine, cytidine and adenosine to the 3' ends of tRNAs. Previously, a temperature-sensitive phenotype in Saccharomyces cerevisiae resulting from a mutation in the CCA1 gene coding for this enzyme was identified. Here, we show that a single guanine-to-adenine transition in cca1-1 generates the temperature-sensitive phenotype. Alignment of the amino acid sequence of S. cerevisiae tRNA nucleotidyltransferase with other tRNA nucleotidyltransferases for which crystal structures have been solved suggests that the resulting Glu-to-Lys substitution is in a ss-turn connecting the structurally and functionally important head and neck domains of the protein. Proteins containing Gln, His or Phe at this position were constructed to further characterize the importance of this residue in enzyme structure and function. As with the Lys variant, the Phe and His variants generate a temperature-sensitive phenotype in isogenic yeast strains, further supporting the role of this position in maintaining the structure and function of this enzyme. Comparative biophysical and biochemical characterization of both the wild-type and variant proteins indicates that amino acid substitutions at this position can result in a structural change in the protein that reduces enzyme activity (both at the permissive and non-permissive temperatures), decreases the melting temperature of the protein and alters its stability at the non-permissive temperature (37 degrees C).

Dual targeting of the tRNA nucleotidyltransferase in plants: not just the signal.

Enzymes involved in tRNA maturation are essential for cytosolic, mitochondrial, and plastid protein synthesis and are therefore localized to these different compartments of the cell. Interestingly, only one isoform of tRNA nucleotidyltransferase (responsible for adding the 3'-terminal cytidine-cytidine-adenosine to tRNAs) has been identified in plants. The present study therefore explored how signals contained on this enzyme allow it to be distributed among the different cell compartments. It is demonstrated that the N-terminal portion of the protein acts as an organellar targeting signal and that differential use of multiple in-frame start codons alters the localization of the protein. Moreover, it is shown that the mature domain has a major impact on the distribution of the protein within the cell. These data indicate that regulation of dual localization involves not only specific N-terminal signals, but also additional factors within the protein or the cell.

Characterization of a gene encoding tRNA nucleotidyltransferase from Candida glabrata.

A gene encoding ATP (CTP):tRNA nucleotidyltransferase (EC2.7.7.25) was isolated from Candida (Torulopsis) glabrata by complementation in Saccharomyces cerevisiae. The predicted amino acid sequence of the protein revealed a large region with high sequence similarity to members of the Class II group of the nucleotidyltransferase superfamily and an N-terminal region characteristic of a mitochondrial targeting sequence. The essential role of the carboxylates within the conserved DXD and RRD motifs was confirmed by mutagenesis. C. glabrata strains bearing truncated CCA1 genes that lacked sequences encoding the putative mitochondrial targeting peptide were unable to grow on non-fermentable carbon sources but were able to grow on a fermentable carbon source. These results suggest that, as in S. cerevisiae, the C. glabrata CCA-adding enzyme is a sorting isozyme that functions in multiple cellular compartments. Mapping of the 5'-ends of primary transcripts of CCA1 revealed multiple transcription start sites located both upstream of and between two in-frame start codons. When the cells were cultured on a non-fermentable carbon source the longer transcripts appeared more abundant, suggesting that the choice of transcription start sites was influenced by carbon source. The shorter transcripts, which lacked sequences encoding the mitochondrial targeting information, were more predominant in cells grown on glucose. These observations suggest that expression of CCA-adding isozymes in C. glabrata may be regulated. The DNA sequence has been assigned GenBank Accession No. AF098803.

In vitro aggregation of the regulated secretory protein chromogranin A.

Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation.