Arc1p was found in a screen for components that interact genetically with Los1p, a nuclear pore‐associated yeast protein involved in tRNA biogenesis. Arc1p is associated with two proteins which were identified as methionyl‐tRNA and glutamyl‐tRNA synthetase (MetRS and GluRS) by a new mass spectrometry method. ARC1 gene disruption leads to slow growth and reduced MetRS activity, and synthetically lethal arc1‐ mutants are complemented by the genes for MetRS and GluRS. Recombinant Arc1p binds in vitro to purified monomeric yeast MetRS, but not to an N‐terminal truncated form, and strongly increases its apparent affinity for tRNAMet. Furthermore, Arc1p, which is allelic to the quadruplex nucleic acid binding protein G4p1, exhibits specific binding to tRNA as determined by gel retardation and UV‐cross‐linking. Arc1p is, therefore, a yeast protein with dual specificity: it associates with tRNA and aminoacyl‐tRNA synthetases. This functional interaction may be required for efficient aminoacylation in vivo.

Two yeast enzymes that catalyze aminoacylation of tRNAs, MetRS and GluRS, form a complex with the protein Arc1p. We show here that association of Arc1p with MetRS and GluRS is required in vivo for effective recruitment of the corresponding cognate tRNAs within this complex. Arc1p is linked to MetRS and GluRS through its amino-terminal domain, while its middle and carboxy-terminal parts comprise a novel tRNA-binding domain. This results in high affinity binding of cognate tRNAs and increased aminoacylation efficiency. These findings suggest that Arc1p operates as a mobile, trans-acting tRNA-binding synthetase domain and provide new insight into the role of eukaryotic multimeric synthetase complexes.

Yeast Los1p, the homolog of human exportin-t, mediates nuclear export of tRNA. Using fluorescence in situ hybridization, we could show that the export of some intronless tRNA species is not detectably affected by the disruption of LOS1. To find other factors that facilitate tRNA export, we performed a suppressor screen of a synthetically lethal los1 mutant and identified the essential translation elongation factor eEF-1A. Mutations in eEF-1A impaired nuclear export of all tRNAs tested, which included both spliced and intronless species. An even stronger defect in nuclear exit of tRNA was observed under conditions that inhibited tRNA aminoacylation. In all cases, inhibition of tRNA export led to nucleolar accumulation of mature tRNAs. Our data show that tRNA aminoacylation and eEF-1A are required for efficient nuclear tRNA export in yeast and suggest coordination between the protein translation and the nuclear tRNA processing and transport machineries.

Saccharomyces cerevisiae cells that carry deletions in both the LOS1 (a tRNA export receptor) and the PUS1 (a tRNA:pseudouridine synthase) genes exhibit a thermosensitive growth defect. A Schizosaccharomyces pombe gene, named spPUS1, was cloned from a cDNA library by complementation of this conditional lethal phenotype. The corresponding protein, spPus1p, shows sequence similarity to S. cerevisiae and murine Pus1p as well as other known members of the pseudouridine synthase family. Accordingly, recombinant spPus1p can catalyze in vitro the formation of pseudouridines at positions 27, 28, 34, 35 and 36 of yeast tRNA transcripts. The sequence and functional conservation of the Pus1p proteins in fungi and mammalian species and their notable absence from prokaryotes suggest that this family of pseudouridine synthases is required for a eukaryote-specific step of tRNA biogenesis, such as nuclear export.

Exit of tRNA from the nucleus was shown, long time ago, to be a saturable and carrier-mediated process. Nevertheless, only recently, progress in the field of nucleocytoplasmic transport gave first insight into the mechanism of tRNA nuclear export. A nuclear export receptor for tRNA (Loslp/Xpo-t), belonging to the importin (karyopherin) family, has been characterized in yeast and mammalian cells. Mature tRNA molecules can associate with Loslp/ Xpo-t and the GTP-bound form of the small GTPase Ran to form an export complex in the nucleus.This complex translocates through the nuclear-pore complexes and dissociates upon GTP hydrolysis in the cytoplasm. GenetLc studies [n yeast have, however, shown that LOS1 is not essential, unless additional stepa in the tRNA biogenesis pathway are impaired, suggesting the existence of additional tRNA nuclear export routes. Furthermore, modification and aminoacylation of tRNA may also be important for efficient transport of tRNA into the cytoplasm.

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Abstract. The immunoreactivity of hypoxia inducible factor 1 · (HIF-1·) has been considered a reliable indicator of the HIF-1 pathway activation in tissue hypoxia. However, HIF-1· immunoreactivity has been evaluated with different antibodies and heterogeneous protocols. The need to interpret contradictory findings requires, among other things, a comparison of the antibodies. This could be accomplished by using identical, well characterized antigenic targets and by decreasing the influence of other variables. We applied most of the commercially available antibodies, and an antibody developed in our laboratories, to the human cervical cancer HeLa cell line and tissue sections from a renal cell carcinoma systematically, and to other tumors selectively. The expression of HIF-1· in HeLa cells was induced by the hypoxia-mimetic DFO. Non-induced HeLa cells were used as 'genuine' negative controls in addition to routine ones. HeLa cells (both induced and not induced) were also examined by immunofluorescence and Western blotting. We found that the antibodies showed immunostaining patterns with remarkable qualitative and quantitative differences, an observation not emphasized in previous literature. Certain antibodies require careful application to avoid specificity issues, and others to avoid low sensitivity problems. Pairing certain antibodies can optimize evaluation of HIF-1· expression. Most previous immunohistochemical studies of HIF-1· have attempted to map hypoxic neoplastic tissues or to demonstrate hypoxia in studies of neoangiogenesis, rather than 'measuring' HIF-1· expression or activation, because this requires a validated immunoassay. Our study thus allows for the development of a controlled and comparative HIF-1· immunoassay, which could be valuable if HIF-1· becomes a therapeutic target.

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