Role of nuclear pools of aminoacyl-tRNA synthetases in tRNA nuclear export

Abstract

Reports of nuclear tRNA aminoacylation and its role in tRNA nuclear export (Lund and Dahlberg, 1998; Sarkar et al., 1999; Grosshans et al., 20001) have led to the prediction that there should be nuclear pools of aminoacyl-tRNA synthetases. We report that in budding yeast there are nuclear pools of tyrosyl-tRNA synthetase, Tys1p. By sequence alignments we predicted a Tys1p nuclear localization sequence and showed it to be sufficient for nuclear location of a passenger protein. Mutations of this nuclear localization sequence in endogenous Tys1p reduce nuclear Tys1p pools, indicating that the motif is also important for nucleus location. The mutations do not significantly affect catalytic activity, but they do cause defects in export of tRNAs to the cytosol. Despite export defects, the cells are viable, indicating that nuclear tRNA aminoacylation is not required for all tRNA nuclear export paths. Because the tRNA nuclear exportin, Los1p, is also unessential, we tested whether tRNA aminoacylation and Los1p operate in alternative tRNA nuclear export paths. No genetic interactions between aminoacyl-tRNA synthetases and Los1p were detected, indicating that tRNA nuclear aminoacylation and Los1p operate in the same export pathway or there are more than two pathways for tRNA nuclear export.

Figure 1

Cellular location of Tys1p-GFP. The location of Tys1p-GFP encoded by YCpTys1-GFP in ts2 cells was determined by autofluorescence. (A) Detection of GFP. v, identifies a vacuole; n, identifies a nucleus. (B) DAPI staining of the DNA in the same cells. (C) Overlap of images A and B. Bar, 10 μm.

Figure 2

Ability of variant TYS1 constructs to complement the ts phenotype of the tys1-1 mutation. ts2 mutant cells with the indicated plasmids were grown and serially diluted, and aliquots were spotted onto complete minus Ura media. Plates were incubated at 23 or 37°C, as indicated, for 3 days.

Figure 3

Location of Tys1p by subcellular fractionation. (A) Western analysis to show the specificity of anti-myc to identify Tys1p-myc proteins. Lane 1, ts2 cells containing vector alone; lane 2, ts2 cells with YCpTYS1-myc; lane 3, ts2 cells with YCpTYS1-nls1-myc; lane 4, ts2 cells with YCpTYS1-nls2-myc. (B) Same blot as for A using antisera 6142 to identify Rna1p. (C) Location of myc-tagged proteins in cell fractions. Lanes 1–3, extracts from cells with YCpTYS1-myc; lanes 4–6, extracts from cells with YCpTYS1-nls1-myc; lanes 7–9, extracts from cells with YCpTYS1-nls2-myc. Lanes 1,4, and 7 have total cell extracts. Lanes 2, 5, and 8 have nuclear-enriched fractions. Lanes 3, 6 and 9 have cytosolic-enriched fractions. (D) The same blot as in C incubated with 32D6 monoclonal anti-Nsp1p to assess the fractionation of an authentic nuclear protein. (E) The same blot as in C incubated with anti-sera 6142 anti-Rna1p to assess the fractionation of a protein primarily located in the cytosol.

Figure 4

Sequence alignment of tyrosyl-tRNA synthetases. Schematic diagram showing alignment of selected eukaryotic cytosolic tyrosyl synthetases with consensus block diagrams of archaeal and eubacterial counterparts. The shaded boxes represent blocks of similarity and do not infer any structural information. The eubacterial shading is deliberately different to reflect dissimilarity from the eukaryotic and archaeal Tys1 proteins. The sequence of a putative ADEPT near the carboxyl terminus is shown below the alignments. Columns to the right indicate the length of the proteins in amino acids and whether cDNA and genomic DNA sequence information is complete (C) or incomplete (I). Cons (n) indicates that a consensus sequence from designated number of organisms was used to generate the block diagram. An x marks the locations of introns. Accession numbers: human (HumYARS), 2665519; Drosophila melanogaster (DmelTys1), 7294108; Caenorhabditis elegans (CelYARS), 7320764; Arabidopsis thaliana (AthYARS), 6560759; rice, expressed sequence tags: AU081543, AU077843, AU081542, AU077844, AQ365016, AQ865733; S. pombe (SpTys1p) 3183174; S. cerevisiae, (ScTys1p), 6321624; C. albicans (CaTys1p), Con4-2782.

Figure 5

Cellular location of Tys1-β-galactosidase fusion proteins. The location of β-galactosidase signal in yeast cells containing plasmids encoding portions of Tys1p fused in-frame to β-galactosidase was determined by indirect immunofluorescence. Left, FITC detection of anti-β-galactosidase. Right, DAPI staining of DNA. (A and A′) Yeast cells containing the parent pFB1-7a vector encoding a cytosolic β-galactosidase. (B and B′) The plasmid pFB1-67a encodes a fusion of the H2B NLS to β-galactosidase. (C and C′ to G and G′) Plasmids encode the indicated wild-type Tys1p amino acids fused in-frame to β-galactosidase. (H and H′) The plasmid encodes changes of four basic amino acids to four acidic amino acids as indicated by bold letters. Bar, 5μm.

Figure 6

Tyrosyl-tRNA synthetase activity in cells with various TYS1 alleles. Extracts were prepared from ts2 mutant cells containing plasmids with various TYS1 alleles and were assayed for the incorporation of [³H]tyrosine and ¹⁴C-amino acids into tRNA. The ³H/¹⁴C ratio of an average of two independent assays at the 10-min time point is shown.

Figure 7

In situ hybridization for cells containing Tys1p nls mutations. ts2 cells containing YCpTYS1-myc (A–D and A′–D′), YCpTYS1-nls1-myc (E–H and E′–H′), or YCpTYS1-nls2-myc (I–L and I′–L′) were studied. Cells in A, E, and I were probed with an oligonucleotide specific for tRNA^Tyr; B, F, and J were probed with an oligonucleotide specific to tRNA^Met; C, G, and K were probed with an oligonucleotide specific for tRNA^Ile; and D, H, and L were probed with oligo(d)T, specific for poly(A) RNA. A′ to L′ are the same cells stained with DAPI to located nuclei. Bar, 5 μm.

Figure 8

Growth of cells containing los1 and aminoacyl-tRNA synthetase mutations. Cells were grown and serially diluted, and aliquots were spotted onto solid rich media and were incubated at 16, 23, 29, or 34°C, as indicated for 3 days. Strains utilized: TYS1 LOS1, SS328; tys1-1 LOS1, ts2; tys1-1 los1:: Kan^r, SSS708; TYS1 los1:: Kan^r, SSS706, derived from SS328; ILS1 LOS1, A364a; ils1-1 LOS1, ts341, derived from A364a; ils1-1 los1:: Kan^r, SSS705, derived from ts341; ILS1 los1:: Kan^r, SSS707, derived from A364a; MES1 LOS1, A364a; mes1-1 LOS1, ts19:3:4, related to A364a; mes1-1 los1:: Kan^r, SSS703, derived from ts19:3:4; MES1 los1:: Kan^r, SSS707.

Figure 9

Two possible models for the different tRNA nuclear export pathways. (A) tRNA aminoacylation in the nucleus and Los1p act in series in one tRNA nuclear export pathway, and alternative pathway(s) remain unidentified. (B) tRNA aminoacylation in the nucleus and Los1p act in parallel in two tRNA nuclear export pathways, and there are other important export pathways yet to be identified.