Sen2p and Sen54p are localized to mitochondria. (A) Sen2p in a wild-type strain, TYSC188 (WT), and an overproducer with pYU042 (OP) was visualized by immunofluorescence with affinity-purified anti-Sen2p antibodies (1st column). Mitochondria and the nucleus were visualized with an anti-Por2p mAb (2nd column) and DAPI (3rd column). Three images, green for Sen2p, red for Por2p, and blue for DAPI, are merged in the 4th column. (B) Sen54p in TYSC188 (WT) and TYSC188/pTYSC161 (OP) was visualized with affinity-purified anti-Sen54p antibodies as in A. The Western blotting in the right most panels demonstrates the specificity of these antibodies. Lane 1, wild-type cells; lane 2, cells only expressing Sen2p-FLAG₃ (A) or Sen54p-FLAG₃ (B). The gel mobility change of the single bands indicates that the antibodies recognize only their target proteins. (C) SEN2-proteinA (Sen2p-protein A) and SEN54-protein A (Sen54p-protein A) strains and their parental strain TYSC188 (no protein A) were subjected to immunofluorescence with anti-protein A antibodies as in A. The Western blotting in the right most panels demonstrates expression of the protein A fusion proteins. Lane 1, wild-type cells; lane 2, cells only expressing Sen2p-protein A; lane 3, cells only expressing Sen54p-protein A.

The TOM70N-SEN54 fusion gene can replace the authentic SEN54. (A) A low-copy plasmid with either no insert (Vec; pRS314), SEN54 (54; p314–54), or TOM70NSEN54 (70N-54; p70N-54) was introduced into a SEN54/Δsen54::HIS3 diploid, and the resulting diploids were sporulated. Their tetrads were dissected and cultured on YPD at 23°C. (B) The localization of Tom70N-Sen54p was examined by immunofluorescence (Sen54p; left). The mitochondria were visualized with an anti-Por2p antibody (Por2p; middle). The right panel shows Western blotting of total extracts prepared from the wild-type cells (lane 1) and the TOM70N-SEN54 cells (lane 2). (C) Crude membranes were prepared from the wild-type cells (54) and the TOM70N-SEN54 cells (70N-54). The membranes were extracted as in Figure 3C. Closed arrowhead, authentic Sen54p; open arrowhead, Tom70N-Sen54p. (D) Total RNAs were prepared from the strains expressing either SEN54 (lane1) or TOM70N-SEN54 (lane2). One microgram of each was analyzed by Northern blotting with probes recognizing unspliced and mature tRNA-Ile^UAU (I^UAU), pre-tRNA-Leu^CAA (L^CAA), or pre-tRNAPro^UGG (P^UGG). On the top panel, the 5.8S and 5S rRNAs on the gel before transfer were visualized by ethidium bromide staining as controls for loading. (E) Cell extracts were prepared from the SEN54 (54) and TOM70NSEN54 (70N-54) cells, and their endonuclease activity was assayed at 30°C for 10 min with pre-tRNA-Phe^GAA as a substrate. The indicated amounts (μg protein) of each total extract were added to 10-μl reaction mixtures. Expected products are represented as in Figure 2B.

Partial deletion of the Sen54p mitochondrial localization signal impaired tRNA splicing. (A) A schematic diagram of sen54 mutants with partial deletion in the mitochondrial localization signal (residues 200–313). The blank area between the bars indicates the deleted region in each mutant. (B) A TRP1 low-copy plasmid harboring the wild-type SEN54 gene or the mutant sen54 genes with the deletion of Δ200-232 or Δ275-313 was introduced into a haploid strain whose chromosomal disruption of SEN54 was complemented with a URA3 plasmid with wild-type SEN54 (none). Mutant genes fused with a TOM70N region at their N-termini were also introduced in the same recipient (70N). Yeast cells dependent on the SEN54 genes on the TRP1 plasmids were selected on 5′-FOA plates, and their growth was compared at the indicated temperatures. (C) The localization of Sen54p in the wild-type (54), sen54Δ200-232 (Δ200), and TOM70N-sen54Δ200-232 (70N-Δ200) cells at 30°C was visualized by immunofluorescence as in Figure 1A. (D) The localization of Sen2p in the wild-type (54) and sen54Δ200-232 (Δ200) cells was visualized as above.

Sen2p, Sen54p, and tRNA splicing endonuclease activity are cofractionated with mitochondria but not with the nucleus. (A) The MSP fraction prepared from wild-type cells was separated on a 20–80% wt/vol sucrose density gradient and was collected from the bottom. In the top panel, the recoveries of Nsp1p (▵), Tim23p (□), and Sec63p (○) were compared with that of the endonuclease activity (•). In the lower panel, the recoveries of Sen2p (pluses), Sen54p (crosses), and the enzyme activity (•) were compared. (B) Fractionations similar to A were performed for SEN2 cells (top) and sen2-3 cells (bottom). The endonuclease activities were assayed, and the products were analyzed by urea-PAGE. To visualize the products of the minor peak fractions, the gel was overexposed. The expected products are schematically represented on the right. (C) Mitochondria and nuclei were prepared from wild-type cells (TYSC188) with Nycodenz gradient and PVP-sucrose gradient centrifugation, respectively. Ten micrograms of total lysate and the purified mitochondria in the mitochondrial preparation (left column; lane L and lane M, respectively) were subjected to Western blotting with the antibodies listed on the left. Similar Western blotting analysis was done for samples of the nuclear preparation (right column; lane L for lysate and lane N for nuclei). Signal ratios (M vs. L, or N vs. L) are listed on the right of each panel.

Sen2p and Sen54p are peripherally associated with the mitochondrial surface. (A) Mitochondrial vesicles prepared from the Nycodenz-purified mitochondria were separated by a sucrose density gradient. Recoveries of marker proteins and Sen2p were quantified by Western blotting. ⋄, Tom70p; □, Tim23p; ▵, Nsp1p; •, Sen2p. The fraction P represents the pellet of the gradient. (B) The intact mitochondria were treated with 0.1 mg/ml proteinase K at 4°C for 20 min in the absence or presence of 0.2%wt/vol Triton X-100 and were analyzed by Western blotting. (C) The mitochondria (T) were extracted with lysis buffer (Buffer), 0.2 M Na₂CO₃ (Na₂CO₃) or lysis buffer with 1% Triton X-100 + 1 M NaCl (Triton) and were separated into a pellet (P) and a supernatant (S) by centrifugation at 100,000 × g for 30 min. Each protein was detected with specific antibodies. (D) The mitochondria were extracted as in C. The supernatants were passed through gel filtration columns to exchange the buffers to lysis buffer with 1% Triton X-100. The pellets were suspended in the same buffer. The endonuclease activity in each fraction was assayed and is represented by the recovery.

sen2-41, a new temperature-sensitive allele of SEN2. (A) The growth of strains with a chromosomal disruption of SEN2, complemented by either wild-type SEN2 or sen2-41 on a low-copy plasmid, was compared on YPD at the indicated temperatures. (B) Wild-type, Δlos1 mutant, and sen2-41 mutant strains were cultured at 23°C (23) and were shifted to 37°C (37) for 4 h. Pre-tRNAs in 1 μg of total RNAs from each culture were detected with specific probes, as in Figure 4D. (C) The tRNA endonuclease activity in the wild-type (wt) and sen2-41 cells (sen2) was assayed as in Figure 4E.

sen2-41 accumulates unspliced pre-tRNAs in the cytosol. (A) Time course of unspliced pre-tRNA accumulation in sen2-41 cells was monitored by FISH. Wild-type cells (1st and 2nd columns) and sen2-41 cells (3rd and 4th columns) were cultured at 23°C, shifted to 37°C and then harvested at 0, 1, 2, 3, and 4 h after the shift. pre-tRNA-Ile^UAU was detected with an intron-specific probe (pre-tRNA), and the position of nucleus was visualized by DAPI staining (DAPI). Bars, 10 μm. (B) Amounts of precursor forms of tRNA-Ile^UAU were monitored by Northern blotting. Total RNA samples were prepared from the wild-type and sen2-41 cells in a similar time course experiment as in A. One microgram of the total RNA was analyzed by Northern blotting with the intron-specific probe. The expected precursor forms are schematically represented on the left. (C) The localization of U14 snoRNA was visualized by FISH. Wild-type (wt) and sen2-41 (sen2) cells were fixed after 3-h exposure at 37°C and stained with a probe against U14 snoRNA (U14 sno) to visualize the nucleolus. DNA was stained with DAPI (DAPI). (D) The localization of mRNA in sen2-41 cells was visualized by FISH with oligo-dT₅₀ probe as in C.

Δlos1 sen2 double mutant accumulates a part of pre-tRNAs in the nucleus. Unspliced precursor (1st and 4th columns) and all (2nd and 5th columns) forms of tRNA-Ile^UAU in wild-type cells (a–c, g–i), sen2-41 cells (m–o, s–u), Δlos1 cells (d–f, j–l), and Δlos1 sen2-41 double mutant cells (p–r, v–x) were visualized by FISH. DNA was detected with DAPI (3rd and 6th columns). Each strain was cultured at 23°C (23°C), and then its aliquot was shifted to 37°C for 3 h (37°C). Bars, 2 μm.