Comparison of Sol and Nag families. (A) Dendrogram showing similarities between selected members of the Sol family and two members of the Nag family. Percentage of similarities to ScSol1p (shown in black) and ScSol3p (shown in red) are overlayed on the CLUSTAL X generated dendrogram (see materials and methods). ScSol1p, ScSol2p, ScSol3p, and ScSol4p, S. cerevisiae Sol family members; CaSol1p and CaSol3p, Candida albicans; SpSol1p, Schizosaccharomyces pombe; Hs6PGL, H. sapiens; Dm6PGL, Drosophila melanogaster; At6PGL1, Arabidopsis thaliana; Tb6PGL, Trypanosoma brucei; Pae6Pgl, Pseudomonas aeruginosa; AnaDevB, Anabaena sp.; and TmaDevB, T. maritima. HsH6PD, H. sapiens, and Pf6PGL, Plasmodium falciparum, are two members of this family that contain both 6Pgl and G6PD domains. HsOscl, human oscillin (GNP1, glucosamine-6-phosphate deaminase), and EcNagB, E. coli, are two members of the Nag family. A schematic is also presented showing the relationships between these two families. Helices are shown in red boxes and sheets are shown in blue boxes. Green boxes represent regions of special importance to the catalytic activity of the Nag family (Rudino-Pinera et al. 2002; Arreola et al. 2003). Protein lengths are indicated to the right. (B) Alignment of Sol and Nag proteins. CLUSTAL X alignment of selected family members, beginning with residue 91 of ScSol1p. Other members are the same as in A. Color coding of amino acids is turned on when at least 10% of the residues in a column are identical or similar on the basis of the BLOSUM 62 matrix. LIVM, blue; YFW, magenta; STAGC, green; DE, red; KRQHN, brown; P, dark blue. Background shading reflects the percentage of identity/similarity within a column: 15–49%, light gray background; 50–74%, yellow background; >74%, yellow background with the consensus line underlined. Similar residues that have higher conservation in the Nag family than in the Sol family are indicated by white type on a dark gray background. Structural information is coded as above, helices are indicated on a red background, and sheets are indicated on a blue background, while a green background represents regions of importance to catalytic activity. Catalytic residues in the Nag family are indicated by asterisks. (C) Structural comparison of the Sol and Nag families. Shown are views of the same face of TmaDevB (1PBT; Kim et al. 2003) and HsOscl (Arreola et al. 2003). Color coding is the same as in A and B. Structures were generated from the crystallographic coordinates with Swiss-Pdb Viewer.

6-Phosphogluconolactonase assay. Yeast glucose-6-phosphate dehydrogenase was incubated with glucose-6-phosphate and the production of NADPH was monitored at 340 nm for 8 min. Cell extract (50 μg protein) and 1.5 units 6-phosphogluconate dehydrogenase were added to the reaction mix and the absorbance at 340 nm was monitored for an additional 10 min. Black open circles, strain MLW115 with vector pRS426; red solid circles, MLW115 + pRSSOL1; green open triangles, MLW115 + WS80 (pRSSOL2); dark-blue solid triangles, MLW115 + WS92 (pRSSOL3); orange open squares, MLW115 + pRSSOL4; gold open triangles, MLW115 + pGST-SOL4; purple solid squares, X2316-3C + pRS426 vector; light-blue solid triangles, no extract. Note that even though GST-SOL4 transcription is regulated by copper, cells grown under noninducing conditions possess significant activity.

Sol1p and Sol2p apparently function in the distribution of tRNA between the nucleus and the cytosol. Fluorescence in situ hybridization analysis of tRNA location in wild-type (X2316-3C), sol1::URA3 (WCS2), sol2::URA3 (WCS7), sol4::hph^r (MLW104), and los1::kanMX4 (YD05055) mutant cells as indicated. (First row) Hybridization employing probe for tRNA^Met. (Second row) The same cells counterstained with DAPI to detect DNA. (Third row) Hybridization employing probe for tRNA^Tyr. (Fourth row) The same cells as in third row, counterstained with DAPI.

Growth and suppression assays. (A) Ability of multi-copy SOL1 and multi-copy SOL4 to suppresses the los1 growth defect. LOS1 and los1::kan^r cells containing pRS426, pRSSOL1, or pRSSOL4 were grown to saturation, serially diluted, and aliquots were spotted onto solid media lacking uracil and incubated at 23°, 34°, or 37° for 3 days. (B) Effect of deletion of SOL genes upon tRNA-mediated ade2-1 nonsense suppression at various temperatures. Strains were patched onto YEPD and then replicate plated onto defined media lacking adenine and incubated at the indicated temperatures for 3 days.

Indirect immunofluorescence location of Sol proteins. Strain SS700 containing pGST-SOL2 or pGST-SOL4, either uninduced (unind) or induced for 1 hr by addition of Cu₂SO₄ to a final concentration of 0.5 mm (ind), YCpSOL1-ET, or pRSSOL1-ET. (Top) Cells were incubated with mouse anti-GST at a dilution of 1:500 or with mouse anti-HA at a dilution of 1:500; Cy3 anti-mouse antibody was used as secondary antibody. (Bottom) The overlay of DNA staining via DAPI and Cy3 detection of the mouse antibodies.