FIG. 5.

Depletion of the essential mitochondrial Isu scaffold proteins causes impairment in the 2-thio modification of cy-tRNA_UUU^Lys2but not of mt-tRNA_UUU^Lys. (A) Isu1 levels were determined by immunological analysis with anti-Isu1 antibody in Gal-ISU1/Δisu2 cells grown with galactose (Gal) or glycerol (Gly) for 48 h. (B) Total tRNAs of Gal-ISU1/Δisu2 cells grown with galactose (Gal) for 12 h or glycerol (Gly) for 49 h and 72 h were examined by APM-Northern analysis. Total tRNAs of Gal-NFS1/YN cells were tested in a similar fashion. The 2-thio modifications of cy-tRNA_UUU^Lys2(left) andmt-tRNA_UUU^Lys(right) were analyzed by the APM-Northern method. The 2-thio-modified and unmodified tRNAs are shown as in Fig. 1C. (C) The 2-thio modifications of cy-tRNA_UUU^Lys2and mt-tRNA_UUU^Lyswere examined with the single deletion mutants Δisu1 and Δisu2. These cells, both of which showed normal growth rates, were grown with glucose for 20 h. Gal-ISU1/Δisu2 cells grown with glucose medium for 48 h to repress the expression of Isu1 in the absence of Isu2 were analyzed in parallel. The same amounts of tRNA samples were examined in a denaturing gel with APM (upper left panel) and without APM (w/o APM). The oligonucleotide probes used were the same as shown in Fig. 1. The right panel indicates the relative proportions of 2-thio-modified tRNAs estimated from APM-Northern analysis shown in the upper left panel, as described in the legend to Fig. 1B.