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. 2003 Jun;23(11):4000–4012. doi: 10.1128/MCB.23.11.4000-4012.2003

FIG. 1.

FIG. 1.

The mature tRNAAla is imported in vitro into potato mitochondria. (A) Cloverleaf structure of the mature A. thaliana cytosolic tRNAAla used as a substrate for standard in vitro import assays. (B) Protection of labeled tRNAAla transcripts from RNase degradation. Standard in vitro import assays of 100-μl volumes containing isolated mitochondria corresponding to 400 μg of proteins and 105 cpm of labeled tRNAAla substrate were performed. Reactions were carried out in the presence of 5 mM ATP. After incubation, RNase A and RNase T1 were added to various final concentrations ranging from 0 to 300 μg/ml and from 0 to 1,500 U/ml, respectively. (C) tRNA import is pH dependent. Standard in vitro import assays of 100 μl containing isolated mitochondria corresponding to 400 μg of proteins and 105 cpm of labeled tRNAAla substrate. Reactions were performed in the presence of 5 mM ATP but at different pHs obtained by adjusting the pH of the standard import mixture either with HCl or with NaOH. After import, the pH of all samples was adjusted to 7.2 with HCl or NaOH before addition of RNase A and RNase T1 to final concentrations of 300 μg/ml and 750 U/ml, respectively. Quantification of the amount of protected RNAs was done using a phosphorimager.