Figure 4.

Mitochondrial import efficiency of RRL-pMORF3 and WGL-pMORF3 under various conditions. A, RRL-pMORF3 (lanes 2 and 3) and WGL-pMORF3 (lanes 7 and 8) were successfully imported into mitochondria. In the presence of valinomycin, the import of RRL-pMORF3 (lanes 4 and 5) and WGL-pMORF3 (lanes 9 and 10) was inhibited. Proteinase K (PK) was used to digest nonimported pMORF3 (lanes 3, 5, 8, and 10). B, Preincubation of RRL-pMORF3 with WGL (lanes 4 and 5) and LE (lane 7) reduced the mitochondrial import efficiency. Import experiments were repeated three times, and statistical differences were based on one-way ANOVA followed by Tukey’s honestly significant difference test. The amount of imported mMORF3 without the addition of WGL or LE (lane 2) was taken as 1. Values marked with different letters are significantly different (P < 0.01). C, Preincubation of RRL-pMORF3 with NaF (lane 3), STY8 (lane 4), or both (lane 5) reduced the mitochondrial import efficiency. The asterisk represents an additional 10 min of incubation with kinase assay buffer before the in vitro kinase assay. Import experiments were repeated three times, and statistical differences were based on one-way ANOVA followed by Tukey’s honestly significant difference test. The amount of imported mMORF3 without STY8 or NaF treatment (lane 2) was taken as 1. Values marked with different letters are significantly different (P < 0.01). D, RRL-pMORF3-T17/S20/T35A (lanes 4 and 5) was imported normally into mitochondria. The amount of imported mMORF3 without NaF treatment (lanes 2 and 5) was taken as 1. In the presence of NaF, the import rate was reduced. Statistical differences at P < 0.01 (**) were based on Student’s t test.