FIGURE 1.

A6-ASRE and A6-ASRE MLS⁽⁻⁾ expression and effect on PF T. brucei growth. (A) A6-ASRE and A6-ASRE MLS⁽⁻⁾ constructs were developed against GUUAUUGG sequences present in the 3′ UTR of ATPase 6 edited mRNA. A6-ASRE construct contains an N-terminal 14 amino acid MLS from dihydroplipoyl dehydrogenase Tb11.01.8470. This sequence is recognized and cleaved upon import (arrowhead) resulting in the retention of five amino acids (residues in bold). The MLS is followed by a Flag epitope tag fused with a T. brucei codon optimized Puf–linker-PIN domain. The A6-ASRE MLS⁽⁻⁾ lacks the mitochondrial localization signal. (B) Northern blot of total cell RNA from A6-ASRE cells taken at 0, 12, or 24 h post tetracycline induction (1 µg/mL) and hybridized with a probe specific for the A6-ASRE sequence. (C) Cell fractionation was carried out on uninduced and induced (1 µg/mL tetracycline, 24 h) cells. Total cellular (T), cytosolic (C), mitochondrial (M), mitochondrial membrane (Mm), and mitochondrial matrix (Ma) fractions were obtained. Cell equivalents were loaded at 1 × 10⁷ for TC, C, M, Mm; while 1 × 10⁸ was loaded for the Ma fraction. Samples were fractionated on SDS-PAGE (10%), and either Coomassie stained (top panel) or transferred for Western blotting and probed with α-Flag (bottom panel). A cross-reacting band of 50 kDa (asterisk) was seen in total cell and cytoplasmic fractions and absent from the mitochondrial fractions (M). An α-Flag reactive band of ∼60 kDa was present in the A6-ASRE-induced cell and fractionated with the mitochondrial matrix (Ma) (arrowhead). (D) Growth of PF T. brucei 29-13 A6-ASRE in the presence (open square) or absence (closed square) of tetracycline (1 µg/mL). Inset shows A6-ASRE expression at 24-h post-induction (arrowhead) and presence of previously described ∼50 kDa cross-reacting band (asterisk). Each lane was loaded with 1 × 10⁷ cell equivalents. (E) Growth of PF T. brucei 29-13 A6-ASRE MLS⁽⁻⁾ in the presence (open square) or absence (closed square) of tetracycline (1 µg/mL). Inset shows A6-ASRE MLS⁽⁻⁾ expression at 24-h post-induction (arrowhead) and presence of described ∼50 kDa cross-reacting protein (asterisk). Each lane was loaded with 1 × 10⁷ cell equivalents.