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Fig. 4. MTS-GFP-DHFR import into mitochondria is inhibited in the presence of aminopterin. (A-C) CHOMTS-GFP-DHFR cells were grown on coverslips in the absence (A) or presence (B and C) of doxicycline. Cells shown in C were treated with aminopterin (50 mM) throughout induction of protein synthesis. Cells were fixed using paraformaldehyde followed by the analysis of GFP-derived fluorescence using a Zeiss LSM 510 confocal microscope. (D) Biochemical analysis of MTS-GFP-DHFR import into mitochondria in the presence and absence of aminopterin. CHOMTS-GFP-DHFR cells were grown in the presence of doxicycline. Where indicated, aminopterin (ao; 50 mM) was added throughout induction of protein synthesis. The various cell lysates were analysed by SDS-PAGE and western blotting using affinity-purified anti-GFP antibodies. Three species of MTS-GFP-DHFR are detected, the precursor (*) as well as an intermediate (
) and the fully processed form (
).
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