Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.
Wild-type apocytochrome c and its hydrophobic segment deleted mutants, named Δ28–39, Δ72–86 and Δ28–29/72–86 were constructed, expressed and highly purified respectively. Insertion ability into phospholipid monolayer, inducing leakage of entrapped fluorescent dye fluorescein sulfonate (FS) from liposomes, and translocation across model membrane system showed that the wild-type apoprotein and Δ28–39 almost exhibited the same characteristics, while mutants with segment 72–86 deletion did not. Furthermore, CD spectra, intrinsic fluorescence emission spectra, and the accessibility of the protein to the fluorescence quenchers: KI, acrylamide and HB demonstrated that the segment 72–86 deletion has a significant effect on the conformational changes of apocytochrome c following its interaction with phospholipid. On the basis of these results it is postulated that the C-terminal hydrophobic segment 72–86 plays an important role in the translocation of apocytochrome c across membrane.