Mitochondrial protein import: modification of sulfhydryl groups of the inner mitochondrial membrane import machinery in Solanum tuberosum inhibits protein import

Protein import into mitochondria involves several components of the mitochondrial outer and inner membranes as well as molecular chaperones located inside mitochondria. Here, we have investigated the effect of sulfhydryl group reagents on import of the in vitro transcribed/translated precursor of the F₁ subunit of the ATP synthase (pF₁) into Solanum tuberosum mitochondria. We have used a reducing agent, dithiothreitol (DTT), a membrane-permeant alkylating agent, N-ethylmaleimide (NEM), a non-permeant alkylating agent, 3-(N-maleimidopropionyl)biocytin (MPB), an SH-group specific agent and cross-linker 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) as well as an oxidizing cross-linker, copper sulfate. DTT stimulated the mitochondrial protein import, whereas NEM, MPB, DTNB and Cu²⁺ were inhibitory. Inhibition by Cu²⁺ could be reversed by addition of DTT. The efficiency of inhibition was higher in energized mitochondria than in non-energized. We have dissected the effect of the SH-group reagents on binding, unfolding and transport of the precursor into mitochondria. Our results demonstrated that the inhibitory effect of NEM, DTNB and Cu²⁺ on the efficiency of import was not due to the interaction of the SH-group reagents with import receptors. Modification of pF₁ with NEM prior to the import resulted in stimulation of import, whereas DTNB and Cu²⁺ were inhibitory. NEM, MPB, DTNB and Cu²⁺ inhibited import of the NEM-modified pF₁ into intact mitochondria. Import of pF₁ through a receptor-independent bypass-route as well as import into mitoplasts were sensitive to DTT, NEM, MPB, DTNB and Cu²⁺ in a similar manner as import into mitochondria. As MPB does not cross the inner membrane, these results indicated that redox and conformational status of SH groups located on the outer surface of the inner mitochondrial membrane were essential for protein import.