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

摘要

Effects of sulfhydryl (SH) group reagents on import of the in vitro transcribed/translated precursor of the F1beta subunit of ATP synthase [adenosinetriphosphatase] (pF1beta) into S. tuberosum mitochondria were investigated. The following sulfhydrylgroup reagents were tested: 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); and copper sulfate, an anoxidizing cross-linker. DTT stimulated mitochondrial protein import, whereas NEM, MPB, DTNB and Cu2+ were inhibitory. Inhibition by Cu2+ was reversed by addition of DTT, the efficiency of inhibition being higher in energized than in non-energized mitochondria. Results demonstrated that inhibitory effects of NEM, DTNB and Cu2+ on import efficiency were not due to interaction of SH-group reagents with import receptors. Modification of pF1beta with NEM prior to protein import resulted in import stimulation, whereas DTNB and Cu2+ were inhibitory. NEM, MPB, DTNB and Cu2+ inhibited import of the NEM-modified pF1beta into intact mitochondria. Import of pF1beta 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 indicate that redox and conformational status of SH groupslocated on the outer surface of the inner mitochondrial membrane are essential for protein import.