Abstract
Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.
Highlights
Nuclear encoded mitochondrial proteins are synthesized in the cytosol and are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes for sorting to one of four compartments: the outer membrane, intermembrane space (IMS),5 inner
Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and IMS, respecchondrial membrane; TEV, tobacco etch virus; BN-PAGE, blue native PAGE; pSu9-DHFR, the presequence of subunit 9 of F0-ATPase fused to mouse dihydrofolate reductase; pHsp60, the precursor to mitochondrial Hsp60; pF1, the precursor to the -subunit of F1-ATPase; AAC, ADP/ATP carrier; PBR, peripheral benzodiazepine receptor; MOPS, 4-morpholinepropanesulfonic acid; WT, wild-type; TOB, topogenesis of mitochondrial outer membrane -barrel proteins; SAM, sorting and assembly machinery
SDS-PAGE followed by immunoblotting with anti-Tom20 or anti-Tom22 antibodies showed that the receptor domain of Tom20TEV or Tom22TEV was completely deleted in 20T or 22T mitochondria, respectively, whereas the amounts of Tom20TEV in 20T and Tom22TEV in 22T before TEV protease treatment were nearly the same as those of Tom20 and Tom22 in wild-type mitochondria (20W and 22W) (Fig. 1B)
Summary
Plasmids—The TOM20 gene containing its own promoter and terminator was amplified by PCR using primers 5Ј-CAA GAC TCG AGC AGA TCT TGC ATT C-3Ј and 5Ј-GCA TGA GCT CAT TTA GGC TAG AGA AAT G-3Ј. SNY1005 was transformed with a TRP1 plasmid harboring the wild-type TOM20 gene (pRS314/ TOM20(WT)) or the gene for Tom20TEV (pRS314/ TOM20(TEV)) to construct 20W and 20T strains, respectively. MNMS1-C was transformed with a TRP1 plasmid harboring the wild-type TOM22 gene (pRS314/TOM22(WT)) or the gene for Tom22TEV (pRS314/TOM22(TEV)) and selected on 5-fluoroorotic acid plates to construct 22W and 22T strains. Cells from 1.2 liters of culture were harvested by centrifugation, resuspended in 100 ml of resuspension buffer (50 mM sodium phosphate, pH 7.8, and 100 mM NaCl), and disrupted by sonication. TEV protease recovered as inclusion bodies was washed twice with 50 ml of resuspension buffer containing 0.5% Triton X-100 and solubilized with 25 ml of 6 M urea buffer (6 M urea, 50 mM sodium phosphate, pH 8.0, and 300 mM NaCl). The column was washed with wash buffer (6 M urea, 50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 10 mM imidazole) and eluted with elution buffer
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