Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.
"In addition to the contribution to carrier protein import, Tom70 has also been implicated in the import of presequence-containing proteins    , though this contribution of Tom70 has been controversial   . In the last years, significant structural insight into Tom70 revealed that the tetratricopeptide repeats (TPR) 1–3 in the N-terminal part of the protein bind to the C-terminal EEVD motif of Hsp70 and Hsp90  . "
"How are chaperone-bound precursors delivered to the receptors Tom70 and Tom20 ? How do the receptors recognize the various precursors, in particular, those with non-canonical MTSs  ? The receptors are only loosely associated with the TOM core complex. "
"Tom20 is best known for recognizing matrix-destined precursors carrying an N-terminal targeting signal which is removed from precursor proteins once they reach the matrix. However, Tom20 also has a degree of overlapping specificity for precursors recognized by Tom70 (Brix et al. 1999; Söllner et al. 1990; Steger et al. 1990). Therefore, such proteins are still imported in mutants lacking Tom70, though with reduced efficiency (Grad et al. 1999; Steger et al. 1990). "
[Show abstract][Hide abstract] ABSTRACT: The alternative oxidase (AOX) of Neurospora crassa transfers electrons from ubiquinol to oxygen. The enzyme is not expressed under normal conditions. However, when the function of the standard electron transport chain is compromised, AOX is induced, providing cells with a means to continue respiration and growth. Induction of the enzyme represents a form of retrograde regulation because AOX is encoded by a nuclear gene that responds to signals produced from inefficiently functioning mitochondria. To identify genes required for AOX expression, we have screened the N. crassa gene knockout library for strains that are unable to grow in the presence of antimycin A, an inhibitor of complex III of the standard electron transport chain. From the 7800 strains containing knockouts of different genes, we identified 62 strains that have reduced levels of AOX when grown under conditions known to induce the enzyme. Some strains have virtually no AOX, whereas others have only a slight reduction of the protein. A broad range of seemingly unrelated functions are represented in the knockouts. For example, we identified transcription factors, kinases, the mitochondrial import receptor Tom70, three subunits of the COP9 signalosome, a monothiol glutaredoxin, and several hypothetical proteins as being required for wild-type levels of AOX production. Our results suggest that defects in many signaling or metabolic pathways have a negative effect on AOX expression and imply that complex systems control production of the enzyme.
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