Article

Distribution of Binding Sequences for the Mitochondrial Import Receptors Tom20, Tom22, and Tom70 in a Presequence-carrying Preprotein and a Non-cleavable Preprotein

Humboldt-Universität zu Berlin, Berlín, Berlin, Germany
Journal of Biological Chemistry (Impact Factor: 4.57). 07/1999; 274(23):16522-30. DOI: 10.1074/jbc.274.23.16522
Source: PubMed

ABSTRACT

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.

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    • "In addition to the contribution to carrier protein import, Tom70 has also been implicated in the import of presequence-containing proteins [22] [23] [24] [25], though this contribution of Tom70 has been controversial [26] [27] [28]. 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 [20] [29]. "
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    ABSTRACT: The translocase of the outer mitochondrial membrane (TOM complex) is the general entry gate into mitochondria for almost all imported proteins. A variety of specific receptors allow the TOM complex to recognize targeting signals of various precursor proteins that are transported along different import pathways. Aside from the well-characterized presequence receptors Tom20 and Tom22 a third TOM receptor, Tom70, binds proteins of the carrier family containing multiple transmembrane segments. Here we demonstrate that Tom70 directly binds to presequence peptides using a dedicated groove. A single point mutation in the cavity of this pocket (M551R) reduces the presequence binding affinity of Tom70 ten-fold and selectively impairs import of the presequence-containing precursor Mdl1 but not the ADP/ATP carrier (AAC). Hence Tom70 contributes to the presequence import pathway by recognition of the targeting signal of the Mdl1 precursor. Copyright © 2015. Published by Elsevier B.V.
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    • "How are chaperone-bound precursors delivered to the receptors Tom70 and Tom20 [46]? How do the receptors recognize the various precursors, in particular, those with non-canonical MTSs [33] [47]? The receptors are only loosely associated with the TOM core complex. "
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    Preview · Article · Feb 2015 · Journal of Molecular Biology
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    • "For targeting from the cytosol to the MT, mitochondrial proteins contain a targeting sequence. Depending on the individual protein, mitochondrial targeting sequences have been identified at three characteristic positions in the protein: the N terminus, internal sequences, and the C terminus (Brix et al., 1999). Of these three subtypes, targeting sequences located at the N terminus of the protein have been best characterized to date. "
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    ABSTRACT: The majority of mitochondrial proteins are encoded in the nuclear genome and imported into mitochondria posttranslationally from the cytosol. An N-terminal presequence functions as the signal for the import of mitochondrial proteins. However, the functional information in the presequence remains elusive. This study reports the identification of critical sequence motifs from the presequence of Arabidopsis thaliana F1-ATPase γ-subunit (pFAγ). pFAγ was divided into six 10-amino acid segments, designated P1 to P6 from the N to the C terminus, each of which was further divided into two 5-amino acid subdivisions. These P segments and their subdivisions were substituted with Ala residues and fused to green fluorescent protein (GFP). Protoplast targeting experiments using these GFP constructs revealed that pFAγ contains several functional sequence motifs that are dispersed throughout the presequence. The sequence motifs DQEEG (P4a) and VVRNR (P5b) were involved in translocation across the mitochondrial membranes. The sequence motifs IAARP (P2b) and IAAIR (P3a) participated in binding to mitochondria. The sequence motifs RLLPS (P2a) and SISTQ (P5a) assisted in pulling proteins into the matrix, and the sequence motif IAARP (P2b) functioned in Tom20-dependent import. In addition, these sequence motifs exhibit complex relationships, including synergistic functions. Thus, multiple sequence motifs dispersed throughout the presequence are proposed to function cooperatively during protein import into mitochondria.
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