The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors.

The preprotein translocase of the outer mitochondrial membrane (Tom) is a multisubunit machinery containing receptors and a general import pore (GIP). We have analyzed the molecular architecture of the Tom machinery. The receptor Tom22 stably associates with Tom40, the main component of the GIP, in a complex with a molecular weight of ϳ400,000 (ϳ400K), while the other receptors, Tom20 and Tom70, are more loosely associated with this GIP complex and can be found in distinct subcomplexes. A yeast mutant lacking both Tom20 and Tom70 can still form the GIP complex when sufficient amounts of Tom22 are synthesized. Besides the essential proteins Tom22 and Tom40, the GIP complex contains three small subunits, Tom5, Tom6, and Tom7. In mutant mitochondria lacking Tom6, the interaction between Tom22 and Tom40 is destabilized, leading to the dissociation of Tom22 and the generation of a subcomplex of ϳ100K containing Tom40, Tom7, and Tom5. Tom6 is required to promote but not to maintain a stable association between Tom22 and Tom40. The following conclusions are suggested. (i) The GIP complex, containing Tom40, Tom22, and three small Tom proteins, forms the central unit of the outer membrane import machinery. (ii) Tom20 and Tom70 are not essential for the generation of the GIP complex. (iii) Tom6 functions as an assembly factor for Tom22, promoting its stable association with Tom40.

Mitochondrial preproteins are imported by a multisubunit translocase of the outer membrane (TOM), including receptor proteins and a general import pore. The central receptor Tom22 binds preproteins through both its cytosolic domain and its intermembrane space domain and is stably associated with the channel protein Tom40 (refs 11-13). Here we report the unexpected observation that a yeast strain can survive without Tom22, although it is strongly reduced in growth and the import of mitochondrial proteins. Tom22 is a multifunctional protein that is required for the higher-level organization of the TOM machinery. In the absence of Tom22, the translocase dissociates into core complexes, representing the basic import units, but lacks a tight control of channel gating. The single membrane anchor of Tom22 is required for a stable interaction between the core complexes, whereas its cytosolic domain serves as docking point for the peripheral receptors Tom20 and Tom70. Thus a preprotein translocase can combine receptor functions with distinct organizing roles in a multidomain protein.

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.Many mitochondrial preproteins are synthesized with amino-terminal extensions, termed presequences, that function as targeting signals to direct the preproteins into mitochondria (1-3). Upon import into the mitochondrial matrix, the positively charged amphipathic presequences are cleaved off by a specific peptidase. Studies with fusion proteins consisting of a presequence and a non-mitochondrial passenger protein demonstrated that a presequence contains sufficient information for specifically directing the import of proteins into mitochondria (4 -6). Extensive studies with fusion proteins and mutagenesis of presequences revealed a predominant role of presequences in targeting of cleavable preproteins (summarized in Refs. 7-9), although a contribution of the mature protein parts was also found with some preproteins (10 -12).Numerous mitochondrial preproteins, however, are synthesized as mature-sized non-cleavable preproteins. In a few cases, such preproteins were shown to contain a positively charged amphipathic targeting signal in the amino-terminal region, comparable to a non-cleaved presequence (13-17), or at an internal po...

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