Distribution of binding sequences for the mitochondrial import receptors Tom20, Tom22, and Tom70 in a presequence-carrying preprotein and a non-cleavable preprotein

Preproteins destined for mitochondria either are synthesized with amino-ter minal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mit ochondrial membrane (designated Tom) contains specific import receptors. Th e cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, hav e been shown to interact with preproteins. Little is known about the intern al targeting information in preproteins and the distribution of binding seq uences for the three import receptors, We have studied the binding of the p urified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound pep tide scans derived from a presequence-carrying cleavable preprotein, cytoch rome c oxidase subunit IV, and a non-cleavable preprotein with internal tar geting information, the phosphate carrier. All three receptor domains are a ble to bind efficiently to linear 13-mer peptides, yet with different speci ficity. Tom20 preferentially binds to presequence segments of subunit IV. T om22 binds to segments corresponding to the carboxyl-terminal part of the p resequence and the amino-terminal part of the mature protein. Tom70 does no t bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom2 0 bind to multiple segments within the phosphate carrier, yet the amino-ter minal 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 char ged amino acids in presequences. Our results demonstrate that linear peptid e segments of preproteins can serve as binding sites for all three receptor s with differential specificity and imply different mechanisms for transloc ation of cleavable and non-cleavable preproteins.