Functional and structural properties of the mitochondrial outer membrane receptor Tom20.
Tom20 is an outer mitochondrial membrane protein that functions as a component of the import receptor complex for cytoplasmically synthesized mitochondrial precursor proteins. The human homologue, hTom20, consists of an N-terminal membrane anchor region predicted between aa5-25 and a soluble cytosolic domain from aa30 to 145. To analyze the properties of hTom20, we have expressed several truncations of the cytosolic domain as fusion proteins with glutathione S-transferase. Our studies reveal that the cytosolic region of hTom20 is a monomeric protein in solution containing two domains which are involved in different functions of the receptor. The N-terminal region is involved in membrane binding (aa30-60) and recognition of the cleavable matrix targeting signals (aa50-90). In addition, we have demonstrated that the receptor recognizes the alpha-helical state of the matrix targeting signal. The dissociation constant for this interaction in the presence of a detergent which induces this secondary structure is 0.6 microM, one-fifth the value in the absence of detergent. In aqueous solution, the region between aa30 and 60 is loosely folded and stabilized against proteolytic cleavage by interaction with detergents or a matrix targeting signal. Our work further shows that the remainder of the cytosolic domain of hTom20, aa60-145, is a compactly folded globular domain containing a region (aa90-145) that is critical for the recognition of proteins bearing internal signal sequences such as the uncoupling protein and porin.
Schleiff
E
E
Department of Biochemistry, McGill University, Montreal, Canada. schleiff@med.mcgill.ca
Turnbull
J L
JL
D003600:Cytosol / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C084008:TOMM20 protein, human