Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis.
Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this 'acid chain' hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20<Tom22 (internal)<Tim23. Precursor binding to the three acidic receptor domains and to the pure cytosolic domain of Tom70 was inhibited by excess targeting peptide, but not by an equally basic control peptide. In this membrane-free and defined system, a precursor pre-bound to the Tom70 or Tom20 domain was transferred efficiently to the Tim23 domain. Transfer was stimulated by the internal Tom22 domain and was much less efficient in the reverse direction. Precursors destined for the outer membrane bound only to Tom20, but not to the internal Tom22 or the Tim23 domain, and a precursor destined for the inner membrane bound only to the Tom20 and the internal Tom22 domain, but not to the Tim23 domain. These results suggest that specific and sequential binding of a targeting signal to strategically situated acidic receptors delivers a precursor across the outer membrane and contributes to intramitochondrial sorting of imported proteins.
Komiya
T
T
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812, Japan.
Rospert
S
S
Koehler
C
C
Looser
R
R
Schatz
G
G
Mihara
K
K
D000143:Acids; D000325:Adrenodoxin / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D019347:Potassium Acetate / Q000494:pharmacology; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D000143:Acids; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C083081:TIM23 protein, S cerevisiae; C489350:TOM20 protein, S cerevisiae; C090857:TOM22 protein, S cerevisiae; C044938:preadrenodoxin; D000325:Adrenodoxin; D005982:Glutathione Transferase; D019347:Potassium Acetate