Targeting of cytochrome b2 into the mitochondrial intermembrane space: specific recognition of the sorting signal.
Cytochrome b2 contains 2-fold targeting information: an amino-terminal signal for targeting to the mitochondrial matrix, followed by a second cleavable sorting signal that functions in directing the precursor into the mitochondrial intermembrane space. The role of the second sorting sequence was analyzed by replacing one, two or all of the three positively charged amino acid residues which are present at the amino-terminal side of the hydrophobic core by uncharged residues or an acidic residue. With a number of these mutant precursor proteins, processing to the mature form was reduced or completely abolished and at the same time targeting to the matrix space occurred. The accumulation in the matrix depended on a high level of intramitochondrial ATP. At low levels of matrix ATP, the mutant proteins were sorted into the intermembrane space like the wild-type precursors.


RESULTS
(i) suggest the existence of one or more matrix components that specifically recognize the second sorting signal and thereby trigger the translocation into the intermembrane space; (ii) indicate that the mutant signals have reduced ability to interact with the recognition component(s) and then embark on the default pathway into the matrix by interacting with mitochondrial hsp70 in conjunction with matrix ATP; (iii) strongly argue against a mechanism by which the hydrophobic segment of the sorting sequence stops translocation in the hydrophobic phase of the inner membrane.
Schwarz
E
E
Institut für Physiologische Chemie der Universität München, FRG.
Seytter
T
T
Guiard
B
B
Neupert
W
W
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001692:Biological Transport; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008727:Methotrexate / Q000494:pharmacology; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate