The signal that sorts yeast cytochrome b2 to the mitochondrial intermembrane space contains three distinct functional regions.
Cytochrome b2, a protein of the yeast mitochondrial intermembrane space, is synthesized with an 80 residue bipartite presequence. The amino-terminal portion resembles a matrix-targeting signal. The carboxy-terminal portion acts as a 'sorting signal' for the intermembrane space and contains a hydrophobic stretch. In order to define this sorting signal, we fused the first 167 residues of the cytochrome b2 precursor to a passenger protein, expressed the fusion protein in yeast and selected for mutations that caused mislocalization of the passenger protein to the matrix. Most mutations mapped within the first 81 amino-terminal residues of the cytochrome b2 moiety. They were located in three regions, all downstream of the matrix-targeting domain: a cluster of three basic residues upstream of the hydrophobic stretch, the hydrophobic stretch itself and the first residue of mature cytochrome b2. The level of missorting caused by mutations within the hydrophobic stretch did not correlate with their effects on hydrophobicity, but appeared to be related to changes in the conformation of this stretch. We conclude that the intermembrane space sorting signal of cytochrome b2 is decoded by protein-protein interactions rather than by simple partitioning into a lipid bilayer.
Biocenter, University of Basel, Switzerland.
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EM
Beasley
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Müller
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Schatz
D000595:Amino Acid Sequence; D001483:Base Sequence; D004274:DNA, Recombinant; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism; D007425:Intracellular Membranes / Q000201:enzymology*; D007770:L-Lactate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D004274:DNA, Recombinant; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D003576:Electron Transport Complex IV