Identification of signals required for import of the soybean F(A)d subunit of ATP synthase into mitochondria.
The requirements for protein import into mitochondria was investigated by using the targeting signal of the F(A)d subunit of soybean mitochondrial ATP synthase attached to two different passenger proteins, its native passenger and soybean alternative oxidase. Both passenger proteins are soybean mitochondrial proteins. Changing hydrophobic residues at positions -24:25 (Phe:Leu), -18:19 (Ile:Leu) and -12:13 (Leu:Ile) of the 31 amino acid cleavable presequence gave more than 50% inhibition of import with both passenger proteins. Some other residues in the targeting signal played a more significant role in targeting of one passenger protein compared to another. Notably changing positive residues (Arg, Lys) had a greater inhibitory affect on import with the native passenger protein, i.e. greater inhibition of import with F(A)d mature protein was observed compared to when alternative oxidase was the mature protein. When using chimeric passenger proteins it was shown that the nature of the mature protein can greatly affect the targeting properties of the presequence. In vivo investigations of the targeting presequence indicated that the presequence of 31 amino acids could not support import of GFP as a passenger protein. However, fusion of the full-length F(A)d coding sequence to GFP did result in mitochondrial localisation of GFP. Using the latter fusion we confirmed the critical role of hydrophobic residues at positions -24:25 and -18:19. These results support the proposal that core mitochondrial targeting features exist in all presequences, but that additional features exist. These features may not be evident with all passenger proteins.
Lee
May-Nee
MN
Plant Molecular Biology Group, Biochemistry and Molecular Biology, School of Biomedical, and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley 6009, WA, Australia.
Whelan
James
J
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D030821:Plants, Genetically Modified; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013025:Glycine max / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D004792:Enzyme Precursors; D008164:Luminescent Proteins; D021382:Protein Sorting Signals; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006180:Proton-Translocating ATPases