The role of protein structure in the mitochondrial import pathway. Analysis of the soluble F1-ATPase beta-subunit precursor.
A series of proteins containing defined internal and presequence deletions in the F1-ATPase beta-subunit precursor have been synthesized in vitro using a linked transcription-translation system. These different forms of the protein have been analyzed by the combination of gel filtration and in vitro mitochondrial import studies. These studies reveal that the soluble F1 beta-subunit precursor (55 kDa) forms a homooligomeric assembly of apparent molecular weight 230,000 on gel filtration analysis. The formation of this tetrameric beta-protein was dependent on the sequence between residues 122 and 144 of the precursor and was independent of the presence of a mitochondrial presequence within the first 19 residues of the precursor. When the tetrameric F1 beta-precursor was partially purified from the translation reaction it was incompetent for import into mitochondria. However, import of the partially purified beta-subunit could be restored by addition of reticulocyte lysate protein. In the absence of the tetramer-forming sequence, the protein behaved as an aggregate complex approximately 400 kDa in size. Formation of the high molecular weight aggregate and import into mitochondria was dependent upon a functional presequence at the amino terminus of the precursor. These studies are discussed in terms of the maintenance of an import competent structure for mitochondrial precursors and role of soluble factors in this process.
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D004792:Enzyme Precursors / Q000032:analysis*; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014176:Protein Biosynthesis; D006180:Proton-Translocating ATPases / Q000096:biosynthesis*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D004792:Enzyme Precursors; D046911:Macromolecular Substances; D006180:Proton-Translocating ATPases