Abstract
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.
Highlights
A series of proteins containing defined internal and presequence deletions in the F1-ATPaseB-subunit precursor have been synthesized in vitro using a linked transcription-translation system
Greater than 90% of the protein content of mitochondria is encoded by nuclear genes (Dujon, 1980; Douglas and Takeda, 1985).In recent studies much has been learned about the mechanism of protein import into mitochondria by characterization and analysis of the transient presequences which are present at theamino terminus of precursor proteins (Honvich et al, 1985, Emr et al, 1986;Hurt andvan Loon, 1986).These short hydrophilic presequences are required for the correct localization of mitochondrial protein in the cell (Hurt et al, 1984; Honvich et al, 1985; Douglas et al, 1984)
During logarithmicgrowth inyeast a pool of mitochondrial precursor proteins presumably located in the cytoplasmic compartment is undetectable
Summary
A series of proteins containing defined internal and presequence deletions in the F1-ATPaseB-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.
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