Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addition of extramitochondrial ATP for import, the FAd ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the FAd protein and alternative oxidase (AOX) with switched presequences revealed that the mature domain of the FAd precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of FAd (pAOX-mFAd) could be imported. Import competence of FAd and pAOX-mFAd correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F1β ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the FAd ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins.
Plant Molecular Biology – Springer Journals
Published: Oct 7, 2004
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