The Amino Terminus of the F₁-ATPase β-Subunit Precursor Functions as an Intramolecular Chaperone To Facilitate Mitochondrial Protein Import

Abstract

Mitochondrial import signals have been shown to function in many steps of mitochondrial protein import. Previous studies have shown that the F₁-ATPase β-subunit precursor (pre-F₁β) of the yeast Saccharomyces cerevisiae contains an extended, functionally redundant mitochondrial import signal at its amino terminus. However, the full significance of this functionally redundant targeting sequence has not been determined. We now report that the extended pre-F₁β signal acts to maintain the precursor in an import-competent conformation prior to import, in addition to its previously characterized roles in mitochondrial targeting and translocation. We found that this extended signal is required for the efficient posttranslational mitochondrial import of pre-F₁pre-F1b both in vivo and in vitro. To determine whether the pre-F₁β signal directly influences precursor conformation, fusion proteins that contain wild-type and mutant forms of the pre-F₁β import signal attached to the model passenger protein dihydrofolate reductase (DHFR) were constructed. Deletions that reduced the import signal to a minimal functional unit decreased both the half-time of precursor folding and the efficiency of mitochondrial import. To confirm that the reduced mitochondrial import associated with this truncated signal was due to a defect in its ability to maintain DHFR in a loosely folded conformation, we introduced structurally destabilizing missense mutations into the DHFR passenger to block precursor folding independently of the import signal. We found that the truncated signal imported this destabilized form of DHFR as efficiently as the intact targeting signal, indicating that the primary defect associated with the minimal signal is an inability to maintain the precursor in a loosely folded conformation. Our results suggest that the loss of this intramolecular chaperone function leads to defects in the early stages of the import process.