THE AMINO-TERMINUS OF THE F1-ATPASE BETA-SUBUNIT PRECURSOR FUNCTIONS AS AN INTRAMOLECULAR CHAPERONE TO FACILITATE MITOCHONDRIAL PROTEIN IMPORT

Mitochondrial import signals have been shown to function in many steps of mitochondrial protein import, Previous studies have shown that the F-1-ATPase beta-subunit precursor (pre-F-1 beta) of the yeast Sacchar omyces cerevisiae contains an extended, functionally redundant mitocho ndrial import signal at its amino terminus, However, the full signific ance of this functionally redundant targeting sequence has not been de termined, We now report that the extended pre-F-1 beta signal acts to maintain the precursor in an import-competent conformation prior to im port, in addition to its previously characterized roles in mitochondri al targeting and translocation. We found that this extended signal is required for the efficient posttranslational mitochondrial import of p re-F-1 beta both in vivo and in vitro. To determine whether the pre-F- 1 beta signal directly influences precursor conformation, fusion prote ins that contain wild-type and mutant forms of the pre-F-1 beta import signal attached to the model passenger protein dihydrofolate reductas e (DHFR) were constructed, Deletions that reduced the import signal to a minimal functional unit decreased both the half-time of precursor f olding and the efficiency of mitochondrial import, To confirm that the reduced mitochondrial import associated with this truncated signal wa s due to a defect in its ability to maintain DHFR in a loosely folded conformation, we introduced structurally destabilizing missense mutati ons into the DHFR passenger to block precursor folding independently o f the import signal, We found that the truncated signal imported this destabilized form of DHFR as efficiently as the intact targeting signa l, indicating that the primary defect associated with the minimal sign al is an inability to maintain the precursor in a loosely folded confo rmation, Our results suggest that the loss of this intramolecular chap erone function leads to defects in the early stages of the import proc ess.