Tight folding of a passenger protein can interfere with the targeting function of a mitochondrial presequence.

The first seven residues of the yeast cytochrome oxidase subunit IV presequence are insufficient to target attached mouse dihydrofolate reductase into isolated yeast mitochondria. However, the targeting function of this truncated presequence can be restored by presenting the fusion protein to isolated mitochondria either as nascent, unfolded chains, or as full-length chains whose dihydrofolate reductase moiety had been destabilized either by urea treatment or by point mutations. The targeting efficiency of a mitochondrial presequence can thus be strongly influenced by the conformation of the attached 'passenger protein'. These results also underscore the difficulty of defining a 'minimal' mitochondrial targeting signal.