Mitochondrial and cytosolic isoforms of yeast fumarase are derivatives of a single translation product and have identical amino termini

We have previously proposed that a single translation product of the FUM1 g ene encoding fumarase is distributed between the cytosol and mitochondria o f Saccharomyces cerevisiae and that all fumarase translation products are t argeted and processed in mitochondria before distribution. Alternative mode ls for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Ed man degradation and mass spectrometry that fumarase cytosolic and mitochond rial isoenzymes have an identical amino terminus that is formed by cleavage by the mitochondrial processing peptidase, (ii) we have generated fumarase mutants in which the second potential translation initiation codon (Met-24 ) has been substituted, yet the protein is processed efficiently and retain s its ability to be distributed between the cytosol and mitochondria, and ( iii) we show that although a signal peptide is required for fumarase target ing to mitochondria the specific fumarase signal peptide and the sequence i mmediately downstream to the cleavage site are not required for the dual di stribution phenomenon. Our results are discussed in light of our model of f umarase targeting and distribution that suggests rapid folding into an impo rt-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.