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 gene encoding fumarase is distributed between the cytosol and mitochondria of Saccharomyces cerevisiae and that all fumarase translation products are targeted and processed in mitochondria before distribution. Alternative models for fumarase distribution have been proposed that require more than one translation product. In the current work (i) we show by using sequential Edman degradation and mass spectrometry that fumarase cytosolic and mitochondrial 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 retains its ability to be distributed between the cytosol and mitochondria, and (iii) we show that although a signal peptide is required for fumarase targeting to mitochondria the specific fumarase signal peptide and the sequence immediately downstream to the cleavage site are not required for the dual distribution phenomenon. Our results are discussed in light of our model of fumarase targeting and distribution that suggests rapid folding into an import-incompetent state and retrograde movement of the processed protein back to the cytosol through the translocation pore.
Sass
E
E
Department of Molecular Biology, Hebrew University Medical School, Jerusalem 91120, Israel.
Blachinsky
E
E
Karniely
S
S
Pines
O
O
D000595:Amino Acid Sequence; D001483:Base Sequence; D003600:Cytosol / Q000201:enzymology*; D017931:DNA Primers; D005649:Fumarate Hydratase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D017931:DNA Primers; D007527:Isoenzymes; D005649:Fumarate Hydratase