Folding of fumarase during mitochondrial import determines its dual targeting in yeast.
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. Thus, fumarase processed in mitochondria returns to the cytosol. In the current work, we (i) generated mutations throughout the coding sequence which resulted in fumarases with altered conformations that are targeted to mitochondria but have lost their ability to be distributed; (ii) showed by mass spectrometry that mature cytosolic and mitochondrial fumarase isoenzymes are identical; and (iii) showed that hsp70 chaperones in the cytosol (Ssa) and mitochondria (Ssc1) can affect fumarase distribution. The results are discussed in light of our model of targeting and distribution, which suggests that rapid folding of fumarase into an import-incompetent state provides the driving force for retrograde movement of the processed protein back to the cytosol through the translocation pore.
Sass
Ehud
E
Department of Molecular Biology, Hebrew University Medical School, Jerusalem 91120, Israel.
Karniely
Sharon
S
Pines
Ophry
O
D000595:Amino Acid Sequence; D001692:Biological Transport; D001710:Biotin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000494:pharmacology; D005649:Fumarate Hydratase / Q000737:chemistry* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005816:Genetic Complementation Test; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D013058:Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D001710:Biotin; D019286:Endopeptidase K; D005649:Fumarate Hydratase; D005690:Galactose