Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.
In an attempt to understand the mechanisms of sorting of mitochondrial inner membrane proteins, we have analyzed the import of subunit 9 of the mitochondrial F1F0-ATPase (Su9) from Neurospora crassa, an integral inner membrane protein. A chimeric protein was used consisting of the presequence and the first transmembrane domain of Su9 fused to mouse dihydrofolate reductase (preSu9(1-112)-DHFR). This protein attains the correct topology across the inner membrane (Nout-Cin) following import. The transmembrane domain becomes first completely imported into the matrix, where after processing of the presequence, it mediates membrane insertion and export of the N-terminal tail. Import and export steps can be experimentally dissected into two distinct events. Translocation of the N-terminal hydrophilic tail out of the matrix was blocked when the presequence was not processed, indicating an important role of the sequences and charges flanking the hydrophobic domain. Furthermore, export was supported by a delta pH and required matrix ATP hydrolysis. Thus the hydrophobic transmembrane domain operates as a membrane insertion signal and not as a stop-transfer signal. Our findings suggest that several aspects of this sorting process have been conserved from their prokaryotic ancestors.
Rojo
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Institut für Physiologische Chemie der Universität München, Germany.
Stuart
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Neupert
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D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism; D021382:Protein Sorting Signals / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; D013762:Tetrahydrofolate Dehydrogenase; D006180:Proton-Translocating ATPases