Michael J Baker

Publications (4)32.36 Total impact

• Article: Impaired Folding of the Mitochondrial Small TIM Chaperones Induces Clearance by the i-AAA Protease.

  Michael J Baker, Ved P Mooga, Bernard Guiard, Thomas Langer, Michael T Ryan, Diana Stojanovski
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  ABSTRACT: The intermembrane space of mitochondria contains a dedicated chaperone network-the small translocase of the inner membrane (TIM) family-for the sorting of hydrophobic precursors. All small TIMs are defined by the presence of a twin CX(3)C motif and the monomeric proteins are stabilized by two intramolecular disulfide bonds formed between the cysteines of these motifs. The conserved cysteine residues within small TIM members have also been shown to participate in early biogenesis events, with the most N-terminal cysteine residue important for import and retention within the intermembrane space via the receptor and disulfide oxidase, Mia40. In this study, we have analyzed the in vivo consequences of improper folding of small TIM chaperones by generating site-specific cysteine mutants and assessed the fate of the incompletely oxidized proteins within mitochondria. We show that no individual cysteine residue is required for the function of Tim9 or Tim10 in yeast and that defective assembly of the small TIMs induces their proteolytic clearance from mitochondria. We delineate a clearance mechanism for the mutant proteins and their unassembled wild-type partner protein by the mitochondrial ATP-dependent protease, Yme1 (yeast mitochondrial escape 1).
  Journal of Molecular Biology 10/2012; · 4.00 Impact Factor
• Article: Structural and functional requirements for activity of the Tim9-Tim10 complex in mitochondrial protein import.

  Michael J Baker, Chaille T Webb, David A Stroud, Catherine S Palmer, Ann E Frazier, Bernard Guiard, Agnieszka Chacinska, Jacqueline M Gulbis, Michael T Ryan
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  ABSTRACT: The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.
  Molecular biology of the cell 12/2008; 20(3):769-79. · 5.98 Impact Factor
• Source

  Available from: Agnieszka Chacinska

  Article: Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase.

  Diana Stojanovski, Dusanka Milenkovic, Judith M Müller, Kipros Gabriel, Agnes Schulze-Specking, Michael J Baker, Michael T Ryan, Bernard Guiard, Nikolaus Pfanner, Agnieszka Chacinska
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  ABSTRACT: The biogenesis of mitochondrial intermembrane space proteins depends on specific machinery that transfers disulfide bonds to precursor proteins. The machinery shares features with protein relays for disulfide bond formation in the bacterial periplasm and endoplasmic reticulum. A disulfide-generating enzyme/sulfhydryl oxidase oxidizes a disulfide carrier protein, which in turn transfers a disulfide to the substrate protein. Current views suggest that the disulfide carrier alternates between binding to the oxidase and the substrate. We have analyzed the cooperation of the disulfide relay components during import of precursors into mitochondria and identified a ternary complex of all three components. The ternary complex represents a transient and intermediate step in the oxidation of intermembrane space precursors, where the oxidase Erv1 promotes disulfide transfer to the precursor while both oxidase and precursor are associated with the disulfide carrier Mia40.
  The Journal of Cell Biology 11/2008; 183(2):195-202. · 10.26 Impact Factor
• Source

  Available from: ulb.ac.be

  Article: Mitochondrial protein-import machinery: correlating structure with function.

  Michael J Baker, Ann E Frazier, Jacqueline M Gulbis, Michael T Ryan
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  ABSTRACT: Most mitochondrial proteins are synthesized in the cytosol, translocated into the organelle and directed along specific sorting pathways. Over the past 20 years, >30 proteins have been identified as having key roles in mitochondrial protein import. Recently, the elucidation of the structures of several import components has provided fresh insight into the import process. Here, we review the different pathways involved in sorting proteins into mitochondrial subcompartments. Along the way, we highlight the available structural information about the protein-import machinery and discuss how these structures correlate with previously ascribed functions. Future challenges for the cell biologists will be to use this structural information to test specific hypotheses addressing the molecular mechanisms of mitochondrial protein import.
  Trends in cell biology 09/2007; 17(9):456-64. · 12.12 Impact Factor