Masatoshi Esaki

Kumamoto University, Kumamoto, Kumamoto Prefecture, Japan

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Publications (28)159.68 Total impact

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    ABSTRACT: The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. A mutation of the conserved aromatic residue or the flanking basic residues in the pore region of the katanin p60 hexameric ring eliminated MT-severing activity but less affected ATPase activity. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 03/2015; 290(18). DOI:10.1074/jbc.M114.614768 · 4.60 Impact Factor
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    ABSTRACT: Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1μm in thickness, and may facilitate new research in cellular structural biology.
    Ultramicroscopy 06/2014; 146C:39-45. DOI:10.1016/j.ultramic.2014.05.008 · 2.75 Impact Factor
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    ABSTRACT: Cdc48p is a highly conserved cytosolic AAA chaperone that is involved in a wide range of cellular processes. It consists of two ATPase domains (D1 and D2), with regulatory regions at the N- and C-terminals. We have recently shown that Cdc48p regulates mitochondrial morphology, in that a loss of the ATPase activity or positive cooperativity in the D2 domain leads to severe fragmentations and aggregations of mitochondria in the cytoplasm. We have now used serial block-face scanning electron microscopy (SBF-SEM), an advanced three-dimensional (3D) electron microscopic technique to examine the structures and morphological changes of mitochondria in the yeast Saccharomyces cerevisiae We found that mutants lacking ATPase activity of Cdc48p showed mitochondrial fragmentations and aggregations, without fusion of the outer membrane. This suggests that the ATPase activity of Cdc48p is necessary for fusion of the outer membranes of mitochondria. Our results also show that SBF-SEM has considerable advantages in morphological and quantitative studies on organelles and intracellular structures in entire cells.
    Journal of Structural Biology 05/2014; 187(2). DOI:10.1016/j.jsb.2014.05.010 · 3.37 Impact Factor
  • Rie Sawamura, Teru Ogura, Masatoshi Esaki
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    ABSTRACT: Bcs1 is atransmembranechaperone in the mitochondrial inner membrane,and is required for the mitochondrial Respiratory Chain Complex III assembly. It has been shown that the highly-conserved C-terminal region of Bcs1 including the AAA ATPase domain in the matrix side is essential for the chaperone function. Here we describe the importance of the N-terminal short segment located in the intermembrane space in the Bcs1 function. Among the N-terminal 44 amino acid residues of yeast Bcs1, the first 37 residues are dispensable whereas a hydrophobic amino acid in the residue 38 is essential for integration of RieskeIron-sulfur Protein into the premature Complex III from the mitochondrial matrix.Substitution of the residue 38 by a hydrophilic amino acid residue affects conformation of Bcs1and interactions with other proteins. Theevolutionarily-conservedshort α helix of Bcs1 in the intermembrane space is an essential element for the chaperone function.
    Biochemical and Biophysical Research Communications 12/2013; 443(3). DOI:10.1016/j.bbrc.2013.12.084 · 2.28 Impact Factor
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    ABSTRACT: During the budding of COPII vesicles from transitional ER (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover, and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain (CCD), which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.
    Molecular biology of the cell 09/2013; 24(21). DOI:10.1091/mbc.E13-04-0185 · 5.98 Impact Factor
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    ABSTRACT: The TOM40 complex is a protein translocator in the mitochondrial outer membrane and consists of several different subunits. Among them, Tom40 is a central subunit that constitutes a protein-conducting channel by forming a β-barrel structure. To probe the nature of the assembly process of Tom40 in the outer membrane, we attached various mitochondrial presequences to Tom40 that possess sorting information for the intermembrane space (IMS), inner membrane, and matrix and would compete with the inherent Tom40 assembly process. We analyzed the mitochondrial import of those fusion proteins in vitro. Tom40 crossed the outer membrane and/or inner membrane even in the presence of various sorting signals. N-terminal anchorage of the attached presequence to the inner membrane did not prevent Tom40 from associating with the TOB/SAM complex, although it impaired its efficient release from the TOB complex in vitro but not in vivo. The IMS or matrix-targeting presequence attached to Tom40 was effective in substituting for the requirement for small Tim proteins in the IMS for the translocation of Tom40 across the outer membrane. These results provide insight into the mechanism responsible for the precise delivery of β-barrel proteins to the outer mitochondrial membrane.
    Molecular biology of the cell 08/2012; 23(20):3936-47. DOI:10.1091/mbc.E12-03-0202 · 5.98 Impact Factor
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    ABSTRACT: Fidgetin is a member of the AAA (ATPases associated with diverse cellular activities) chaperones. It is well-known that the specific function of a given AAA protein primarily depends upon its subcellular localization and interacting partners. FIGL-1, a Caenorhabditis elegans homolog of mammalian fidgetin, is localized in the nucleus. Here, we identified that the N-terminal PKRVK sequence of FIGL-1 functions as a monopartite nuclear localization signal. Nuclear localization of FIGL-1 is required for its function. We also found that FIGL-1 specifically interacted with SMO-1, a C. elegans homolog of small ubiquitin-like modifier (SUMO), using a yeast two-hybrid assay. Furthermore, the direct physical interaction between FIGL-1 and SMO-1 was demonstrated by pull-down assay using purified proteins as well as immunoprecipitation assay using lysates from epitope-tagged SMO-1-expressing worms. Binding of FIGL-1 to SMO-1 is required for its function. The depletion of FIGL-1 and SMO-1 resulted in developmental defects in C. elegans. Taken altogether, our results indicate that FIGL-1 is a nuclear protein and that in concert with SMO-1, FIGL-1 plays an important role in the regulation of C. elegans development.
    Journal of Structural Biology 05/2012; 179(2):143-51. DOI:10.1016/j.jsb.2012.04.022 · 3.37 Impact Factor
  • Masatoshi Esaki, Teru Ogura
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    ABSTRACT: Cdc48p/p97 is a cytosolic essential AAA chaperone, which regulates multiple cellular reactions in a ubiquitin-dependent manner. We have recently shown that Cdc48p exhibits positively cooperative ATPase activity and loss of the positive cooperativity results in yeast cell death. Here we show that loss of the positive cooperativity of the yeast Cdc48p ATPase activity led to severe mitochondrial aggregation. The actin cytoskeleton and distribution of the ER-mitochondria tethering complex (ERMES) were eliminated from the cause of the mitochondrial aggregation. Instead, a mitochondrial outer membrane protein Fzo1p, which is required for mitochondrial fusion, and components of ERMES, which is involved in mitochondrial morphology, were remarkably stabilized in the Cdc48p mutants. In the last couple of years, it was shown that Vms1p functions as a cofactor of Cdc48p for the function of protein degradation of mitochondrial outer membrane proteins. Nevertheless, we found that Vms1p was not involved in the Cdc48p-dependent mitochondrial aggregation and loss of Vms1p did not significantly affect degradation rates of proteins anchored to the mitochondrial outer membrane. These results suggest that Cdc48p controls mitochondrial morphology by regulating turnover of proteins involved in mitochondrial morphology in a Vms1p-independent manner.
    Journal of Structural Biology 05/2012; 179(2):112-20. DOI:10.1016/j.jsb.2012.04.017 · 3.37 Impact Factor
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    ABSTRACT: Spastin belongs to the meiotic subfamily, together with Vps4/SKD1, fidgetin and katanin, of AAA (ATPases associated with diverse cellular activities) proteins, and functions in microtubule severing. Interestingly, all members of this subgroup specifically contain an additional α-helix at the very C-terminal end. To understand the function of the C-terminal α-helix, we characterised its deletion mutants of SPAS-1, a Caenorhabditis elegans spastin homologue, in vitro and in vivo. We found that the C-terminal α-helix plays essential roles in ATP binding, ATP hydrolysing and microtubule severing activities. It is likely that the C-terminal α-helix is required for cellular functions of members of meiotic subgroup of AAA proteins, since the C-terminal α-helix of Vps4 is also important for assembly, ATPase activity and in vivo function mediated by ESCRT-III complexes.
    Journal of Structural Biology 04/2012; 179(2):138-42. DOI:10.1016/j.jsb.2012.04.010 · 3.37 Impact Factor
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    ABSTRACT: p97 is composed of two conserved AAA (ATPases associated with diverse cellular activities) domains, which form a tandem hexameric ring. We characterized the ATP hydrolysis mechanism of CDC-48.1, a p97 homolog of Caenorhabditis elegans. The ATPase activity of the N-terminal AAA domain was very low at physiological temperature, whereas the C-terminal AAA domain showed high ATPase activity in a coordinated fashion with positive cooperativity. The cooperativity and coordination are generated by different mechanisms because a noncooperative mutant still showed the coordination. Interestingly, the growth speed of yeast cells strongly related to the positive cooperativity rather than the ATPase activity itself, suggesting that the positive cooperativity is critical for the essential functions of p97.
    Journal of Biological Chemistry 03/2011; 286(18):15815-20. DOI:10.1074/jbc.M110.201400 · 4.60 Impact Factor
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    ABSTRACT: Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.
    Proceedings of the National Academy of Sciences 01/2011; 108(1):91-6. DOI:10.1073/pnas.1014918108 · 9.81 Impact Factor
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    Masatoshi Esaki, Teru Ogura
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    ABSTRACT: Cdc48p/p97 is a highly conserved essential AAA protein that is required for many cellular processes, and is identified as a causative gene for an autosomal dominant human disorder, inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia (IBMPFD). Cdc48p/p97 is composed of an N-terminal domain, followed by two AAA domains (D1 and D2) whose ATPase activities have been characterized extensively. In this study, effects of mutations on the essential functions of yeast Cdc48p/p97 in vivo were systematically analyzed. IBMPFD-related mutations do not affect the essential functions of Cdc48p/p97. Loss of ATPase activity of D2 leads to loss of function of the protein in vivo. In contrast, ATPase activity of D1 per se is not essential, but a mutation locking D1 in an ATP-bound form is exceptionally lethal. Site-directed and random mutagenesis analyses suggest that the ATP-bound form of D1 changes an inter-domain interaction, thereby perturbing an essential function of Cdc48p/p97.
    Biochemistry and Cell Biology 02/2010; 88(1):109-17. DOI:10.1139/o09-116 · 2.35 Impact Factor
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    ABSTRACT: Mitochondrial protein traffic requires precise recognition of the mitochondrial targeting signals by the import receptors on the mitochondrial surface including a general import receptor Tom20 and a receptor for presequence-less proteins, Tom70. Here we took a proteome-wide approach of mitochondrial protein import in vitro to find a set of presequence-containing precursor proteins for recognition by Tom70. The presequences of the Tom70-dependent precursor proteins were recognized by Tom20, whereas their mature parts exhibited Tom70-dependent import when attached to the presequence of Tom70-independent precursor proteins. The mature parts of the Tom70-dependent precursor proteins have the propensity to aggregate, and the presence of the receptor domain of Tom70 prevents their aggregate formation. Therefore Tom70 plays the role of a docking site for not only cytosolic chaperones but also aggregate-prone substrates to maintain their solubility for efficient transfer to downstream components of the mitochondrial import machineries.
    Journal of Biological Chemistry 09/2009; 284(46):31635-46. DOI:10.1074/jbc.M109.041756 · 4.60 Impact Factor
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    ABSTRACT: Mutations of human spastin, an AAA (ATPases associated with diverse cellular activity) family protein, cause an autosomal dominant form of hereditary spastic paraplegia, which is characterized by weakness, spasticity and loss of the vibratory sense in the lower limbs. Recently, it has been reported that spastin displays microtubule-severing activity. We also previously reported that Caenorhabditis elegans spastin homologue SPAS-1 displays microtubule severing. However, the detailed molecular mechanism of microtubule severing remains unknown. Here, we describe that SPAS-1 forms a stable hexamer in a concentration-dependent manner and that ATPase activity of SPAS-1 is greatly stimulated by microtubules. Furthermore, MTBD (microtubule-binding domain) of SPAS-1 is essential for binding to microtubules. Taken these results together, we propose that MTBD of SPAS-1 plays a critical role in enrichment of SPAS-1 to microtubules, where SPAS-1 is concentrated and able to form a stable hexamer, subsequently its ATPase activity is stimulated. On the other hand, our mutational analyses revealed that the conserved aromatic and basic amino acid residues in the pore region are important for microtubule severing. We also detected the direct interaction of the extremely acidic C-terminal polypeptide of tubulin with SPAS-1. Consequently, we propose that the central pore residues are important for the recognition of substrates.
    Genes to Cells 08/2009; 14(8):925-40. DOI:10.1111/j.1365-2443.2009.01320.x · 2.86 Impact Factor
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    ABSTRACT: Polyglutamine (polyQ)-expanded proteins are associated with cytotoxicity in some neurodegenerative disorders such as Huntington's disease. We have reported that the aggregation of the polyQ-expanded protein is partially suppressed by co-expression of either of two homologs of an AAA chaperone p97, CDC-48.1 or CDC-48.2, in Caenorhabditis elegans, but how p97 regulates the aggregation of polyQ-expanded proteins remains unclear. Here we present direct evidence that CDC-48.1 and CDC-48.2 suppress the aggregation of a huntingtin (Htt) exon1 fragment containing an expanded polyQ repeat in vitro. CDC-48.1 and CDC-48.2 bound the Htt exon1 fragment directly, and suppressed the formation of SDS-insoluble aggregates of Htt fragments containing 53 glutamine residues (HttQ53) independently of nucleotides. CDC-48.1 and CDC-48.2 also modulated the oligomeric states of HttQ53 during the aggregate formation. In the absence of CDC-48.1 and CDC-48.2, HttQ53 formed 70-150 kDa oligomers, whereas 300-500 kDa oligomers as well as 70-150 kDa oligomers accumulated in the presence of CDC-48.1 and CDC-48.2. Taken together, these results suggest that p97 plays a protective role in neurodegenerative disorders by directly suppressing the protein aggregation as a molecular chaperone.
    Genes to Cells 09/2008; 13(8):827-38. DOI:10.1111/j.1365-2443.2008.01214.x · 2.86 Impact Factor
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    ABSTRACT: Newly synthesized mitochondrial precursor proteins have to become unfolded by the mitochondrial Hsp70 (mtHsp70) import motor to cross the mitochondrial membranes. To assess the mechanism of unfolding of precursor proteins by mtHsp70, we designed a system to measure step sizes of the mtHsp70 import motor, which are distances at which the motor system moves along polypeptide chains during a single turnover of ATP. We made a series of fusion proteins consisting of a mitochondrial presequence containing the first mtHsp70 binding site, a spacer sequence containing an Hsp70 avoidance segment followed by the second mtHsp70 binding site, and different folded mature domains. Analyses of the dependence of the import rates of those fusion proteins on the lengths of Hsp70 avoidance segments allowed us to estimate the step sizes, which differ for different mature domains and different lengths of the spacers. These results suggest that the mtHsp70 import motor functions at least as a molecular Brownian ratchet to unfold mitochondrial precursor proteins.
    Journal of Biological Chemistry 09/2008; 283(40):27325-32. DOI:10.1074/jbc.M805249200 · 4.60 Impact Factor
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    ABSTRACT: Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.
    Journal of Biological Chemistry 03/2008; 283(7):3799-807. DOI:10.1074/jbc.M708339200 · 4.60 Impact Factor
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    ABSTRACT: AAA (ATPase associated with various cellular activities) proteins remodel substrate proteins and protein complexes upon ATP hydrolysis. Substrate remodelling is diverse, e.g. proteolysis, unfolding, disaggregation and disassembly. In the oligomeric ring of the AAA protein, there is a conserved aromatic residue which lines the central pore. Functional analysis indicates that this conserved residue in AAA proteases is involved in threading unfolded polypeptides. Katanin and spastin have microtubule-severing activity. These AAA proteins also possess a conserved aromatic residue at the central pore, suggesting its importance in their biological activity. We have constructed pore mutants of these AAA proteins and have obtained in vivo and in vitro results indicating the functional importance of the pore motif. Degradation of casein by the Escherichia coli AAA protease, FtsH, strictly requires ATP hydrolysis. We have constructed several chimaeric proteases by exchanging domains of FtsH and its homologues from Caenorhabditis elegans mitochondria, and examined their ATPase and protease activities in vitro. Interestingly, it has been found that some chimaeras are able to degrade casein in an ATP-independent manner. The proteolysis is supported by either ATP[S] (adenosine 5'-[gamma-thio]triphosphate) or ADP, as well as ATP. It is most likely that substrate translocation in these chimaeras occurs by facilitated diffusion. We have also investigated the roles of C. elegans p97 homologues in aggregation/disaggregation of polyglutamine repeats, and have found that p97 prevents filament formation of polyglutamine proteins in an ATP-independent fashion.
    Biochemical Society Transactions 03/2008; 36(Pt 1):68-71. DOI:10.1042/BST0360068 · 3.24 Impact Factor
  • Masatoshi Esaki, Yang Liu, Benjamin S Glick
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    ABSTRACT: In Pichia pastoris, coat protein complex II (COPII) vesicles form at discrete transitional ER (tER) sites. Analyzing COPII coat proteins in this yeast will help to reveal the mechanisms of tER organization. Here, we show that like Saccharomyces cerevisiae, P. pastoris contains essential SEC23 and SEC24 genes, as well as the non-essential SEC24 homolog LST1. In addition, P. pastoris contains a novel non-essential SEC23 homolog that we have designated SHL23. The products of all four genes are concentrated at tER sites. Deletion of SHL23 does not disrupt tER morphology. As judged by two-hybrid analysis, Sec23p associates with both Sec24p and Lst1p, whereas Shl23p associates selectively with Lst1p. These results suggest that P. pastoris COPII vesicles contain an Shl23p/Lst1p complex that is absent in S. cerevisiae.
    FEBS Letters 11/2006; 580(22):5215-21. DOI:10.1016/j.febslet.2006.08.058 · 3.34 Impact Factor
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    ABSTRACT: Many newly synthesized proteins have to become unfolded during translocation across biological membranes. We have analyzed the effects of various stabilization/destabilization mutations in the Ig-like module of the muscle protein titin upon its import from the N terminus or C terminus into mitochondria. The effects of mutations on the import of the titin module from the C terminus correlate well with those on forced mechanical unfolding in atomic-force microscopy (AFM) measurements. On the other hand, as long as turnover of the mitochondrial Hsp70 system is not rate-limiting for the import, import of the titin module from the N terminus is sensitive to mutations in the N-terminal region but not the ones in the C-terminal region that affect resistance to global unfolding in AFM experiments. We propose that the mitochondrial-import system can catalyze precursor-unfolding by reducing the stability of unfolding intermediates.
    Proceedings of the National Academy of Sciences 01/2006; 102(50):17999-8004. DOI:10.1073/pnas.0504495102 · 9.81 Impact Factor

Publication Stats

811 Citations
159.68 Total Impact Points

Institutions

  • 2008–2014
    • Kumamoto University
      • Department of Molecular Cell Biology
      Kumamoto, Kumamoto Prefecture, Japan
  • 2013
    • Princeton University
      • Department of Molecular Biology
      Princeton, New Jersey, United States
  • 1999–2012
    • Nagoya University
      • Department of Chemistry
      Nagoya, Aichi, Japan
  • 2005–2006
    • University of Chicago
      • Department of Molecular Genetics & Cell Biology
      Chicago, Illinois, United States
  • 2004
    • Chubu University
      • Department of Environmental Biology
      Касугай, Aichi, Japan