Abstract
Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Δ18 in addition to the endogenous wild-type Tim44. Tim44Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.
Bibliography
Merlin, A., Voos, W., Maarse, A. C., Meijer, M., Pfanner, N., & Rassow, J. (1999). The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation. The Journal of Cell Biology, 145(5), 961â972.
References
84
Referenced
38
10.1016/0076-6879(95)60144-9
/ Methods Enzymol / Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae. by Alconada (1995)10.1016/S0092-8674(00)81320-3
/ Cell / Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria by Bauer (1996)10.1016/S0092-8674(05)80013-3
/ Cell / The MIM complex mediates preprotein translocation across the mitochondrial inner membrane and couples it to the mt-Hsp70/ ATP driving system by Berthold (1995){'key': '2023072900334273100_B4', 'first-page': '7364', 'article-title': 'The essential yeast protein MIM44 (encoded by MPI1) is required at an early step of preprotein translocation across the mitochondrial inner membrane', 'volume': '13', 'author': 'Blom', 'year': '1993', 'journal-title': 'Mol Cell Biol'}
/ Mol Cell Biol / The essential yeast protein MIM44 (encoded by MPI1) is required at an early step of preprotein translocation across the mitochondrial inner membrane by Blom (1993)10.1111/j.1432-1033.1995.tb20813.x
/ Eur J Biochem / Functional and physical interactions of components of the yeast mitochondrial inner-membrane import machinery (MIM) by Blom (1995)10.1016/0076-6879(87)54076-9
/ Methods Enzymol / 5-Fluoroorotic acid as a selective agent in yeast molecular genetics by Boeke (1987)10.1093/emboj/16.9.2205
/ EMBO (Eur Mol Biol Organ) J / Multiple interactions of components mediating preprotein translocation across the inner mitochondrial membrane by Bömer (1997)10.1093/emboj/17.15.4226
/ EMBO (Eur Mol Biol Organ) J / Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins by Bömer (1998)10.1016/S0968-0004(96)80163-0
/ Trends Biochem Sci / Post-translational protein translocation: not all hsc70s are created equal by Brodsky (1996)10.1016/S0092-8674(00)80928-9
/ Cell / The Hsp70 and Hsp60 chaperone machines by Bukau (1998)10.1379/1466-1268(1998)003<0028:SFAEOD>2.3.CO;2
/ Cell Stress Chaperones / Structure, function and evolution of DnaJ: conservation and adaptation of chaperone function by Cheetham (1998)10.1083/jcb.137.7.1483
/ J Cell Biol / The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae. by Corsi (1997)10.1016/0968-0004(94)90281-X
/ Trends Biochem Sci / DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70 by Cyr (1994)10.1016/S0021-9258(18)33624-X
/ J Biol Chem / Import of proteins into mitochondria: energy-dependent, two-step processing of the intermembrane space enzyme cytochrome b2by isolated yeast mitochondria by Daum (1982)10.1006/jmbi.1997.1131
/ J Mol Biol / Role of mitochondrial GrpE and phosphate in the ATPase cycle of matrix Hsp70 by Dekker (1997)10.1093/emboj/16.17.5408
/ EMBO (Eur Mol Biol Organ) J / The Tim core complex defines the number of mitochondrial translocation contact sites and can hold arrested preproteins in the absence of matrix Hsp70-Tim44 by Dekker (1997)10.1074/jbc.271.39.23960
/ J Biol Chem / Purification and biochemical properties of Saccharomyces cerevisiae's Mge1p, the mitochondrial cochaperone of Ssc1p by Deloche (1996)10.1128/jb.179.19.6066-6075.1997
/ J Bact / Structure-function analysis of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli. by Deloche (1997)10.1074/jbc.272.45.28539
/ J Biol Chem / Purification and biochemical properties of Saccharomyces cerevisiaeMdj1p, the mitochondrial DnaJ homologue by Deloche (1997)10.1128/MCB.18.4.2023
/ Mol Cell Biol / The carboxy-terminal domain of Hsc70 provides binding sites for a distinct set of chaperone cofactors by Demand (1998){'key': '2023072900334273100_B21', 'first-page': '3288', 'article-title': 'Topology and functional domains of Sec63p, an ER membrane protein required for secretory protein translocation', 'volume': '12', 'author': 'Feldheim', 'year': '1992', 'journal-title': 'Mol Cell Biol'}
/ Mol Cell Biol / Topology and functional domains of Sec63p, an ER membrane protein required for secretory protein translocation by Feldheim (1992)10.1002/j.1460-2075.1995.tb07222.x
/ EMBO (Eur Mol Biol Organ) J / Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1 by Freeman (1995)10.1083/jcb.123.1.109
/ J Cell Biol / A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins by Gambill (1993)10.1093/emboj/17.22.6497
/ EMBO (Eur Mol Biol Organ) J / Unfolding of preproteins upon import into mitochondria by Gaume (1998)10.1016/0378-1119(88)90185-0
/ Gene / New yeast-Escherichia coli shuttle vectors constructed with in vitromutagenized yeast lacking six-base pair restriction sites by Gietz (1988)10.1016/0092-8674(95)90444-1
/ Cell / Can Hsp70 proteins act as force-generating motors? by Glick (1995)10.1002/pro.5560021112
/ Protein Sci / Import of cytochrome b2to the mitochondrial intermembrane space: the tightly folded heme-binding domain makes import dependent upon matrix ATP by Glick (1993)10.1073/pnas.95.11.6108
/ Proc Natl Acad Sci USA / Role of the J-domain in the cooperation of hsp40 with hsp70 by Greene (1998)10.1126/science.276.5311.431
/ Science / Crystal structure of the nucleotide exchange factor GrpE bound to the ATPase domain of the molecular chaperone DnaK by Harrison (1997)10.1038/381571a0
/ Nature / Molecular chaperones in cellular protein folding by Hartl (1996)10.1021/bi00016a033
/ Biochemistry / 1H and 15N magnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coliDnaJ (1-78) by Hill (1995)10.1002/pro.5560050421
/ Protein Sci / The mitochondrial protein import motor: dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis by Horst (1996)10.1023/A:1022470303365
/ J Bioenerg Biomembr / The mitochondrial import pathway: are precursors imported through membrane channels? by Jensen (1997)10.1038/348137a0
/ Nature / Requirement for hsp70 in the mitochondrial matrix for translocation and folding of precursor proteins by Kang (1990)10.1016/S0968-0004(98)01215-8
/ Trends Biochem Sci / The J-domain family and the recruitment of chaperone power by Kelley (1998)10.1073/pnas.91.26.12818
/ Proc Natl Acad Sci USA / Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane by Kronidou (1994)10.1128/MCB.15.12.7098
/ Mol Cell Biol / Mitochondrial GrpE modulates the function of matrix Hsp70 in translocation and maturation of preproteins by Laloraya (1995)10.1073/pnas.88.7.2874
/ Proc Natl Acad Sci USA / Escherichia coliDnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK by Liberek (1991)10.1083/jcb.137.2.377
/ J Cell Biol / Tim23, a protein import component of the mitochondrial inner membrane, is required for normal activity of the multiple conductance channel, MCC by Lohret (1997)10.1083/jcb.114.4.623
/ J Cell Biol / Characterization of SIS1, a Saccharomyces cerevisiaehomologue of bacterial dnaJ proteins by Luke (1991)10.1002/j.1460-2075.1992.tb05446.x
/ EMBO (Eur Mol Biol Organ) J / MPI1, an essential gene encoding a mitochondrial membrane protein, is possibly involved in protein import into yeast mitochondria by Maarse (1992)10.1093/emboj/16.22.6727
/ EMBO (Eur Mol Biol Organ) J / Active unfolding of precursor proteins during mitochondrial protein import by Matouschek (1997)10.1006/jmbi.1996.0762
/ J Mol Biol / Mge1 functions as a nucleotide release factor for Ssc1, a mitochondrial Hsp70 of Saccharomyces cerevisiae. by Miao (1997)10.1006/bbrc.1994.1468
/ Biochem Biophys Res Commun / YGE1p, a eukaryotic GrpE homolog, is localized in the mitochondrial matrix and interacts with mitochondrial Hsp70 by Nakai (1994)10.1146/annurev.biochem.66.1.863
/ Annu Rev Biochem / Protein import into mitochondria by Neupert (1997)10.1016/S0021-9258(17)36007-6
/ J Biol Chem / Nucleotide sequence of the Escherichia colidnaJ gene and purification of the gene product by Ohki (1986)10.1016/S0960-9822(95)00033-9
/ Curr Biol / Protein sorting: pulling in the proteins by Pfanner (1995)10.1016/S0960-9822(06)00048-0
/ Curr Biol / Mitochondrial biogenesis: the Tom and Tim machine by Pfanner (1997)10.1146/annurev.cellbio.13.1.25
/ Annu Rev Cell Dev Biol / Mitochondrial preprotein translocase by Pfanner (1997){'key': '2023072900334273100_B50'}
10.1016/S0955-0674(96)80027-5
/ Curr Opin Cell Biol / Approaching the mechanism of protein transport across the ER membrane by Rapoport (1996)10.1016/0014-5793(91)81157-4
/ FEBS Lett / Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space by Rassow (1991){'key': '2023072900334273100_B53'}
10.1083/jcb.127.6.1547
/ J Cell Biol / Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44 by Rassow (1994)10.1016/0962-8924(95)80013-7
/ Trends Cell Biol / Partner proteins determine multiple functions of Hsp70 by Rassow (1995)10.1006/jmbi.1998.2455
/ J Mol Biol / The preprotein translocase of the mitochondrial inner membrane: function and evolution by Rassow (1999)10.1016/0092-8674(94)90317-4
/ Cell / Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding by Rowley (1994)10.1016/0092-8674(95)90089-6
/ Cell / Protein translocation across mitochondrial membranes: what a long, strange trip it is by Ryan (1995)10.1128/MCB.18.1.178
/ Mol Cell Biol / Characterization of the mitochondrial inner membrane translocase complex: the Tim23p hydrophobic domain interacts with Tim17p but not with other Tim23p molecules by Ryan (1998)10.1083/jcb.109.6.2665
/ J Cell Biol / A yeast gene essential for protein assembly in the endoplasmic reticulum and the nucleus has homology to DnaJ, an E. coliheat shock protein by Sadler (1989)10.1016/0003-2697(91)90094-A
/ Anal Biochem / Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form by Schägger (1991)10.1074/jbc.271.50.31763
/ J Biol Chem / The protein import system of mitochondria by Schatz (1996)10.1073/pnas.89.24.11930
/ Proc Natl Acad Sci USA / Identification of a 45-kD protein at the protein import site of the yeast mitochondrial inner membrane by Scherer (1992)10.1083/jcb.129.4.979
/ J Cell Biol / A yeast DnaJ homologue, Scj1p, can function in the endoplasmic reticulum with BiP/ Kar2p via a conserved domain that specifies interaction with Hsp70s by Schlenstedt (1995)10.1038/371768a0
/ Nature / Mitochondrial Hsp70/MIM44 complex facilitates protein import by Schneider (1994)10.1002/j.1460-2075.1996.tb00966.x
/ EMBO (Eur Mol Biol Organ) J / The nucleotide exchange factor MGE exerts a key function in the ATP-dependent cycle of mtHsp70-Tim44 interaction driving mitochondrial protein import by Schneider (1996)10.1091/mbc.4.11.1145
/ Mol Biol Cell / Genetic interactions between KAR2 and SEC63, encoding eukaryotic homologues of DnaK and DnaJ in the endoplasmic reticulum by Scidmore (1993)10.1016/0092-8674(93)90287-Z
/ Cell / Eucaryotic DnaJ homologs and the specificity of Hsp70 activity by Silver (1993)10.1073/pnas.89.9.3770
/ Proc Natl Acad Sci USA / What drives the translocation of proteins? by Simon (1992)10.1021/bi00713a022
/ Biochemistry / Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles by Sims (1974)10.1016/S0091-679X(08)61689-1
/ Methods Cell Biol / Analysis of mitochondrial protein import using translocation intermediates and specific antibodies by Söllner (1991)10.1073/pnas.91.24.11343
/ Proc Natl Acad Sci USA / NMR structure determination of the Escherichia coliDnaJ molecular chaperone: secondary structure and backbone fold of the N-terminal region (residues 2-108) containing the highly conserved J domain by Szyperski (1994)10.1126/science.7973708
/ Science / The role of Hsp70 in conferring unidirectionality on protein translocation into mitochondria by Ungermann (1994)10.1002/j.1460-2075.1996.tb00409.x
/ EMBO (Eur Mol Biol Organ) J / The ΔΨ- and Hsp70/MIM44-dependent reaction cycle driving early steps of protein import into mitochondria by Ungermann (1996)10.1074/jbc.272.31.19594
/ J Biol Chem / Functional interaction of the auxilin J domain with the nucleotide- and substrate-binding modules of Hsc70 by Ungewickell (1997)10.1074/jbc.270.50.29848
/ J Biol Chem / The mitochondrial protein import machinery. Role of ATP in dissociation of the Hsp70-Mim44 complex by von Ahsen (1995)10.1083/jcb.123.1.119
/ J Cell Biol / Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import in heat shock protein 70 in the matrix by Voos (1993){'key': '2023072900334273100_B78', 'first-page': '6627', 'article-title': 'Mitochondrial GrpE is present in a complex with Hsp70 and preproteins in transit across membranes', 'volume': '14', 'author': 'Voos', 'year': '1994', 'journal-title': 'Mol Cell Biol'}
/ Mol Cell Biol / Mitochondrial GrpE is present in a complex with Hsp70 and preproteins in transit across membranes by Voos (1994)10.1002/j.1460-2075.1996.tb00627.x
/ EMBO (Eur Mol Biol Organ) J / Differential requirement for the mitochondrial Hsp70-Tim44 complex in unfolding and translocation of preproteins by Voos (1996)10.1073/pnas.95.1.144
/ Proc Natl Acad Sci USA / Characterization of mammalian translocase of inner mitochondrial membrane (Tim44) isolated from diabetic newborn mouse kidney by Wada (1998)10.1006/jmbi.1997.1267
/ J Mol Biol / Mdj2p, a novel DnaJ homolog in the mitochondrial inner membrane of the yeast Saccharomyces cerevisiae. by Westermann (1997)10.1002/j.1460-2075.1995.tb07351.x
/ EMBO (Eur Mol Biol Organ) J / The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria by Westermann (1995)10.1128/MCB.16.12.7063
/ Mol Cell Biol / Role of the mitochondrial DnaJ homolog Mdj1p as a chaperone for mitochondrially synthesized and imported proteins by Westermann (1996)10.1016/0092-8674(93)90646-8
/ Cell / The yeast SIS1 protein, a DnaJ homolog, is required for the initiation of translation by Zhong (1993)
Dates
Type | When |
---|---|
Created | 23 years, 1 month ago (July 26, 2002, 12:45 p.m.) |
Deposited | 2 years, 1 month ago (July 28, 2023, 8:34 p.m.) |
Indexed | 1 year, 1 month ago (July 1, 2024, 4:28 p.m.) |
Issued | 26 years, 2 months ago (May 31, 1999) |
Published | 26 years, 2 months ago (May 31, 1999) |
Published Online | 26 years, 2 months ago (May 31, 1999) |
Published Print | 26 years, 2 months ago (May 31, 1999) |
@article{Merlin_1999, title={The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation}, volume={145}, ISSN={1540-8140}, url={http://dx.doi.org/10.1083/jcb.145.5.961}, DOI={10.1083/jcb.145.5.961}, number={5}, journal={The Journal of Cell Biology}, publisher={Rockefeller University Press}, author={Merlin, Alessio and Voos, Wolfgang and Maarse, Ammy C. and Meijer, Michiel and Pfanner, Nikolaus and Rassow, Joachim}, year={1999}, month=may, pages={961–972} }