The Hsp70 peptide-binding domain determines the interaction of the ATPase domain with Tim44 in mitochondria

Strub, Andreas; Röttgers, Karin; Voos, Wolfgang

doi:10.1093/emboj/21.11.2626

Cited by 44 publications

(35 citation statements)

References 38 publications

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“…Although deletion of Ssc1p is lethal, disruption of Ecm10 has no apparent mitochondrial phenotype. Ecm10 is expressed at a lower level compared with Ssc1p but even its overexpression did not rescue the deletion of Ssc1p (25,26). In contrast, Mdj2 is obviously capable of replacing Tim14 if expressed at a level as high as that of Tim14.…”

Section: Discussionmentioning

confidence: 94%

The Import Motor of the Yeast Mitochondrial TIM23 Preprotein Translocase Contains Two Different J Proteins, Tim14 and Mdj2

Mokranjac

Sichting

Popov‐Čeleketić

et al. 2005

Journal of Biological Chemistry

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The import motor of the mitochondrial (mt)TIM23 complex drives translocation of presequence-containing preproteins across the mitochondrial inner membrane in an ATP-dependent manner. Tim44 is the central component of the motor. It recruits mtHsp70, which binds the incoming preproteins. The J protein Tim14 stimulates the ATPase activity of mtHsp70 and thereby enables efficient binding of mtHsp70 to preproteins. Tim16 is a J-like protein that forms a stable subcomplex with Tim14 and recruits it to the translocase. All subunits of the TIM23 translocase but one are essential for yeast cell viability. Yeast cells contain a close homologue of Tim14, Mdj2. In contrast to Tim14, its deletion leads to no obvious growth defect. In the present study we analyzed Mdj2 and compared it with Tim14. Mdj2 forms a complex with Tim16 and is recruited to the TIM23 translocase. It stimulates the ATPase activity of mtHsp70 to the same extent that Tim14 does. Mdj2 is expressed at a lower level compared with Tim14, and its complex with Tim16 is less stable. However, overexpressed Mdj2 fully restores the growth of cells lacking Tim14. We conclude that Mdj2 is a functional J protein and a component of the mitochondrial import motor.Translocation and sorting of precursor proteins into the various mitochondrial subcompartments are achieved by the concerted action of translocases present in both mitochondrial membranes (for reviews on protein import into mitochondria see Refs. 1-4). After crossing the outer membrane via the TOM complex, presequence-containing preproteins are directed to the TIM23 complex in the inner membrane. The TIM23 complex mediates their translocation across or insertion into the inner membrane. In doing so it uses two energy sources, the membrane potential and ATP. The TIM23 complex can be functionally and structurally subdivided into the membraneintegrated translocation channel and the import motor, which is bound to it at the matrix face. Tim50 is the first component of the TIM23 complex that interacts with the incoming polypeptides as soon as they emerge from the outlet of the TOM channel (5-7). They are then delivered to the translocation channel of the TIM23 complex, which is most likely formed by Tim17 and Tim23. The amino acid sequences of the membrane integrated parts of Tim17 and Tim23 are related, suggesting a common evolutionary origin. Tim23 has an additional N-terminal domain of ϳ100 amino acid residues that is exposed to the intermembrane space. It apparently cooperates with Tim50 in recognition of presequences. The N-terminal intermembrane space-exposed domain of Tim17 is much shorter but also appears to have an essential function in protein translocation, most likely in the gating of the channel (8). For completion of translocation into the matrix, the membrane-integrated part of the translocase cooperates with the import motor (9). The import motor is made up from at least five subunits, Tim14/ Pam18, Tim16/Pam16, Tim44, Mge1, and mtHsp70.

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Section: Discussionmentioning

confidence: 94%

The Import Motor of the Yeast Mitochondrial TIM23 Preprotein Translocase Contains Two Different J Proteins, Tim14 and Mdj2

Mokranjac

Sichting

Popov‐Čeleketić

et al. 2005

Journal of Biological Chemistry

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“…Tim44, which serves as a tether of Ssc1 for the import channel, shows a weak similarity to the J domain of Sec63, and a deletion of this region disrupts the interaction of Tim44 with Ssc1 in mitochondria (17). Although controversial, some recent reports indicate that Tim44 interacts with the ATPase domain of Ssc1, as does the J domain of known J proteins (18,19). However, regardless of whether it acts as a J protein partner of Ssc1, Tim44 regulates the interaction of Ssc1 with the import channel.…”

mentioning

confidence: 99%

J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

D’Silva

Schilke

Walter

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

171

133

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The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import. M ost proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes and must therefore be imported across the outer and inner mitochondrial membranes. Translocation across the inner membrane occurs through the inner membrane channel and is driven by the membrane potential and an import motor (1-3). Three critical components of this motor are the major Hsp70 molecular chaperone of mitochondria (mtHsp70; Ssc1 in yeast); the peripheral inner membrane protein Tim44, which tethers Ssc1 to the import channel; and the nucleotide release factor for Ssc1, Mge1. Multiple cycles of Ssc1 binding to and release from translocating polypeptide, driving the import process, are required for import of proteins into the mitochondrial matrix (1-3).Ssc1, like other Hsp70s, contains a C-terminal domain that binds short segments of unfolded polypeptides rich in hydrophobic amino acids (4, 5). The N-terminal ATPase domain regulates this binding through interaction with adenine nucleotides. In turn, binding of the peptide segment to the C-terminal domain stimulates the ATPase activity of the N terminus. In the ATP-bound state, interaction with peptide substrate is unstable, with very fast on and off rates. In the ADP-bound state, the interaction is relatively stable, with slow on and off rates. Therefore, it is thought that the ATP-bound form of Hsp70 initiates interaction with polypeptide substrate, which is then stabilized by the hydrolysis of ATP. Exchange of ATP for bound ADP results in the release of peptide, thus completing the cycle.Hsp70s rarely, if ever, function independently. Rather, they function with cochaperones. J proteins, named because of the presence of the signature J domain that interacts with the ATPase domain, stimulate Hsp70's ATPase activity, thus facilitating interaction with substrate polypeptides (6, 7). This activity is a critical feature of J...

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“…Using this approach we were able to analyze the behavior of the mutated proteins in the native mitochondrial environment. 36 As they contained the identical presequence, all constructs imported with similar efficiencies into isolated mitochondria. After completion of import and removal of excess preprotein by protease treatment, the mitochondria were lysed under native conditions and substrate binding studies and immunoprecipitation experiments were performed.…”

Section: Resultsmentioning

confidence: 99%

The Putative Helical Lid of the Hsp70 Peptide-binding Domain is Required for Efficient Preprotein Translocation into Mitochondria

Strub

Zufall

Voos

2003

Journal of Molecular Biology

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The Hsp70 peptide-binding domain determines the interaction of the ATPase domain with Tim44 in mitochondria

Cited by 44 publications

References 38 publications

The Import Motor of the Yeast Mitochondrial TIM23 Preprotein Translocase Contains Two Different J Proteins, Tim14 and Mdj2

The Import Motor of the Yeast Mitochondrial TIM23 Preprotein Translocase Contains Two Different J Proteins, Tim14 and Mdj2

J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

The Putative Helical Lid of the Hsp70 Peptide-binding Domain is Required for Efficient Preprotein Translocation into Mitochondria

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