The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation

Lim, Joo Hyun; Martin, Falk; Guiard, Bernard; Pfanner, Nikolaus; Voos, Wolfgang

doi:10.1093/emboj/20.5.941

Cited by 77 publications

(63 citation statements)

References 60 publications

(114 reference statements)

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“…Affinity-purification of antibodies (8), ATPase assays (8), co-IP of Ssc1-Tim44 complex (9), mitochondria purification (24), and in vitro import assays (25,26) were carried out as described. For import assays, mitochondria isolated from cells grown at 30°C were preincubated at 37°C for 10 min to induce the mutant phenotype before the addition of precursor protein at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

D’Silva

Schilke

Walter

et al. 2005

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

108

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Translocation of proteins across the mitochondrial inner membrane is an essential process requiring an import motor having mitochondrial Hsp70 (mtHsp70) at its core. The J protein partner of mtHsp70, Pam18, is an integral part of this motor, serving to stimulate the ATPase activity of mtHsp70. Pam16, an essential protein having an inactive J domain that is unable to stimulate mtHsp70's ATPase activity, forms a heterodimer with Pam18, but its function is unknown. We set out to test the importance of three properties of Pam16: (i) a stable interaction between Pam16 and Pam18, (ii) the inability of Pam16's degenerate J domain to stimulate Ssc1's ATPase domain, and (iii) the innately lower stimulatory activity of the Pam16:Pam18 heterodimer, compared to Pam18 alone. Neither substantial reduction in the ability of Pam18 to stimulate Ssc1's ATPase activity, nor the presence of an active J domain in Pam16, had deleterious effects on cell growth, indicating the lack of importance of two of these biochemical properties. However, a stable interaction between Pam16's degenerate J domain and Pam18's J domain was found to be critical for function. Alterations that destabilized the Pam16:Pam18 heterodimer had deleterious effects on cell growth and mitochondrial protein import; intragenic suppressors that restored robust growth also restored heterodimer stability. Our results support the idea that Pam16's J-like domain strongly interacts with Pam18's J domain, leading to a productive interaction of Pam18 with mtHsp70 at the import channel. mitochondria ͉ translocation ͉ Hsp40 ͉ Pam18 ͉ heterodimer

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Section: Methodsmentioning

confidence: 99%

Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

D’Silva

Schilke

Walter

et al. 2005

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

108

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“…In contrast to previous mitochondrial degradation assays using radiolabeled preproteins in small amounts, we challenged the proteolytic system with large amounts of substrate proteins (27), an assay system that allowed us to determine the kinetics of the degradation reaction. Two reporter proteins with different folding stabilities were accumulated in the mitochondrial matrix to compare the energy requirement of their degradation and to examine the involvement of the chaperones Hsp78 and Mcx1 in the process.…”

mentioning

confidence: 99%

The ClpB Homolog Hsp78 Is Required for the Efficient Degradation of Proteins in the Mitochondrial Matrix

Röttgers

Zufall²,

Guiard

et al. 2002

Journal of Biological Chemistry

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Molecular chaperones perform vital functions in mitochondrial protein import and folding. In yeast mitochondria, two members of the Clp/Hsp100 chaperone family, Hsp78 and Mcx1, have been identified as homologs of the bacterial proteins ClpB and ClpX, respectively. In this report we employed a novel quantitative assay system to assess the role of Hsp78 and Mcx1 in protein degradation within the matrix. Mitochondria were preloaded with large amounts of two purified recombinant reporter proteins exhibiting different folding stabilities. Proteolysis of the imported substrate proteins depended on the mitochondrial level of ATP and was mediated by the matrix protease Pim1/LON. Degradation rates were found to be independent of the folding stability of the reporter proteins. Mitochondria from hsp78⌬ cells exhibited a significant defect in the degradation efficiency of both substrates even at low temperature whereas mcx1⌬ mitochondria showed wild-type activity. The proteolysis defect in hsp78⌬ mitochondria was independent from the aggregation behavior of the substrate proteins. We conclude that Hsp78 is a genuine component of the mitochondrial proteolysis system required for the efficient degradation of substrate proteins in the matrix.

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“…Apart from driving polypeptide movement through the translocation channel, mtHsp70 is also required for the unfolding of stably folded preprotein domains (26). Active unfolding implies the generation of an inward-directed translocation force on preproteins in transit (27,29). As was previously observed for the conditional mutant ssc1-2, in the absence of a translocation force import becomes limited by (low) spontaneous unfolding rates.…”

Section: Discussionmentioning

confidence: 84%

“…All results are representative for at least five independent experiments. sensitive Ssc1-2 protein that is characterized by increased and prolonged interactions with imported proteins (27,47,53). Increased trapping of matrix proteins by mtHsp70 interferes with an efficient translocation reaction because the release of substrate proteins becomes limiting.…”

Section: Discussionmentioning

confidence: 99%

Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force

Becker

Krayl

Strub

et al. 2009

Journal of Biological Chemistry

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The essential mitochondrial Hsp70 (mtHsp70) is required for the import of mitochondrial preproteins into the matrix compartment. The translocation-specific activity of mtHsp70 is coordinated by its interaction with specific partner proteins, forming the import motor complex that provides the energy for unfolding and complete translocation of precursor polypeptide chains. A major biochemical characteristic of Hsp70-type chaperones is their nucleotide-regulated affinity to polypeptide substrates. To study the role of this allosteric regulation in the course of preprotein translocation, we have generated specific mtHsp70 mutations located within or close to the interface between the nucleotide-binding and the substrate-binding domains. Mitochondria isolated from the mtHsp70 mutants displayed severely reduced import efficiencies in vitro. Two of the mutants exhibited strong growth defects in vivo and were significantly impaired in the generation of an inward-directed, ATPdependent import force on precursor proteins in transit. The biochemical properties of these two mutant proteins were consistent with defects in the transfer of conformational signals to the substrate-binding domain, resulting in a prolonged and enhanced interaction with imported substrate proteins. Furthermore, interference with the allosteric mechanism resulted in defects of translocation-specific partner protein interaction. We conclude that even a partial disruption of the interdomain communication in the mtHsp70 chaperone results in an almost complete breakdown of its translocation-driving properties.Chaperones of the 70-kDa family (Hsp70) are ubiquitous proteins that occur in all species (except archaea) and fulfill crucial cellular functions under normal and stress conditions (1). Eukaryotic cells contain several Hsp70 paralogs that are generally involved in all stages of protein biogenesis, ranging from biosynthesis at the ribosomes, intracellular transport, and eventually proteolysis (2-4). Mitochondria of the model organism Saccharomyces cerevisiae contain three types of Hsp70 proteins, encoded by the genes SSC1, SSQ1, and SSC3 (ECM10) (5). Ssc3 is expressed at very low levels under normal growth conditions and its cellular function is so far unknown. Ssq1 has been shown to assist the assembly of iron/sulfur cluster cofactors in mitochondria. The most abundant and functionally important mitochondrial Hsp70 is Ssc1, generally referred to as mtHsp70. It performs most of the typical chaperone functions in terms of protein folding and quality control for polypeptides localized in the mitochondrial matrix compartment (5-7). In addition, Ssc1 is required for the import of all mitochondrial precursor proteins destined for the matrix (8). In yeast cells, deletion of SSC1 results in a lethal phenotype, a property that has been directly attributed to its crucial role in the import process. Mitochondrial preprotein translocation comprises a series of consecutive steps (9): after synthesis at cytosolic ribosomes, the N-terminal targeting signal of a p...

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The mitochondrial Hsp70-dependent import system actively unfolds preproteins and shortens the lag phase of translocation

Cited by 77 publications

References 60 publications

Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

The ClpB Homolog Hsp78 Is Required for the Efficient Degradation of Proteins in the Mitochondrial Matrix

Impaired Interdomain Communication in Mitochondrial Hsp70 Results in the Loss of Inward-directed Translocation Force

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