Mitochondrial Protein Import: Molecular Basis of the ATP-dependent Interaction of MtHsp70 with Tim44

Abstract

Protein translocation across the mitochondrial inner membrane is driven by cycles of binding and release of mitochondrial heat shock protein 70 (mtHsp70) in the matrix. The peripheral inner membrane protein, Tim44, recruits mtHsp70 in an ATP-dependent manner to the import sites. We show that DnaK, the closely related Hsp70 of Escherichia coli, when targeted to the matrix of yeast mitochondria, interacts in a specific manner with Tim44. The interaction is, however, not regulated by ATP, and DnaK cannot support protein translocation. We used truncated mtHsp70s and chimeric proteins consisting of segments of mtHsp70 and DnaK to analyze which portions of mtHsp70 bind and functionally interact with Tim44. We show that Tim44 interacts with the ␤-stranded core of the peptide binding domain of mtHsp70 and of DnaK. The ␣-helices A and B of the peptide binding domain of mtHsp70 are required to transmit the nucleotide state of the ATPase domain to the peptide binding domain. Tim44, by interacting in this way with the peptide binding domain, is proposed to coordinate the binding of mtHsp70 to the incoming preprotein and the subsequent release of the mtHsp70-preprotein complex from the TIM23 complex, the translocase of the inner membrane.The major mtHsp70, 1 Ssc1p in yeast, mediates translocation of proteins across the inner membrane of mitochondria. Ssc1p cooperates with Tim44, a peripheral inner membrane protein associated on the matrix with the TIM23 complex, which constitutes the preprotein conducting import channel across the inner membrane (1-7). Tim44 is a dimer that recruits two molecules of Ssc1p to the import channel (6,8). It was proposed that Tim44, Ssc1p, and its co-chaperone Mge1p are all parts of an ATP-dependent molecular motor that drives translocation of preproteins by a "hand over hand" mode (6, 7). Ssc1p binds to segments of a translocating preprotein as they emerge from the import channel. It is then released from the inner membrane, and the Ssc1p-preprotein complex can move further into the matrix. A central question regarding this pathway is how Tim44 can sense when to release mtHsp70 that has bound a preprotein. To address this question it is important to determine which portions of mtHsp70 interact with Tim44.Molecular chaperones of the Hsp70 family are composed of two domains, an N-terminal ATPase domain of about 45 kDa that is connected by a short linker to a peptide binding domain of ϳ25 kDa (9). The first part of the peptide binding domain consists of eight anti-parallel ␤-strands that form the substrate binding pocket (10). The ␤-stranded core is followed by an ␣-helical portion consisting of five helices, ␣A-␣E, and a C terminus of unknown structure. ␣B consists of two halves connected by a flexible hinge. ␣A and the first half of ␣B are packed against the ␤-stranded core. The second half of ␣B and ␣C-␣E correspond to a compact ␣-helical subdomain (11). To efficiently close the substrate binding pocket, the ␣-helical subdomain folds like a lid over the ␤-stranded core. Hsp70s bind unfolded poly...

A GTP-driven motor moves proteins across the outer envelope of chloroplasts

“…A further treatment of chloroplasts with protease (thermolysin) results in complete degradation of Toc159f to a 52-kDa fragment (8). Thermolysin-treated chloroplasts still exhibit a residual import capacity (34), which could indicate a second translocation pathway across the outer envelope (35) or a slow basal import that functions by diffusion through the Toc75 channel and chaperone action to move the precursor inside (20)(21)(22). Toc159f is, however, still active and can function as a precursor-interacting protein (18).…”

“…Translocation across the chloroplastic Tic or the mitochondrial translocase of the inner membrane complex is most likely driven by ATPdependent action of molecular chaperones (20)(21)(22). Here we present a reconstituted system of protein import into proteoliposomes by using Toc subunits that allows the elucidation of single steps of the translocation process in a detailed manner.…”

Overexpression of Mitochondrial Hsp70/Hsp75 Protects Astrocytes against Ischemic Injury in vitro

“…Both facilitating import, and facilitating and maintaining proper mitochondrial protein conformation likely contribute to protection by Hsp75. As both import and facilitation of folding depend on the ATPase activity of Hsp75 (Moro et al, 2002), it will be interesting to determine the extent to which its neuroprotective ability also requires the ATPase domain and ATPase activity.…”

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…Similar to the ratchet mechanism of Kar2 for precursor import into the ER, the binding and release of Ssc1 from Tim44 at the inner mitochondrial membrane facilitate the movement of the precursor in the forward direction (399). Specifically, Tim44 interacts with the ␤-stranded portion of the peptide-binding domain of Ssc1, which is postulated to position the substratebinding domain near the outlet of the import channel to make Ssc1 available immediately for precursor protein binding (307). Genetic and biochemical experiments also showed Ssc1 to interact with the integral membrane protein Tim17 in an ATPase-dependent manner (27).…”