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

Abstract

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...

ATPase Domain and Interdomain Linker Play a Key Role in Aggregation of Mitochondrial Hsp70 Chaperone Ssc1

“…The importance of this region for Ssc1 function has been confirmed by observations made with an E240A/V241A mutant. The mutant protein was not functional in intact yeast cells (42). This segment in the ATPase domain, however, is probably not the only region crucial for aggregation of Ssc1, because the Ssc1K ins variant was observed to aggregate in E. coli at higher temperatures (data not shown).…”

Chaperones and Proteases of Mitochondria: From Protein Folding and Degradation to Mitophagy

“…While the basic mechanism relies on a Brownian ratchet model, where the translocation process is driven by diffusion, it has been experimentally proven that the complete import of preproteins containing conformational restriction, i.e., folded C-terminal domains, requires a more active mechanism [35]. The generation of a pulling force needs three prerequisites, (i) a conformational change in the chaperone, driven by nucleotide hydrolysis, combined with (ii) a stable and functional interaction with the translocase and (iii) the preprotein polypeptide [36,37]. All these prerequisites are fulfilled in case of the import motor complex, consisting of Ssc1, Tim44, Pam18, and Mge1.…”

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

“…Isolated mitochondria were suspended in import buffer and preincubated in presence of 2 mM ATP for 15 min at 25°C. Mitochondria were reisolated and lysed, and protein extracts were incubated with ATP-agarose or RCMLA-Sepharose, as described (28). Samples were analyzed by SDS-PAGE and immunoblot.…”

“…Co-Immunoprecipitation of Imported Proteins-Co-immunoprecipitations of imported proteins with mtHsp70 using immobilized antibodies against Ssc1 were performed as described (28). To examine the interaction of Ssc1 with Zim17, radiolabeled Zim17-WT, -3a, -3b, and Mdh1 were imported into isolated WT mitochondria for 20 min at 25°C.…”

“…The radiolabeled precursor protein Su9(86)-DHFR was imported into isolated yeast mitochondria according to established procedures (28). For the analysis of the inwardly directed translocation activity of Ssc1 ("pulling assay"), radiolabeled b 2 (167) ⌬ -DHFR was arrested as translocation intermediates with an import in the presence of the dihydrofolate reductase (DHFR) ligand methotrexate (MTX) at 5 M. The precursors were pretreated with MTX, and the import reaction was performed for 15 min at 25°C.…”