Impaired interdomain communication in mitochondrial Hsp70 results in the loss of inward-directed translocation force.
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, ATP-dependent 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.
Becker
Dorothea
D
Institut für Biochemie und Molekularbiologie, Universität Bonn, Nussallee 11, D-53115 Bonn; Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg.
Krayl
Martin
M
Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg.
Strub
Andreas
A
Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg.
Li
Yanfeng
Y
Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, D-79104 Freiburg.
Mayer
Matthias P
MP
ZMBH, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany.
Voos
Wolfgang
W
Institut für Biochemie und Molekularbiologie, Universität Bonn, Nussallee 11, D-53115 Bonn. Electronic address: wolfgang.voos@uni-bonn.de.
D000494:Allosteric Regulation; D019943:Amino Acid Substitution; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047468:Immunoprecipitation; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D029701:Saccharomyces cerevisiae Proteins