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© The Rockefeller University Press, 0021-9525/1999//961 $5.00
The Journal of Cell Biology, Volume 145, Number 5, , 1999 961-972

The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation

Alessio Merlin^*, Wolfgang Voos^*, Ammy C. Maarse, Michiel Meijer, Nikolaus Pfanner^*, and Joachim Rassow^*

^* Institut für Biochemie und Molekularbiologie, Universität Freiburg, D-79104 Freiburg, Germany; and Institute for Molecular Cell Biology, BioCentrum Amsterdam, 1098 SM Amsterdam, The Netherlands

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44₁₈ in addition to the endogenous wild-type Tim44. Tim44₁₈ is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44₁₈ together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44₁₈ in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

Key Words: mitochondria • inner membrane • protein translocation • Tim44 • Hsp70

Abbreviations used in this paper: EGS, ethylene glycolbis succinimidylsuccinate; mt, mitochondrial; Su9-DHFR, fusion protein between presequence of F₀-ATPase subunit 9 and dihydrofolate reductase; Tim44, 44-kD protein of the mitochondrial inner membrane import machinery.

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