Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering - PubMed (original) (raw)

Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mitochondrial Hsp70 tethering

Dirk Schiller et al. Mol Cell Biol. 2008 Jul.

Abstract

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by the action of the import motor, which is associated with the translocon on the matrix side of the membrane. It is well established that an essential peripheral membrane protein, Tim44, tethers mitochondrial Hsp70 (mtHsp70), the core of the import motor, to the translocon. This Tim44-mtHsp70 interaction, which can be recapitulated in vitro, is destabilized by binding of mtHsp70 to a substrate polypeptide. Here we report that the N-terminal 167-amino-acid segment of mature Tim44 is sufficient for both interaction with mtHsp70 and destabilization of a Tim44-mtHsp70 complex caused by client protein binding. Amino acid alterations within a 30-amino-acid segment affected both the release of mtHsp70 upon peptide binding and the interaction of Tim44 with the translocon. Our results support the idea that Tim44 plays multiple roles in mitochondrial protein import by recruiting Ssc1 and its J protein cochaperone to the translocon and coordinating their interactions to promote efficient protein translocation in vivo.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Interaction of Tim44 fragments with Ssc1. (A) Diagram of Tim44 and two fragments used in this study. Tim4443-209, N terminus of mature Tim44; Tim44210-431, C terminus. The arrow indicates the site of cleavage by the matrix processing protease (MPP) to generate mature Tim44. (B) (Top) Tim4443-209 and full-length Ssc1 were incubated together at a molar ratio of 2:1 (Tim44 to Ssc1) for 60 min in the presence of ATP. (Bottom) Same as top panel, except that the Tim4443-209-Ssc1 mixture was incubated for an additional 5 min with substrate peptide P5 prior to analysis. The mixtures were subjected to chromatography, and eluted fractions were analyzed by SDS-PAGE and immunoblotting using Tim44- or Ssc1-specific antibodies. Migration of Ssc1 alone (short dashes) and Tim4443-209 alone (long dashes) is indicated by vertical lines. (C) Tim44210-431 and full-length Ssc1 were incubated together in the presence of ATP (+Ssc1). As a control, Tim44210-431 alone was subjected to chromatography (−Ssc1). Analysis of samples was done as described for panel B.

FIG. 2.

FIG. 2.

Mutational analysis of TIM44. (A) Overview of the phenotypes of tim44 mutants. (Top) Eight deletion mutants lacking 12 to 17 amino acids within the interval of residues 68 to 202 of Tim44 (dotted rectangles). Deletion mutations causing phenotypic effects are underlined (see panel B). (Bottom) Alanine scanning of the segment containing residues 126 to 209. Twenty-one quadruple mutants substituting four alanines for four consecutive native residues were constructed. Amino acid substitutions leading to growth defects are underlined. Residue R180, which is critical for Tim44's in vivo function, is labeled with an asterisk. ts: temperature sensitive. (B) Growth phenotypes of _tim44_Δ strains carrying deletions within the coding sequence for the N-terminal segment of Tim44. Tenfold serial dilutions of cells were plated on the indicated medium. (Left) Medium containing 5-FOA. _tim44_Δ strains expressing the indicated plasmid-encoded mutant and wild-type (WT) TIM44 genes were expressed at 23°C for 4 days. (Right) Strains recovered from plating on 5-FOA were spotted onto rich glucose-based medium (rich) and incubated for 3 days (23°C) or 2 days (30°C and 37°C). (C) Protein expression. Cell extracts of _tim44_Δ strains expressing both the indicated mutant and WT TIM44 genes were analyzed by SDS-PAGE and immunoblotting using Tim44-specific antibodies. Cells harboring both WT and mutant TIM44 genes on different plasmids were grown at 23°C in minimal medium to select for the presence of both plasmids. See Materials and Methods for details. (D) Growth phenotypes of TIM44 amino acid substitution mutants. Cells were plated onto rich medium and incubated at the indicated temperatures for 3 days.

FIG. 3.

FIG. 3.

Translocation defect of TIM44 mutants. (A) Accumulation of a preprotein in vivo. Cell extracts from tim44 mutant strains grown at 23°C and subsequently shifted to 37°C for 6 h were analyzed by SDS-PAGE and immunoblotting using Hsp60-specific antibodies. p, premature Hsp60; m, mature Hsp60. (B) Import of the recombinant precursor cytochrome _b_2(220)Δ19-DHFR into tim44 mitochondria. Radiolabeled cytochrome _b_2(220)Δ19-DHFR was incubated with energized mitochondria for the indicated times. Following protease treatment to remove unimported precursor, cytochrome _b_2(220)Δ19-DHFR was detected by SDS-PAGE and autoradiography. (C) Steady-state protein levels in tim44 mitochondria. Five and 10 μg of total mitochondrial protein was subjected to SDS-PAGE and immunoblotting using antibodies against the indicated components of the TIM23 translocase and the PAM. (D) Fractionation of tim44 mitochondria. Purified mitochondria were fractionated by sonication followed by ultracentrifugation. The distributions of the respective Tim44 variants in the total (T) and soluble (S) fractions and the pellet containing the membranes (P) were monitored using Tim44-specific antibodies. As controls, the presence of the matrix-soluble Mge1 and the inner membrane protein Tim23 was also assessed. (E) Solubility of mutant Tim44 proteins after temperature upshift. Energized mitochondria were incubated for 30 min at the indicated temperatures. After solubilization with detergent, mitochondrial lysates were subjected to ultracentrifugation to separate them into supernatant (S) and pellet (P) fractions that were subsequently subjected to SDS-PAGE and immunoblotting using antibodies against Tim44, Mge1, and Tim23.

FIG. 4.

FIG. 4.

Interaction of Tim44 variants with Ssc1. (A) In organellar. Mitochondrial lysates prepared in the presence (+) or absence (−) of ATP were subjected to immunoprecipitation with Tim44-specific antibodies, followed by immunoblotting with the indicated antibodies. (B) In vitro. (Top) Indicated Tim44 proteins (closed symbols) and Ssc1 (open circles) were incubated in the presence of ATP and then subjected to size-exclusion chromatography. Eluted fractions were analyzed by SDS-PAGE and immunoblotting using Tim44- or Ssc1-specific antibodies. (Bottom) Same as top panels, except that peptide substrate (P5) was added after formation of the Tim44-Ssc1 complex, prior to chromatography.

FIG. 5.

FIG. 5.

Association of the import motor with the TIM23 translocase. (A) Mitochondria were solubilized by treatment with digitonin. Solubilized material was subjected to immunoprecipitation using Tim23-specific antibodies cross-linked to protein A beads. Precipitates were analyzed by SDS-PAGE and immunoblotting using antibodies specific for the indicated proteins (IP). A total of 4% solubilized material was used as a loading control (load). (B) Signals were quantified by densitometry and plotted as percentages of the wild-type control reaction value.

Similar articles

Cited by

References

    1. Bauer, M. F., K. Gempel, A. S. Reichert, G. A. Rappold, P. Lichtner, K. D. Gerbitz, W. Neupert, M. Brunner, and S. Hofmann. 1999. Genetic and structural characterization of the human mitochondrial inner membrane translocase. J. Mol. Biol. 28969-82. - PubMed
    1. Bauer, M. F., C. Sirrenberg, W. Neupert, and M. Brunner. 1996. Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria. Cell 8733-41. - PubMed
    1. Becker, L., M. Bannwarth, C. Meisinger, K. Hill, K. Model, T. Krimmer, R. Casadio, K. N. Truscott, G. E. Schulz, N. Pfanner, and R. Wagner. 2005. Preprotein translocase of the outer mitochondrial membrane: reconstituted Tom40 forms a characteristic TOM pore. J. Mol. Biol. 3531011-1020. - PubMed
    1. Boeke, J. D., F. LaCroute, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197345-346. - PubMed
    1. Bomer, U., A. C. Maarse, F. Martin, A. Geissler, A. Merlin, B. Schonfisch, M. Meijer, N. Pfanner, and J. Rassow. 1998. Separation of structural and dynamic functions of the mitochondrial translocase: Tim44 is crucial for the inner membrane import sites in translocation of tightly folded domains, but not of loosely folded preproteins. EMBO J. 174226-4237. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources