Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1998 Sep;18(9):5256-62.
doi: 10.1128/MCB.18.9.5256.

Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins

D Rapaport  1 , K P Künkele, M Dembowski, U Ahting, F E Nargang, W Neupert, R Lill
Affiliations

Dynamics of the TOM complex of mitochondria during binding and translocation of preproteins

D Rapaport et al. Mol Cell Biol. 1998 Sep.

Abstract

Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex of Neurospora crassa and Saccharomyces cerevisiae mitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Tom40 forms an oligomeric structure in the outer membrane of N. crassa and yeast mitochondria. (A) Untreated OMV (circles) and trypsin-treated OMV (squares) were solubilized in buffer G. A third sample was solubilized before treatment with trypsin (triangles). All samples were applied to a Superose 6 column and chromatographed and fractions were collected as described in Materials and Methods. Tom40 was detected by immunostaining and quantitated by densitometry. The peak of elution of various marker proteins of the indicated molecular masses is marked by arrows. a.u., arbitrary units. (B) Mitochondria were isolated from a yeast strain expressing a hexahistidinyl-tagged version of Tom40 (Tom40his6). The organelles were solubilized in buffer B (50 mM Tris-HCl [pH 7.4], 200 mM KCl, 10 mM imidazole, and 0.5% Triton X-100) containing 7 M urea where indicated. The extract (Load) was applied to a Ni-NTA affinity resin. Bound and unbound material was analyzed by immunostaining using antibodies against Tom40 and Tom20. Wild-type Tom40 does not bind to the Ni-NTA affinity resin (not shown).
FIG. 2
FIG. 2
Tom40 forms homo-oligomers and interacts with Tom6. The indicated cross-linking reagents (see Materials and Methods) were added to intact mitochondria (A) or OMV (B). Samples were incubated for 30 min at 25°C before the cross-linkers were quenched. Proteins were analyzed by SDS-PAGE under nonreducing conditions and immunostaining with antibodies against Tom40. (C) The Tom40-containing 45-kDa band is a cross-linking adduct of Tom40 and Tom6. OMV were incubated with the cross-linker EDC or DSP for 30 min. Aliquots of each sample were analyzed by immunostaining with antibodies (Ab) against Tom40 and Tom6. Tom6 is only weakly stained due to its poor blotting efficiency. (D) Tom40 cross-linking products in yeast mitochondria. Isolated yeast mitochondria were treated with the indicated cross-linkers and analyzed by immunostaining for Tom40 as described for panel A. (E) The Tom40 cross-linking products are formed by using purified N. crassa TOM complex (21). As described for panel A, cross-linkers were added to the purified TOM complex and samples were incubated for 90 min at 0°C. Further analysis was performed as described for panel A. (F) The isoelectric point of the Tom40 cross-linking products is identical to that of the Tom40 monomer. Cross-linking with DSG was performed as described for panel B, using OMV. The sample was separated in the first dimension by isoelectric focusing and in the second dimension by SDS-PAGE (2). The pI values and the molecular masses of marker proteins are indicated.
FIG. 3
FIG. 3
The cytosolic domains of the surface receptors modulate the structure of the Tom40 oligomer. (A) Cross-linking with DSP was performed as described in the legend to Fig. 2A by using untreated OMV (−) or OMV that were treated with trypsin (60 μg/ml) for 15 min on ice (before). With one sample, trypsin treatment was performed after the cross-linking reaction (after). Further analysis by immunostaining of Tom40 was performed as described in the legend to Fig. 2A. (B) Mutations introduced into the cytosolic domain of N. crassa Tom22. A schematic representation of the cytosolic domain of Tom22 is shown and the three blocks of negative charges (I, II, and III) are boxed. Above and below the boxes, the wild-type (WT) and mutant (MUT) residues, respectively, are given for the positions indicated inside the box. The lower panel presents the N. crassa strains expressing Tom22 mutant proteins with various combinations of the three mutated blocks. The net negative charge of the cytosolic domain is given on the right. (C) Mutations in the cytosolic domain of Tom22 alter the oligomeric structure of Tom40. OMV isolated from the various Tom22 strains were incubated without (−) or with (+) DSG. Further analysis of Tom40-containing cross-linking products was performed as described in the legend to Fig. 2A. (D) The mutations in the cytosolic domain of Tom22 affect the sensitivity of Tom22 and Tom40 to proteolytic attack. OMV isolated from the indicated Tom22 strains were treated with different concentrations of proteinase K (15 min at 0°C). After the addition of phenylmethylsulfonyl fluoride, further analysis and immunostaining for Tom40 and Tom22 were performed as described in the legend to Fig. 2A. The positions of Tom22 and its C-terminal 12-kDa fragment are indicated (18). WT, wild-type strain.
FIG. 4
FIG. 4
Structural alterations in the Tom40 oligomer upon binding and movement of preproteins from the cis to the trans side of the outer membrane. (A) OMV were incubated with (+) or without (−) pSu9-DHFR in the presence of NADPH and MTX for 10 min at 0°C. The samples containing pSu9-DHFR were treated with low- or high-salt buffer (20 or 120 mM KCl, respectively). After reisolation of the OMV by centrifugation and resuspension in SEMK buffer containing NADPH and MTX, DSG was added. One of the samples lacking pSu9-DHFR was also treated with DSG, while the other one remained untreated. Further analysis and immunostaining against Tom40 were performed as described in the legend to Fig. 2A. (B) Preprotein binding to cis and trans sites alters the cross-linking pattern of Tom40. The indicated concentrations of pSu9-DHFR were added to OMV under conditions leading to specific binding to the cis or trans sites (see references and 31). Cross-linking was performed with DSG, and samples were analyzed by immunostaining for Tom40 as described in the legend to Fig. 2A. (C) Binding of preproteins does not cause dissociation of the Tom40 oligomer. OMV were incubated for 20 min at 0°C with various concentrations of pSu9-DHFR. The cross-linkers DSG, DSP, and DPDPB were added as indicated for 30 min at 25°C. Further treatment and analysis by immunostaining for Tom40 was performed as described in the legend to Fig. 2A. (D) Presequence peptides alter the structure of Tom40. Isolated mitochondria were incubated with the indicated concentrations of the presequence peptides pCoxIV (residues 1 to 22 of the precursor of subunit IV of yeast cytochrome oxidase [11]) and pF1β (residues 1 to 32 of the precursor of the β subunit of yeast F1-ATPase) or with the control peptide CH4 (N-terminal 25 residues of N. crassa cytochrome c heme lyase [7]) for 15 min at 0°C. DSG was added, and further treatment and analysis by immunostaining of Tom40 were performed as described in the legend to Fig. 2A.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Alconada A, Kübrich M, Moczko M, Hönlinger A, Pfanner N. The mitochondrial receptor complex: the small subunit Mom8b/Isp6 supports association of receptors with the general insertion pore and transfer of preproteins. Mol Cell Biol. 1995;15:6196–6205. - PMC - PubMed
    1. Bjellqvist B, Sanchez J C, Pasquali C, Ravier F, Paquet N, Frutiger S, Hughes G J, Hochstrasser D. Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins. Electrophoresis. 1993;14:1375–1378. - PubMed
    1. Brix J, Dietmeier K, Pfanner N. Differential recognition of preproteins by the purified cytosolic domains of the mitochondrial import receptors Tom20, Tom22, and Tom70. J Biol Chem. 1997;272:20730–20735. - PubMed
    1. Crowley K S, Liao S, Worrell V E, Reinhart G D, Johnson A E. Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore. Cell. 1994;78:461–471. - PubMed
    1. Daum G, Böhni P C, Schatz G. Import of proteins into mitochondria: cytochrome b2 and cytochrome c peroxidase are located in the intermembrane space of yeast mitochondria. J Biol Chem. 1982;257:13028–13033. - PubMed

Publication types

MeSH terms

LinkOut - more resources